Polymorphisms in Toll-Like Receptor 4 Underlie Susceptibility to Tumor Induction by the Mouse Polyomavirus

PERA/Ei (PE) mice are susceptible to tumor induction by polyomavirus (Py), while C57BR/cdJ (BR) mice are resistant. Antigen-presenting cells from BR mice respond to the virus with interleukin-12 (IL-12) and those from PE mice with IL-10. These polarized cytokine responses underlie the development of effective antitumor immunity in BR mice and the lack thereof in PE mice. An ex vivo cytokine production assay using spleen cells from infected [PE × BR] F2 mice together with a genome-wide SNP (single-nucleotide polymorphism)-based QTL (quantitative trait locus) analysis was used to map the determinant of cytokine production to a region of chromosome 4 carrying the Toll-like receptor 4 (TLR4) gene. Genotyping of infected F2 mice showed concordance of TLR4 allele-specific DNA sequences with the cytokine profile. Cytokine responses elicited by Py are MyD88 dependent. Bacterial lipopolysaccharide (LPS), a known TLR4 ligand, induced the same polarized responses as the virus in these host strains. Spleen cells from C3H/HeJ and C57BL/10ScNJ LPS-nonresponsive mice challenged in vitro with Py showed an impaired IL-12 response but were unaffected in IL-10 production. TLR4s of strains PE and BR differ by 3 amino acid substitutions, 2 in the extracellular domain and 1 in the intracellular domain. cDNAs encoding the TLR4s signaled equally to an NF-κB reporter in 293 cells in a ligand-independent manner. When introduced into TLR2/TLR4 double-knockout macrophages, the TLR4 cDNA from BR mice conferred a robust IL-12 response to Py and no IL-10 response. The TLR4 cDNA from PE mice failed to confer a response with either cytokine. These results establish TLR4 as a key mediator of the cytokine response governing susceptibility to tumor induction by Py.     

Treatment-independent miRNA signature in blood of wilms tumor patients

ABSTRACT: BACKGROUND: Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared themiRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOPprotocol 2001. RESULTS: We did not find a significant difference between miRNA signature of both groups. However both, Wilmstumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. Thesignature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients afterchemotherapy an accuracy of 97.0%, each as compared to healthy controls. CONCLUSION: Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of fourweeks preoperative chemotherapy treatment.     

Controlling and sampling adult sand flies with a fumigant containing permethrin and deltamethrin

The efficacy of a new smoke-generating formulation (fumigant, MidMos Solutions Ltd., GB), containing the active ingredients permethrin and deltamethrin, was evaluated against adult sand flies in an apartment (280 m(3)), a semi-open large animal shelter (enclosing an area of 300 m(2)), a closed Bedouin animal tent (104 m(3)), and a garden (141 m(2)) enclosed by a stone wall. In each location, four cages with approx. 100 Phlebotomus papatasi were exposed to the fumigant 0.5 m and 2.0 m above ground for 15 and 60 min. Controls were kept in untreated similar rooms and there were two repetitions. In the apartment and the animal tent, a single cartridge caused 100% mortality within 15 min. In the large animal shelter, one fumigant caused mortality of 86% in the lower cages and 75% in the upper cages after 15 min. After 60 min, mortality was 94 and 87%, respectively. With two fumigants, mortality was 98.5 and 91% after 15 min and after 60 min all sand flies were dead. In the garden, one fumigant caused mortality of 93% in the lower cages and 85.5% in the upper cages after 15 min. After 60 min the mortality was 98 and 92%, respectively. With two fumigants, all flies were dead within 15 min.     

The association between distance to water pipes and water bodies positive for anopheline mosquitoes (Diptera: Culicidae) in the urban community of Malindi, Kenya.

The increasing risk of mosquito-borne diseases in African urban environments has been partly attributed to failed planning and resource underdevelopment. Though engineered systems may reduce mosquito proliferation, there are few studies describing this relationship. This study investigates how engineered systems such as roads and piped water systems affect the odds of anopheline immatures (i.e., larvae and pupae) occurring in water bodies located in Malindi, Kenya. Anopheles gambiae s.s. (Giles), An. arabiensis (Patton), and An. merus (Dointz) were identified in urban Malindi, with Anopheles gambiae s.s. being the predominant species identified. The Breslow-Day test was used to explore interactions among independent variables. Logistic regression was used to test whether water bodies positive for anopheline immatures are associated with engineered systems, while controlling for potential confounding and interaction effects associated with urban water body characteristics. Water bodies more than 100 m from water pipes were 13 times more likely to have anopheline immatures present, compared to water bodies that were less than 100 m from water pipes (OR = 13.54, 95% CI: 3.15-58.23). Roads were not significantly associated with water bodies positive for anopheline immatures. Statistical interaction was detected between water body substrate type and distance to water pipes. This study provides insight into how water pipes influence the distribution of water bodies positive with immature anophelines in urban environments.     

Larval fish assemblages and geostrophic circulation in Bahia de La Paz and the surrounding southwestern region of the Gulf of California

We analyze spatial–temporal relationship between larvalfish assemblages and geostrophic surface flow in Bahíade La Paz and the neighboring Gulf of California (May, Julyand October 2001 and February 2002). The analysis of fish larvaedistribution in relation to geostrophic circulation and hydrographyis an innovative interdisciplinary approach for the understandingof fish larvae ecology. The Bray–Curtis Index definedtwo larval fish assemblages with spatial–temporal variations:coastal assemblage dominated by epipelagic coastal species (e.g.Sardinops caeruleus) and oceanic assemblages—oceanic andtransitional oceanic assemblages both dominated by mesopelagicspecies (e.g. Vinciguerria lucetia and Benthosema panamense)but with different larval abundance. The assemblage variationsappear to be related to water exchange between the bay and theGulf through the North Mouth. During July–October, thegeostrophic flow through the entrance is strong, and the oceanicassemblages spread in the whole bay, whereas during February–Maywhen the geostrophic transport is weak, the coastal assemblageis distributed over the whole bay. The strong summer–autumnwater interchange between bay and gulf is in agreement withthe annual evolution of the surface water properties insidethe bay, from high-salinity Gulf of California Water duringwinter–spring to fresher Tropical Surface Water duringsummer–autumn, when the highest species number was recorded.     

Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system

The purpose of our study was to create functional muscle tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional muscle-neuronal cultures were established by co-cultivating primary rat skeletal muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) -subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR -subunit to the -subunit without neural agrin activity.A.D.B. and J.P.B. contributed equally to this studyThis work was supported by a major grant from the State of Baden-Württemberg within the scope of the Valley TEC (Valley Tissue Engineering Center)     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.