Identification of KCNJ15 as a Susceptibility Gene in Asian Patients with Type 2 Diabetes Mellitus

Recent advances in genome research have enabled the identification of new genomic variations that are associated with type 2 diabetes mellitus (T2DM). Via fine mapping of SNPs in a candidate region of chromosome 21q, the current study identifies potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15) as a new T2DM susceptibility gene. KCNJ15 is expressed in the β cell of the pancreas, and a synonymous SNP, rs3746876, in exon 4 (C566T) of this gene, with T allele frequency among control subjects of 3.1%, showed a significant association with T2DM affecting lean individuals in three independent Japanese sample sets (p = 2.5 × 10⁻⁷, odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.76–3.67) and with unstratified T2DM (p = 6.7 × 10⁻⁶, OR = 1.76, 95% CI = 1.37–2.25). The diabetes risk allele frequency was, however, very low among Europeans in whom no association between this variant and T2DM could be shown. Functional analysis in human embryonic kidney 293 cells demonstrated that the risk allele of the synonymous SNP in exon 4 increased KCNJ15 expression via increased mRNA stability, which resulted in the higher expression of protein as compared to that of the nonrisk allele. We also showed that KCNJ15 is expressed in human pancreatic β cells. In conclusion, we demonstrated a significant association between a synonymous variant in KCNJ15 and T2DM in lean Japanese patients with T2DM, suggesting that KCNJ15 is a previously unreported susceptibility gene for T2DM among Asians.     

A Spread-Deficient Cytomegalovirus for Assessment of First-Target Cells in Vaccination

Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-ΔM94 is apathogenic and protective against wild-type challenge even in highly susceptible IFNαβR^−/− mice. MCMV-ΔM94 was able to induce a robust CD4⁺ and CD8⁺ T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8⁺ T cells in vivo. Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV-ΔM94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.The human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes severe disease primarily in the immunocompromised or immunologically immature individual. Reactivation from latent infection can occur when the balance between viral maintenance and immune control is disturbed (56). HCMV is able to productively infect a large variety of different cell types and is thus associated with a broad spectrum of diseases. In adults, the main risk groups are immunocompromised patients such as transplant recipients and AIDS or tumor patients (73). Further, congenital HCMV infection represents the most common infectious cause of brain damage in children (20). Despite extensive efforts, no effective vaccine against HCMV is available to date, although its development is considered to be of greatest importance (70). Design of a protective HCMV vaccine must serve complex purposes of safety and immunogenicity due to the diversity of disease manifestations and the heterogeneous immune status of the patient groups (63).The continuous passage of HCMV on fibroblasts generated the Towne strain, which was used in trials as a live attenuated vaccine. The attenuation was associated with randomly acquired mutations leading to the loss of antigens and receptors for tropism as well as genes with known or unknown functions (10). Vaccination with the Towne strain mitigated CMV disease in seronegative renal transplant recipients and protected against a low-dose virulent HCMV challenge in volunteers (51) but failed to confer protection against infection by natural contact (1, 22). Due to the undefined nature of the attenuation of the Towne strain, the focus for vaccine design shifted to a selective but well-investigated set of viral antigens and their linked effector mechanism.Other strategies included adoptive transfer of T-cell clones that protected transplant recipients (58) and transferred anti-HCMV antibodies to prevent congenital HCMV transmission (47). Further, vaccination of mice with a recombinant adenovirus expressing gB of murine CMV (MCMV), the major target for neutralizing antibodies (8), protected against MCMV disease (66). Currently, a subunit vaccine targeting both B- and T-cell responses is under investigation but has yet to prove its potential as a suitable HCMV vaccine (current clinical trials are listed at http://clinicaltrials.gov).In order to generate a CMV vaccine, further aspects deserve attention. Animal experiments proved that, although antibodies limit dissemination of recurrent virus, they were found to be not sufficient to control primary infection (32). Moreover, CD4⁺ as well as CD8⁺ T cells contain CMV infection by recognition of a multitude of T-cell epitopes (31, 57).A large number of HCMV genes counteract innate and adaptive host immune responses (reviewed in reference 62). Efficient antigen processing is critical for protection against CMV disease in the presence of viral immune evasion proteins (28). Recently, we generated an attenuated MCMV by deletion of gene regions known to encompass such immune-evasive genes (11). The resulting vaccine, Δm01-17+m144-158-MCMV, lacked 32 genes dispensable for in vitro replication and induced a protective immune response against wild-type MCMV (MCMV-wt). Although this vaccine was strongly attenuated in vivo, the possibility that such a mutant might cause harm under severe immunosuppressive conditions cannot be excluded. Prolonged replication of attenuated vaccines in immunocompromised patients has indeed been observed (37). Therefore, virus spread needs to be completely excluded while maintaining full immunogenic properties. A similar approach has so far been applied only to alphaherpesviruses in animal models (reviewed in reference 16). In these studies, the deletion of genes essential for viral DNA replication (46) or packaging (35) gave rise to mutants in which the replication cascade was terminated at different checkpoints. The antibody response to such vaccines was stronger the later the block during the viral morphogenesis was introduced (3, 46). This concept led to protective vaccines against wild-type challenge in animal models (15, 18). Some of these are now entering clinical trials (http://clinicaltrials.gov). In this study a vaccine against betaherpesvirus was evaluated in its natural host. We constructed an MCMV that cannot spread from the first infected cell. For this purpose, we deleted the gene M94, which is conserved in all herpesvirus subfamilies. The gene M94 of MCMV represents the homolog of UL94 in HCMV, which is essential (17). The encoded protein, pM94, is part of the virion (34) and, like pUL94, expressed at late time points of infection (64, 77). Thus, the deletion of M94 should not affect the expression of other viral genes.Here, we describe the properties of the MCMV-ΔM94 mutant. The MCMV-ΔM94 vaccine was well tolerated even by immune-deficient mice highly susceptible to MCMV. Vaccination induced robust humoral and cellular immune responses and conferred protection against wild-type virus challenge. MCMV-ΔM94 infection is restricted to the first-target cell, and the impact of infection of different cell types on induction of adaptive immune responses was examined. Using Cre-loxP-based conditional expression of a model antigen, we observed that mainly endothelial cells (EC) but not hepatocytes (Hc) contribute to the induction of virus-specific CD8⁺ T cells. The genome of MCMV-ΔM94 was found to be maintained in mice for at least 1 year, indicating that first-target cells of the vaccine are long-lived and could serve as an antigen reservoir for prolonged activation of the immune system.     

Being a parasitoid of parasites: host finding in the tick wasp Ixodiphagus hookeri by odours from mammals

The parasitic wasp Ixodiphagus hookeri Howard (Hymenoptera: Encyrtidae) parasitizes larvae and nymphs of a number of tick species worldwide. Ticks themselves are parasitic on vertebrate hosts. To study the specificity and reliability of vertebrate odours used by I. hookeri for host location, we conducted bioassays in a four‐chamber olfactometer. Wasps were arrested by carbon dioxide and by odours from roe deer faeces and odours from hair of roe deer and wild boar. Odours from faeces of cattle, rabbit, and field mouse as well as odours from hair of cattle and field mouse had no effect. Odours from faeces of the host tick species Ixodes ricinus L. (Acari: Ixodidae) were attractive only up to a distance of 1 cm. Thus, I. hookeri reacts to general (carbon dioxide) and specific vertebrate odours from wild boar and deer. Examination of freshly shot specimens demonstrates that deer and wild boars are infested with a sufficient number of tick nymphs to tap the full reproductive potential of an I. hookeri female, which makes cues from these mammal species reliable. These results indicate that I. hookeri locates its hosts using specific and reliable mammal odours and that ticks are parasitized on their vertebrate hosts. The implications of this host‐finding strategy and its benefits for the parasitoid are discussed.     

Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters

Cephalosporin C amidase was covalently attached using a protein loading of 7.0–200 mg protein/g dry carrier on four epoxy‐activated Sepabeads differing in particle size and pore diameter. Initial‐rate kinetic analysis showed that for Sepabeads with small pore diameters (30–40 nm), the apparent K_M of the amidase for hydrolysis of cephalosporin C at 37°C and pH 8.0 increased ～3‐fold in response to increased particle size (～120–400 µm) and increased amount of immobilized enzyme (7.0–70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150–250 nm), the K_M was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K_M on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH‐sensitive dye fluorescein, and Ru(II) tris(4,7‐diphenyl‐1,10‐phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal‐to‐noise ratio compared to fluorescence intensity‐based measurements using singly labeled particles. The difference at apparent steady state (ΔpH) between bulk (external pH) and intraparticle pH (internal pH) was as large as ～0.6 units. The ΔpH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier‐bound biocatalyst. Biotechnol. Bioeng. 2010;106: 528–540. © 2010 Wiley Periodicals, Inc.     

Model-based boosting in high dimensions

SUMMARY: The R add-on package mboost implements functional gradient descent algorithms (boosting) for optimizing general loss functions utilizing componentwise least squares, either of parametric linear form or smoothing splines, or regression trees as base learners for fitting generalized linear, additive and interaction models to potentially high-dimensional data. AVAILABILITY: Package mboost is available from the Comprehensive R Archive Network (http://CRAN.R-project.org) under the terms of the General Public Licence (GPL).     

Physical and chemical interactions between aphids and plants

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca(2+) influx, possibly mediated by mechano sensitive Ca(2+) channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.     

The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion

Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel alpha subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv alpha subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.     

plprot: a comprehensive proteome database for different plastid types

Different plant plastid types contain a distinct protein complement for specialized functions and metabolic activities. plprot was established as a plastid proteome database to provide information about the proteomes of chloroplasts, etioplasts and undifferentiated plastids. The current version of plprot features 2,043 protein entries and consists of two modules. Module one contains a BLAST search option and provides comparative information on the proteomes of different plastid types. The second module contains four searchable databases, three for each individual plastid type and one comprehensive composite database that provides the results of plastid proteome analyses from different laboratories. plprot is accessible at http://www.plprot.ethz.ch.