Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.     

Topotecan enhances immune clearance of gliomas

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3⁺ T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3⁺ T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-γ, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling. Jun Wei and Guillermo DeAngulo are Co-lead authors.     

<Emphasis Type="Italic">Marinobacterium marisflavi</Emphasis> sp. nov., Isolated from a Costal Seawater

A marine bacterium designated strain IMCC4074^T was isolated from surface seawater collected off Incheon Port, the Yellow Sea, and subjected to a polyphasic taxonomy. The strain was Gram-negative, chemoheterotrophic, slightly halophilic, strictly aerobic, and motile rods. Based on 16S rRNA gene sequence comparisons, the strain was most closely related to Marinobacterium litorale KCTC 12756^T (93.9%) and shared low 16S rRNA gene sequence similarities with members of the genus Marinobacterium (91.8–93.9%) and the genus Neptunomonas (93.4%) in the order Oceanospirillales. Phylogenetic analyses showed that this marine isolate formed an independent phyletic line within the genus Marinobacterium clade. The DNA G+C composition of the strain was 56.0 mol% and the predominant constituents of the cellular fatty acids were C_16:0 (28.0%), C_16:1 ω7c and/or iso-C_15:0 2-OH (19.3%), C_18:1 ω7c (17.8%), and C_17:1 cyclo (12.5%), which differentiated the strain from other Marinobacterium species. Based on the taxonomic data collected in this study, only a distant relationship could be found between strain IMCC4074^T and other members of the genus Marinobacterium, thus the strain represents a novel species of the genus Marinobacterium, for which the name Marinobacterium marisflavi sp. nov. is proposed. The type strain of Marinobacterium marisflavi is IMCC4074^T (= KCTC 12757^T = LMG 23873^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IMCC4074^T is EF468717. An erratum to this article can be found at     

Salubrinal, an inhibitor of protein synthesis, promotes deep slow wave sleep

Previous work showed that sleep is associated with increased brain protein synthesis and that arrest of protein synthesis facilitates sleep. Arrest of protein synthesis is induced during the endoplasmic reticulum (ER) stress response, through phosphorylation of eukaryotic initiation factor 2alpha (p-eIF2alpha). We tested a hypothesis that elevation of p-eIF2alpha would facilitate sleep. We studied the effects of intracerebroventricular infusion of salubrinal (Salub), which increases p-eIF2alpha by inhibiting its dephosphorylation. Salub increased deep slow wave sleep by 255%, while reducing active waking by 49%. Delta power within non-rapid eye movement (NREM) sleep was increased, while power in the sigma, beta, and gamma bands during NREM was reduced. We found that Salub increased expression of p-eIF2alpha in the basal forebrain (BF) area, a sleep-wake regulatory brain region. Therefore, we quantified the p-eIF2alpha-immunolabeled neurons in the BF area; Salub administration increased the number of p-eIF2alpha-expressing noncholinergic neurons in the caudal BF. In addition, Salub also increased the intensity of p-eIF2alpha expression in both cholinergic and noncholinergic neurons, but this was more widespread among the noncholinergic neurons. Our findings support a hypothesis that sleep is facilitated by signals associated with the ER stress response.     

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer’s disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.     

Nebularine Affects Plant Growth and Development but does not Interfere with Cytokinin Signaling

Nebularine is known for its high cytotoxicity in animals, whereas in plants it was originally believed to be an anticytokinin. In this study we show that in classical cytokinin bioassays, nebularine antagonized cytokinin function in senescence and callus biotests but not in the Amaranthus bioassay. Nebularine reversed the inhibitory effect of cytokinin on lateral root formation in Arabidopsis seedlings, and when applied alone caused increased lateral root formation and shortening of the main root. Systematic spraying of Arabidopsis plants with nebularine led to yellowing and formation of purple pigments, local drying, and withering, although younger plants showed a greater resilience. Comparison of nebularine cytotoxicity in plant and animal cells showed that the growth of tobacco BY-2 cells was inhibited with only about tenfold lower efficacy than mammalian cell lines. Most importantly, direct binding assay with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 showed that nebularine did not compete for binding with the natural cytokinin trans-zeatin. Although nebularine reduced cytokinin-induced expression of the cytokinin reporter ARR5:GUS in planta, the same effect was observed for DR5:GUS, an auxin reporter gene. Taken together, the results indicate that the mode of action of nebularine does not involve cytokinin signaling and that the anticytokinin-like effect is rather a consequence of the inhibition of various processes as described for animal systems.     

Waste recycling: utilization of coffee grounds and kitchen waste in vermicomposting

Vermicomposting using Lumbricus rubellus for 49 days was conducted after 21 days of pre-composting. Three different combination of treatments were prepared with eight replicates for each treatment namely cow dung: kitchen waste in 30:70 ratio (T(1)), cow dung: coffee grounds in 30:70 ratio (T(2)), and cow dung: kitchen waste: coffee grounds in 30:35:35 ratio (T(3)). The multiplication of earthworms in terms of numbers and weight were measured at the end of vermicomposting. Consequently, only T(2) showed significant increase (from it initial stage) compared to other treatments. The presence of coffee grounds in T(2) and T(3) showed higher percentage of nutrient elements in vermicompost produced. The data reveal that coffee grounds can be decomposed through vermicomposting and help to enhance the quality of vermicompost produced rather than sole use of kitchen waste in vermicomposting.     

GENETICS OF INCIPIENT SPECIATION IN DROSOPHILA MOJAVENSIS: II. HOST PLANTS AND MATING STATUS INFLUENCE CUTICULAR HYDROCARBON QTL EXPRESSION AND G × E INTERACTIONS

We performed a quantitative trait locus (QTL) analysis of epicuticular hydrocarbon variation in 1650 F₂ males from crosses of Baja California and mainland Mexico populations of Drosophila mojavensis cultured on two major host cacti. Principal component (PC) analysis revealed five PCs that accounted for 82% of the total epicuticular hydrocarbon variation. Courtship trials with mainland females were used to characterize hydrocarbon profiles of mated and unmated F₂ males, and logistic regression analysis showed that cactus substrates, two PCs, and a PC by cactus interaction were associated with mating success. Multiple QTLs were detected for each hydrocarbon PC and seven G × E (cactus) interactions were uncovered for the X, second, and fourth chromosomes. Males from the courtship trials and virgins were used, so "exposure to females" was included as a factor in QTL analyses. "Exposed" males expressed significantly different hydrocarbon profiles than virgins for most QTLs, particularly for the two PCs associated with mating success. Ten QTLs showed G × E (exposure) interactions with most resulting from mainland genotypes expressing altered hydrocarbon amounts when exposed to females compared to Baja genotypes. Many cactus × exposure interaction terms detected across QTL and all PCs confirmed that organ pipe-reared males expressed significantly lower hydrocarbon amounts when exposed to females than when reared on agria cactus. Epicuticular hydrocarbon variation in D. mojavensis is therefore a multigenic trait with some epistasis, multiple QTLs exhibited pleiotropy, correlated groups of hydrocarbons and cactus substrates determined courtship success, and males altered their hydrocarbon profiles in response to females.