Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

Transformation of pecan and regeneration of transgenic plants

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of Elliott, Wichita, and Schley were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line Elliott6, 3 from Schley5/3, and 3 from Wichita9. Transgenic embryos of Wichita9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3′ of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3′ 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3′ portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.Several plant viruses have been developed as tools for various biotechnology applications, including expression platforms for protein production in plants (1, 2, 6) and as gene silencing systems as part of reverse genetics approaches toward understanding host plant gene function (4, 5). For both of these applications, nonviral sequences conferring the desired function are cloned into the virus genome in order to be expressed during replication in plants. One advantage of using viruses engineered with nonviral sequences is flexibility in manipulating these “extra” sequences, which are not essential for viral replication or movement (1). However, recombinant viruses also tend to lose these sequences, causing instability at the insertion site and resulting in loss of function of the recombinant viral vector. The relatively high error rates of viral replicases (7, 14, 24) and the propensity for recombination events (9) contribute to the instability often seen with some viral vector systems.Recombination events in RNA viruses typically result in joining of two noncontiguous RNA segments (16). These could be sequences from two separate RNA molecules or distant regions of the same molecule. Retention by viruses of favorable sequences is selection driven and eliminates sequences that are unnecessary or negatively affect fitness (11, 31), hence making recombination critical to virus evolution (13, 29). Although phylogenetic analyses predict that recombination events have affected evolution for essentially all groups of RNA viruses (3), some viruses appear to be more prone to recombination than others. For example, plant-infecting supergroup II viruses of the family Tombusviridae appear to undergo frequent recombination, as is supported by the many well-characterized defective-interfering (DI) RNAs of Tomato bushy stunt virus (TBSV) (10, 30). The TBSV DI RNAs, derived entirely from the parental viral RNA, are not replication competent alone and depend on the parent virus to replicate them in trans. Recent developments of in vitro systems (19, 21) have further enhanced dissection of recombination mechanisms giving rise to TBSV DI RNAs.Of the proposed mechanisms for viral recombination (12, 20), the copy choice or template-switching mechanism is the most widely reported (8, 16, 18). This occurs when the viral replicase and its attached nascent polynucleotide chain switches viral RNA templates (or jumps locations on the same template) when making cRNA. Some properties for preferred donor/acceptor sites (sequences in the RNA molecule at which viral replicase switches from one template to another) are known for various RNA viruses (3, 20, 27), suggesting that recombination is not entirely random.The previously described TBSV vectors were efficient silencers of host genes but only while the inserted sequences were retained (23). Thus, optimizing viral vectors requires a better understanding of factors responsible for recombination and consequent loss of insert sequences. In order to address possible recombination mechanisms, we used previously characterized sequence-specific microRNA (miRNA)-guided cleavage determinants as parts of our TBSV vectors. We introduced the miRNA target site sequences for miR171 or miR159 3′ to the silencing inserts of our TBSV vectors (23). After plant inoculation, we analyzed recombinant virus sequences, determining specific recombination patterns, and demonstrated miRNA-mediated recombination events in vivo for the recombinant TBSV vectors. We also showed miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage for recombinant TBSV RNA and evidence supporting the TBSV RNA-dependent RNA polymerase (RdRp) switching templates during cRNA synthesis.