Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

PRAF1: a Golgi complex transmembrane protein that interacts with viruses.

Prenylated Rab acceptor domain family member 1 (PRAF1), a transmembrane protein whose precise function is unknown, localizes to the Golgi complex, post-Golgi vesicles, lipid rafts, endosomes, and the plasma membrane. VAMP2 and Rab3A are SNARE proteins that interact with PRAF1, and, as part of a SNARE complex, PRAF1 may function in the regulation of docking and fusion of transport vesicles both in the Golgi complex and at the plasma membrane. Alternately, PRAF1 may function as a sorting protein in the Golgi complex. In addition to interacting with SNARE proteins, PRAF1 interacts with rotaviral, retroviral, and herpes viral proteins. The function of viral protein interaction is unknown, but PRAF1 may enhance rotaviral and retroviral assembly. In contrast, PRAF1 may inhibit the herpes virus life cycle.     

Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.     

Assessing the trypanocidal potential of natural and semi-synthetic diketopiperazines from two deep water marine-derived fungi

Human African trypanosomiasis (HAT, commonly known as African sleeping sickness) is categorized as a neglected disease, as it afflicts >50,000 people annually in sub-saharan Africa, and there are few formal programs in the world focused on drug discovery approaches for this disease. In this study, we examined the crude extracts of two fungal strains (Aspergillus fumigatus and Nectria inventa) isolated from deep water sediment which provided >99% growth inhibition at 1 μg/mL of Trypanosoma brucei, the causative parasite of HAT. A collection of fifteen natural products was supplemented with six semi-synthetic derivatives and one commercially available compound. Twelve of the compounds, each containing a diketopiperazine core, showed excellent activity against T. brucei (IC₅₀ = 0.002–40 μM), with selectivity over mammalian cells as great as 20-fold. The trypanocidal diketopiperazines were also tested against two cysteine protease targets Rhodesain and TbCatB, where five compounds showed inhibition activity at concentrations less than 20 μM. A preliminary activity pattern is described and analyzed.     

The Glycoprotein B Disintegrin-Like Domain Binds Beta 1 Integrin To Mediate Cytomegalovirus Entry

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact β1 integrin ectodomain. Furthermore, a physical association between gB-DLD and β1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.Like many other herpesviruses, human cytomegalovirus (HCMV) is an opportunistic pathogen that is able to asymptomatically infect the human population with high incidence throughout the world. Primary infection is followed by a life-long latent phase that may reactivate and cause disease during the immunosuppression experienced by AIDS patients and organ transplant recipients (14, 52). HCMV disease is also a cause of significant morbidity and mortality during primary congenital infections (66). Currently there is no effective HCMV vaccine, and HCMV antiviral therapies, such as ganciclovir, are highly toxic and unsuitable for treating pregnant women in the congenital setting (92).HCMV disease can manifest itself in most organ systems and tissue types. Pathology from HCMV-infected individuals reveals that HCMV can infect most cell types, including fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, stromal cells, monocytes/macrophages, neutrophils, neuronal cells, and hepatocytes (20, 25, 77, 83, 87). The broad intrahost organ and tissue tropism of HCMV is paralleled in vitro with the virus'' ability to bind and fuse with nearly every vertebrate cell type tested (40, 62, 78). However, full productive infection is limited to secondary strains of fibroblasts and endothelial cells. The ability of HCMV to enter such a diverse range of cell types is indicative of multiple cell-specific receptors, broadly expressed receptors, or a complex entry pathway in which a combination of both cell-specific and broadly expressed cellular receptors are utilized.The genes that encode envelope glycoprotein B (gB) and gH are essential (37), play several key roles during virus entry and egress, and are conserved throughout the Herpesviridae (reviewed in reference 80). A soluble form of gB truncated at the transmembrane domain (gBs) binds to permissive cells specifically, blocks virus entry, and is sufficient to trigger signal transduction events that result in the activation of an interferon-responsive pathway that is also activated by HCMV virions (10, 12, 13).HCMV entry requires initial tethering of virions to cell surface heparan sulfate proteoglycans (HSPGs) (22, 80). The HCMV envelope contains at least two separate glycoprotein complexes with affinities for heparan sulfate: gB (22) and the gM/gN complex (48). The gM/gN complex is more abundant than gB within the envelope (88) and binds heparin with higher affinity (49). Thus, the gM/gN complex is thought to be the primary heparin-binding component of the HCMV envelope.Virus-cell tethering via HSPGs is followed by a more stable interaction and subsequent signal transduction cascades. This interaction was proposed to be mediated via cell surface epidermal growth factor receptor (EGFR) (17, 95). These data, however, conflicted with more recent reports that demonstrate EGFR is not explicitly required for infection (21, 42). Platelet-derived growth factor receptor (PDGFR) has also been reported to function as an attachment receptor that functions to activate signaling cascades required for infection (79). The relative contribution of signaling and virus-host cell attachment for each of these growth factor receptors remains to be further characterized. The possibility also exists that additional attachment receptors still remain unidentified.Integrins are expressed on the cell surfaces of all vertebrate cells, a characteristic that parallels the promiscuity of HCMV entry. Additionally, β1 integrins are capable of mediating many of the same signal transduction pathways that are triggered during HCMV entry into host cells. Upon binding and fusing with host cell surfaces, HCMV triggers changes in Ca²⁺ homeostasis (36) and the activation of phospholipases C and A2, as well as an increased release of arachidonic acid and its metabolites (2). Additionally, mitogen-activated protein kinase (MAPK) (44, 45), phosphatidylinositol-3-OH kinase (PI3-K) (46), and G proteins are activated (73). Indeed, it was shown that HCMV entry led to an activation of integrin signaling pathways that reorganized the actin cytoskeleton (31) and phosphorylated β1 and β3 integrin cytoplasmic domains (31), focal adhesion kinase (FAK) (31), and Src (94). Integrin antibody blocking studies in combination with HCMV infectivity assays in β1 integrin-null GD25 cells identified α2β1, α6β1, and αVβ3 integrins as HCMV “postattachment” entry receptors (31). Certain integrin signaling events could be triggered by both HCMV and a soluble version of gB and require the expression of β1 integrin, identifying this specific viral ligand in integrin engagement (31).ADAM family members are multifunctional proteins that contain a metalloproteinase domain involved in ectodomain shedding and a disintegrin module of approximately 90 amino acids that confers RGD-independent integrin binding (43, 81, 99). The minimum component of the disintegrin module required for integrin engagement is the 12- to 13-amino-acid disintegrin loop, for which a consensus sequence has been described: RX₆DLXXF (29). The 20-amino-acid stretch encompassing the gB disintegrin-like domain is highly conserved, with greater than 98% amino acid identity among HCMV clinical isolates. Additionally, this domain is present in most gammaherpesviruses and all betaherpesviruses, suggesting that integrin engagement may be a conserved feature for most of the Herpesviridae. Synthetic peptides of the gB disintegrin loop block virus fusion (tegument delivery) but not virus attachment (31). This fact suggests a disintegrin-mediated molecular mechanism of herpesvirus-integrin engagement. Glycoprotein H (gH) has also been identified as an αVβ3 integrin ligand (94). However, gH contains no previously identified integrin recognition motifs, and the αVβ3 integrin heterodimer does not typically engage ADAM family proteins.Herein, we explore the molecular mechanism of integrin engagement by HCMV envelope gB. We provide multiple lines of evidence that demonstrate a physical interaction between the gB disintegrin module with β1 integrin. Furthermore, this interaction has significant consequences to the viral life cycle, since a soluble version of the gB disintegrin module efficiently blocks HCMV infection at a postattachment step during entry into multiple in vivo cell targets. Similarly, polyclonal antibodies directed against the gB disintegrin-like domain neutralize HCMV infectivity. These data identify the molecular mechanism of an HCMV ligand-receptor interaction required for virus-host fusion.     

Human cytomegalovirus envelope glycoproteins B and H are necessary for TLR2 activation in permissive cells

Human CMV (HCMV) is a ubiquitous member of the Herpesviridae family and an opportunistic pathogen that poses significant health risks for immunocompromised patients. HCMV pathogenesis is intimately tied to the immune status of the host, thus characterization of the innate immune response to HCMV infection is critical for understanding disease progression. Previously, we identified TLR2 as a host factor that detects and initiates inflammatory cytokine secretion in response to HCMV independent of viral replication. In this study, we show that two entry-mediating envelope gp, gp B (gB) and gp H (gH), display determinants recognized by TLR2. Neutralizing Abs against TLR2, gB and gH inhibit inflammatory cytokine responses to HCMV infection, suggesting that inflammatory cytokine stimulation by HCMV is mediated by interactions between these envelope gp and TLR2. Furthermore, both gB and gH coimmunoprecipitate with TLR2 and TLR1, indicating that these envelope gp directly interact with TLR2 and that a TLR2/TLR1 heterodimer is a functional sensor for HCMV. Because our previous studies were conducted in model cell lines, we also show that TLR2 is expressed by HCMV permissive human fibroblast cell strains, and that TLR2 is a functional sensor in these cells. This study further elucidates the importance and potency of envelope gp as a class of molecules displaying pathogen-associated molecular patterns that are recognized with immediate kinetics by TLRs in permissive cells.     

Hydrologic flowpaths influence inorganic and organic nutrient leaching in a forest soil

Hydrologic pathways through soil affect element leaching by determining the relative importance of biogeochemical processes such as sorption and decomposition. We used stable hydrogen isotopes of water (δD) to examine the influence of flowpaths on soil solution chemistry in a mature spruce–hemlock forest in coastal Oregon, USA. Soil solutions (50 cm depth, n = 13) were collected monthly for 1 year and analyzed for δD, major ions and dissolved organic carbon (DOC) and nitrogen (DON). We propose that the variability of δD can be used as an index of flowpath length and contact time. Throughfall variability in δD was much greater than soil solution variability, illustrating that soil solution integrates the variation in inputs. Lysimeters with greater variation in δD presumably have a greater proportion of flow through rapid flowpaths such as macropores. The variation in soil solution δD for individual lysimeters explained up to 53% of the variation in soil solution chemistry, and suggests that flowpaths influence leaching of some constituents. Soil solutions from lysimeters with greater δD variation had higher DOC and DON (r ² = 0.51 and 0.37, respectively), perhaps because transport via macropores reduces interaction of DOM with the soil matrix. In contrast, nitrate concentrations were highest in lysimeters with a small variation in δD, where long contact time and low DOC concentrations may yield higher net nitrification. Our results demonstrate the utility of stable isotopes to link flowpaths and soil solution chemistry, and illustrate how the spatial complexity of soils can influence ecosystem-level nutrient losses.     

Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase

The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of 14 subunits distributed between a peripheral V1 subcomplex and an integral membrane V0 subcomplex. Genome-wide screens have led to the identification of the newest yeast V-ATPase subunit, Vma9p. Vma9p (subunit e) is a small hydrophobic protein that is conserved from fungi to animals. We demonstrate that disruption of yeast VMA9 results in the failure of V1 and V0 V-ATPase subunits to assemble onto the vacuole and in decreased levels of the subunit a isoforms Vph1p and Stv1p. We also show that Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER), which then localizes to the limiting membrane of the vacuole. All V0 subunits and V-ATPase assembly factors are required for Vma9p to efficiently exit the ER. In the ER, Vma9p and the V0 subunits interact with the V-ATPase assembly factor Vma21p. Interestingly, the association of Vma9p with the V0-Vma21p assembly complex is disrupted with the loss of any single V0 subunit. Similarly, Vma9p is required for V0 subunits Vph1p and Vma6p to associate with the V0-Vma21p complex. In contrast, the proteolipids associate with Vma21p even in the absence of Vma9p. These results demonstrate that Vma9p is an integral membrane subunit of the yeast V-ATPase V0 subcomplex and suggest a model for the arrangement of polypeptides within the V0 subcomplex.     

Factors Influencing Realized Sex Ratios in Fig Wasps: Double Oviposition and Larval Mortalities

Pollinator fig wasps (Agaonidae) are a model system for studies of sex ratio evolution. They lay their eggs in galled ovules within figs. Only one adult emerges from each gall, suggesting that only one egg is always laid per ovule, but if double oviposition occurs then the assumption that adult (realised) sex ratios of fig wasps are representative of primary sex ratios may be violated. Many galls also fail to produce any wasps. If they initially contained eggs then differential mortality rates may also modify realized sex ratios. We investigated whether Kradibia (= Liporrhopalum) tentacularis foundresses in Ficus montana figs avoid laying in ovules that already contain eggs. Comparisons of oviposition frequencies and wasp emergence frequencies showed that most galls that failed to produce wasps will have had eggs laid in them, but few occupied ovules contained two eggs. Realised sex ratios therefore do not necessarily reflect primary sex ratios in this species, but double oviposition is not responsible.