Autosomal interactions and mechanisms of pyrethroid resistance in house flies, Musca domestica

Five BC? lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.     

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(−)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC₅₀) values for the R-form of 2HG varied from approximately 25 μM for the histone N^ɛ-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.     

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact ^1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible^8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays¹⁰. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.Download video file.^{(47M, mov)}     

The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence

Background

Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga.

Principal Findings

A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support.

Conclusion

All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.     

The 3' UTR variants in the GRP78 are not associated with overall survival in resectable hepatocellular carcinoma

Background

Elevated glucose-regulated protein 78 (GRP78) levels in tissues have been known to be related with poor prognosis in hepatocellular carcinoma (HCC) patients. Though the variants in the 3′ untranslated region (UTR) of GRP78 gene were not associated with HCC risk, we wonder whether these polymorphisms affect survival of HCC patients.

Methodology/Principal Findings

Blood samples of HCC patients were maintained in our specimen bank between 1996 to 2003. DNA from 576 unrelated and resectable patients with HCC was typed for rs16927997 (T>C), rs1140763 (T>C) and rs12009 (T>C) by TaqMan assays. The Kaplan-Meier method and log-rank test were used to estimate overall survival. Linkage disequilibrium (LD) analysis identified a total of 3 haplotypes and 6 diplotypes in this region. The distribution of haplotype was not related to the clinical characteristics. Univariate analysis showed that the allele, genotype, haplotype and diplotype did not effect the survival. None of the clinical features show a significant association (P _correced>0.05) with overall patient outcome in multiple comparisons.

Conclusions/Significance

There is no noteworthy influence of 3′ UTR variants in the GRP78 on prognosis of resectable HCC in the Chinese population.     

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.     

A Chinese Creutzfeldt-Jakob disease patient with E196K mutation in PRNP

Genetic Creutzfeldt-Jakob disease (gCJD) is caused by a range of mutations in the prion protein gene (PRNP) and account for approximately 10–15% of overall human prion diseases worldwide. They are different with disease onset, disease duration, clinical signs and diagnostic findings. Here we reported a 71 year-old female with an E196K mutation in one PRNP allele, while the codon 129 was a methionine homozygous genotype. The patient started with non-specific symptoms, but displayed rapidly progressive disturbances of speech, memory, cognitive and physical movement. No periodic activity was recorded at electroencephalography (EEG) during the entire disease course. Retrospective investigation of her family members did not reveal similar neurological disorders. Total clinical course was about seven months.Key words: Creutzfeldt-Jakob disease, PRNP, E196K