Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ~1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR-mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease.     

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10⁻²⁰⁴) and 10 loci for sphingolipids (smallest P-value = 3.10×10⁻⁵⁷). After a correction for multiple comparisons (P-value<2.2×10⁻⁹), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.     

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims

Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.

Methods

MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.

Results

Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.

Conclusions

MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.     

Response of anaerobic ammonium-oxidizing bacteria to hydroxylamine

Anaerobic ammonium oxidation is a recent addition to the microbial nitrogen cycle, and its metabolic pathway, including the production and conversion of its intermediate hydrazine, is not well understood. Therefore, the effect of hydroxylamine addition on the hydrazine metabolism of anaerobic ammonium-oxidizing (anammox) bacteria was studied both experimentally and by mathematical modeling. It was observed that hydroxylamine was disproportionated biologically in the absence of nitrite into dinitrogen gas and ammonium. Little hydrazine accumulated during this process; however, rapid hydrazine production was observed when nearly all hydroxylamine was consumed. A mechanistic model is proposed in which hydrazine is suggested to be continuously produced from ammonium and hydroxylamine (possibly via nitric oxide) and subsequently oxidized to N(2). The electron acceptor for hydrazine oxidation is hydroxylamine, which is reduced to ammonium. A decrease in the hydroxylamine reduction rate, therefore, leads to a decrease in the hydrazine oxidation rate, resulting in the observed hydrazine accumulation. The proposed mechanism was verified by a mathematical model which could explain and predict most of the experimental data.     

Correlation of low molecular weight advanced glycation end products and nitric oxide metabolites with chronic complications in type 1 diabetic patients

Advanced glycation end products (AGEs) are involved in the occurrence of vascular complications in diabetes. The present study was undertaken to investigate the level of low-molecular weight products of AGEs (LMW-AGEs) in relation to microvascular complications in type 1 diabetes, and the possible relationship with nitric oxide (NO) as a marker of endothelial function. Patients with normal renal function (NRF) were classified into two groups: (1) without, and (2) with diabetic neuropathy; and patients with renal impairment also into two groups: (3) diabetic renal disease, and (4) end-stage renal disease. The fluorescence of LMW-AGEs and measurement of NO metabolites was assessed in 277 serum samples. In addition, multiple regression analysis was performed. In group 1, LMW-AGEs level (9.3±1.1 AF%) was higher than in the control group (2.4±0.3 AF%). A trend in the increase of LMW-AGEs with neuropathy (29.7±5.5 AF%, group 2), and further with renal impairment (47.0±8.0, group 3 and 137.8±25.5 AF%, group 4), was observed. In multivariate regression analysis LMW-AGEs were associated with NO metabolites in group 2. In NRF patients, diabetic neuropathy was significantly correlated with LMW-AGEs and NO metabolites, independently of serum creatinine and duration of diabetes. This relationship suggests that the NO and LMW-AGEs’ actions (possibly synergistic) in endothelial activation possess a role in the initiation and development of diabetic microvascular complications.     

Extremely low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity

We previously reported that exposure to extremely low-frequency electromagnetic fields (ELFEFs) increases the expression and function of voltage-gated Ca2+)channels and that Ca2+ influx through Ca(v)1 channels plays a key role in promoting the neuronal differentiation of neural stem/progenitor cells (NSCs). The present study was conducted to determine whether ELFEFs influence the neuronal differentiation of NSCs isolated from the brain cortices of newborn mice by modulating Ca(v)1-channel function. In cultures of differentiating NSCs exposed to ELFEFs (1 mT, 50 Hz), the percentage of cells displaying immunoreactivity for neuronal markers (beta-III-tubulin, MAP2) and for Ca(v)1.2 and Ca(v)1.3 channels was markedly increased. NSC-differentiated neurons in ELFEF-exposed cultures also exhibited significant increases in spontaneous firing, in the percentage of cells exhibiting Ca2+ transients in response to KCl stimulation, in the amplitude of these transients and of Ca2+ currents generated by the activation of Ca(v)1 channels. When the Ca(v)1-channel blocker nifedipine (5 microM) was added to the culture medium, the neuronal yield of NSC differentiation dropped significantly, and ELFEF exposure no longer produced significant increases in beta-III-tubulin- and MAP2-immunoreactivity rates. In contrast, the effects of ELFEFs were preserved when NSCs were cultured in the presence of either glutamate receptor antagonists or Ca(v)2.1- and Ca(v)2.2-channel blockers. ELFEF stimulation during the first 24 h of differentiation caused Ca(v)1-dependent increases in the number of cells displaying CREB phosphorylation. Our data suggest that ELFEF exposure promotes neuronal differentiation of NSCs by upregulating Ca(v)1-channel expression and function.     

Testing Magnetic Orientation in a Solitary Subterranean Rodent Ctenomys talarum (Rodentia: Octodontidae)

To test for the hypothesis that Ctenomys talarum can use the earth's magnetic field for spatial orientation, we carried out field and laboratory experiments to analyse if C. talarum burrows present any geomagnetic orientation in their natural habitat, if C. talarum show any spontaneous directional preference when starting to excavate their burrows and if this subterranean rodent is capable to use the earth's magnetic field to orient towards a goal in a complex maze. No correlation between the burrowing direction and the earth's magnetic field was found. We could not find any evidence for any spontaneous directional preference when starting to excavate the burrows in C. talarum. The change of the horizontal vector of the geomagnetic field did not affect the ability of this rodent to orient towards a goal in an artificial labyrinth. Explanations for these results and other possible mechanisms of orientation that could be used by C. talarum are discussed.     

Carotenoid S(1) state in a recombinant light-harvesting complex of Photosystem II.

The carotenoid species lutein, violaxanthin, and zeaxanthin are crucial in the xanthophyll-dependent nonphotochemical quenching occurring in photosynthetic systems of higher plants, since they are involved in dissipation of excess energy and thus protect the photosynthetic machinery from irreversible inhibition. Nonetheless, important properties of the xanthophyll cycle carotenoids, such as the energy of their S(1) electronic states, are difficult to study and were only recently determined in organic solvents [Polívka, T. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 4914. Frank, H. A. (2000) Biochemistry 39, 2831]. In the present study, we have determined the S(1) energies of three carotenoid species, violaxanthin, lutein, and zeaxanthin, in their LHCII (peripheral light-harvesting complex of photosystem II) protein environment by constructing recombinant Lhcb1 (Lhc = light-harvesting complex) proteins containing single carotenoid species. Within experimental error the S(1) energy is the same for all three carotenoids in the monomeric LHCII, 13,900 +/- 300 cm(-1) (720 +/- 15 nm), thus well below the Q(y)() transitions of chlorophylls. In addition, we have found that, although the S(1) lifetimes of violaxanthin, lutein, and zeaxanthin differ substantially in solution, when incorporated into the LHCII protein, their S(1) states have in fact the same lifetime of about 11 ps. Despite the similar spectroscopic properties of the carotenoids bound to the LHCII, we observed a maximal fluorescence quenching when zeaxanthin was present in the LHCII complex. On the basis of these observations, we suggest that, rather than different photochemical properties of individual carotenoid species, changes in the protein conformation induced by binding of carotenoids with distinct molecular structures are involved in the quenching phenomena associated with Lhc proteins.