Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression

Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.     

Accounting for Population Structure in Gene-by-Environment Interactions in Genome-Wide Association Studies Using Mixed Models

Although genome-wide association studies (GWASs) have discovered numerous novel genetic variants associated with many complex traits and diseases, those genetic variants typically explain only a small fraction of phenotypic variance. Factors that account for phenotypic variance include environmental factors and gene-by-environment interactions (GEIs). Recently, several studies have conducted genome-wide gene-by-environment association analyses and demonstrated important roles of GEIs in complex traits. One of the main challenges in these association studies is to control effects of population structure that may cause spurious associations. Many studies have analyzed how population structure influences statistics of genetic variants and developed several statistical approaches to correct for population structure. However, the impact of population structure on GEI statistics in GWASs has not been extensively studied and nor have there been methods designed to correct for population structure on GEI statistics. In this paper, we show both analytically and empirically that population structure may cause spurious GEIs and use both simulation and two GWAS datasets to support our finding. We propose a statistical approach based on mixed models to account for population structure on GEI statistics. We find that our approach effectively controls population structure on statistics for GEIs as well as for genetic variants.     

A Layer‐Structured Electrode Material Reformed by a PO4‐O2 Hybrid Framework toward Enhanced Lithium Storage and Stability

The surface framework of LiCoO₂ is modified through a surface treatment called phosphidation, which suppressed the unwanted phase transition occurring above 4.2 V. The surface instability of LiCoO₂ toward organic electrolytes is simultaneously improved by changing its surface structure from an O₂‐based framework to a PO₄‐based framework that can protect against HF attack during cycling. Phosphidated LiCoO₂ is successfully synthesized that showed greater stability in its bulk and surface structures. The phosphidated form enables faster Li⁺ diffusion and prevents irreversible phase transitions, especially when charged above 4.2 V, and consequently demonstrates excellent cycling performance and rate capabilities. The improved kinetics and stability resulting from phosphidation make LiCoO₂ highly suitable as a high‐voltage cathode material for use in lithium ion batteries.     

Induction of RANTES and CCR5 through NF-κB Activation via MAPK Pathway in Aged Rat Gingival Tissues

Chemokine and chemokine receptor expression in gingival tissues plays a central role in periodontal disease during aging. In the present study, we explored the modulation of chemokines and chemokine receptors expression in aging rat gingival tissues. In the 24-month-old (Old) rat gingival tissues, RANTES and CCR5 mRNA and protein levels were 2–4 fold increased over those of the 6-month-old (Young) rats. The Old rats had considerable enhancement of all three of the studied MAPK activities: extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. These results suggest that age-related increases in RANTES and CCR5 expression are associated with increased IκBα, nuclear NF-κB, and MAPK activity in gingival tissues.     

Rare Variant Association Testing Under Low-Coverage Sequencing

Deep sequencing technologies enable the study of the effects of rare variants in disease risk. While methods have been developed to increase statistical power for detection of such effects, detecting subtle associations requires studies with hundreds or thousands of individuals, which is prohibitively costly. Recently, low-coverage sequencing has been shown to effectively reduce the cost of genome-wide association studies, using current sequencing technologies. However, current methods for disease association testing on rare variants cannot be applied directly to low-coverage sequencing data, as they require individual genotype data, which may not be called correctly due to low-coverage and inherent sequencing errors. In this article, we propose two novel methods for detecting association of rare variants with disease risk, using low coverage, error-prone sequencing. We show by simulation that our methods outperform previous methods under both low- and high-coverage sequencing and under different disease architectures. We use real data and simulation studies to demonstrate that to maximize the power to detect associations for a fixed budget, it is desirable to include more samples while lowering coverage and to perform an analysis using our suggested methods.     

Sterically crowded triazenides as novel ancillary ligands in copper chemistry

We have synthesized copper salts MN₃RR′ derived from the biphenyl- or m-terphenyl-substituted triazenes Tph₂N₃H (1a) and Dmp(Tph)N₃H (1b) (Dmp = 2,6-Mes₂C₆H₃ with Mes = 2,4,6-Me₃C₆H₂; Tph = 2-TripC₆H₄ with Trip = 2,4,6-i-Pr₃C₆H₂). The homoleptic copper triazenide [CuN₃Tph₂] (2a) was obtained in high yield from the metallation of 1a with mesityl copper in n-heptane, while the complex [CuN₃(Dmp)Tph] (2b) was generated by the same method in situ only. Reaction of 2a with triphenylphosphane gave the 2:1 adduct [CuN₃Tph₂(PPh₃)₂] (3a), regardless of the used complex/donor ratio, while reaction of 2a or 2b with a stochiometric amount of t-butylisonitrile afforded the 1:1 adducts [Tph₂N₃CuCNtBu] (4a) and [Dmp(Tph)N₃CuCNtBu] (4b). All new compounds (except 2b) have been characterized by ¹H NMR, ¹³C NMR and IR spectroscopy, elemental analysis, melting point (not 2a), and X-ray crystallography. The IR spectroscopic examination of the ν(CN) stretch in the isonitrile adducts 4a and 4b revealed the weaker donor character of the supporting triazenido ligands compared to related β-diketiminato ligands.     

Characterization of Genome-Methylome Interactions in 22 Nuclear Pedigrees

Genetic polymorphisms can shape the global landscape of DNA methylation, by either changing substrates for DNA methyltransferases or altering the DNA binding affinity of cis-regulatory proteins. The interactions between CpG methylation and genetic polymorphisms have been previously investigated by methylation quantitative trait loci (mQTL) and allele-specific methylation (ASM) analysis. However, it remains unclear whether these approaches can effectively and comprehensively identify all genetic variants that contribute to the inter-individual variation of DNA methylation levels. Here we used three independent approaches to systematically investigate the influence of genetic polymorphisms on variability in DNA methylation by characterizing the methylation state of 96 whole blood samples in 52 parent-child trios from 22 nuclear pedigrees. We performed targeted bisulfite sequencing with padlock probes to quantify the absolute DNA methylation levels at a set of 411,800 CpG sites in the human genome. With mid-parent offspring analysis (MPO), we identified 10,593 CpG sites that exhibited heritable methylation patterns, among which 70.1% were SNPs directly present in methylated CpG dinucleotides. We determined the mQTL analysis identified 49.9% of heritable CpG sites for which regulation occurred in a distal cis-regulatory manner, and that ASM analysis was only able to identify 5%. Finally, we identified hundreds of clusters in the human genome for which the degree of variation of CpG methylation, as opposed to whether or not CpG sites were methylated, was associated with genetic polymorphisms, supporting a recent hypothesis on the genetic influence of phenotypic plasticity. These results show that cis-regulatory SNPs identified by mQTL do not comprise the full extent of heritable CpG methylation, and that ASM appears overall unreliable. Overall, the extent of genome-methylome interactions is well beyond what is detectible with the commonly used mQTL and ASM approaches, and is likely to include effects on plasticity.     

A Genome-Wide Association Study Identifies Potential Susceptibility Loci for Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital and heterogeneous disorder characterized by the absence of intramural nervous plexuses along variable lengths of the hindgut. Although RET is a well-established risk factor, a recent genome-wide association study (GWAS) of HSCR has identified NRG1 as an additional susceptibility locus. To discover additional risk loci, we performed a GWAS of 123 sporadic HSCR patients and 432 unaffected controls using a large-scale platform with coverage of over 1 million polymorphic markers. The result was that our study replicated the findings of RET-CSGALNACT2-RASGEF1A genomic region (_rawP = 5.69×10⁻¹⁹ before a Bonferroni correction; _corrP = 4.31×10⁻¹³ after a Bonferroni correction) and NRG1 as susceptibility loci. In addition, this study identified SLC6A20 (_adjP = 2.71×10⁻⁶), RORA (_adjP = 1.26×10⁻⁵), and ABCC9 (_adjP = 1.86×10⁻⁵) as new potential susceptibility loci under adjusting the already known loci on the RET-CSGALNACT2-RASGEF1A and NRG1 regions, although none of the SNPs in these genes passed the Bonferroni correction. In further subgroup analysis, the RET-CSGALNACT2-RASGEF1A genomic region was observed to have different significance levels among subgroups: short-segment (S-HSCR, _corrP = 1.71×10⁻⁵), long-segment (L-HSCR, _corrP = 6.66×10⁻⁴), and total colonic aganglionosis (TCA, _corrP>0.05). This differential pattern in the significance level suggests that other genomic loci or mechanisms may affect the length of aganglionosis in HSCR subgroups during enteric nervous system (ENS) development. Although functional evaluations are needed, our findings might facilitate improved understanding of the mechanisms of HSCR pathogenesis.