Six sequence variants on chromosome 9p21.3 are associated with a positive family history of myocardial infarction: a multicenter registry

Background

Recent genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) and myocardial infarction (MI). The 9p21.3 locus was verified by numerous replication studies to be the first common locus for CAD and MI. In the present study, we investigated whether six single nucleotide polymorphisms (SNP) rs1333049, rs1333040, rs10757274, rs2383206, rs10757278, and rs2383207 representing the 9p21.3 locus were associated with the incidence of an acute MI in patients with the main focus on the familial aggregation of the disease.

Methods

The overall cohort consisted of 976 unrelated male patients presenting with an acute coronary syndrome (ACS) with ST-elevated (STEMI) as well as non-ST-elevated myocardial infarction (NSTEMI). Genotyping data of the investigated SNPs were generated and statistically analyzed in comparison to previously published findings of matchable control cohorts.

Results

Statistical evaluation confirmed a highly significant association of all analyzed SNP's with the occurrence of MI (p < 0.0001; OR: 1.621-2.039). When only MI patients with a positive family disposition were comprised in the analysis a much stronger association of the accordant risk alleles with incident disease was found with odds ratios up to 2.769.

Conclusions

The findings in the present study confirmed a strong association of the 9p21.3 locus with MI particularly in patients with a positive family history thereby, emphasizing the pathogenic relevance of this locus as a common genetic cardiovascular risk factor.

     

The two-domain NK1 fragment of plasminogen: folding, ligand binding, and thermal stability profile

The two-domain fragment N+K1 (rNK1) [Glu(1)-Glu(163)] of human plasminogen was expressed in E. coli as a hexahistidine-tagged fusion protein and chromatographically purified. The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1). The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands. The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1. Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module. Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum. Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N). Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain. In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1. Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C.     

Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase,a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD⁺-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.     

Long-term forest fire ecology and dynamics in southern Switzerland

1 Pollen and charcoal analysis at two lakes in southern Switzerland revealed that fire has had a prominent role in changing the woodland composition of this area for more than 7000 years.
2 The sediment of Lago di Origlio for the period between 5100 and 3100  bc cal. was sampled continuously with a time interval of about 10 years. Peaks of charcoal particles were significantly correlated with repeated declines in pollen of Abies , Hedera , Tilia , Ulmus , Fraxinus excelsior t., Fagus and Vitis and with increases in Alnus glutinosa t., shrubs (e.g. Corylus , Salix and Sambucus nigra t.) and several herbaceous species. The final disappearance of the lowland Abies alba stands at around 3150  bc cal. may be an example of a fire-caused local extinction of a fire-intolerant species.
3 Forest fires tended to diminish pollen diversity. The charcoal peaks were preceded by pollen types indicating human activity. Charcoal minima occurred during periods of cold humid climate, when fire susceptibility would be reduced.
4 An increase of forest fires at about 2100  bc cal. severely reduced the remaining fire-sensitive plants: the mixed-oak forest was replaced by a fire-tolerant alder–oak forest. The very strong increase of charcoal influx, and the marked presence of anthropogenic indicators, point to principally anthropogenic causes.
5 We suggest that without anthropogenic disturbances Abies alba would still form lowland forests together with various deciduous broadleaved tree taxa.     

The BD FACSPresto Point of Care CD4 Test Accurately Enumerates CD4+ T Cell Counts

Objective

Currently 50% of ART eligible patients are not yet receiving life-saving antiretroviral therapy (ART). Financial constraints do not allow most developing countries to adopt a universal test and offer ART strategy. Decentralizing CD4+ T cell testing may, therefore, provide greater access to testing, ART, and better patient management. We evaluated the technical performance of a new point-of-care CD4+ T cell technology, the BD FACSPresto, in a field methods comparison study.

Methods

264 HIV-positive patients were consecutively enrolled and included in the study. The BD FACSPresto POC CD4+ T cell technology was placed in two rural health care facilities and operated by health care facility staff. We compared paired finger-prick and venous samples using the BD FACSPresto and several existing reference technologies, respectively.

Results

The BD FACSPresto had a mean bias of 67.29 cells/ul and an r² of 0.9203 compared to the BD FACSCalibur. At ART eligibility thresholds of 350 and 500 cells/ul, the sensitivity to define treatment eligibility were 81.5% and 77.2% and the specificities were 98.9% and 100%, respectively. Similar results were observed when the BD FACSPresto was compared to the BD FACSCount and Alere Pima. The coefficient of variation (CV) was less than 7% for both the BD FACSCalibur and BD FACSPresto. CD4+ T cell testing by nurses using the BD FACSPresto at rural health care facilities showed high technical similarity to test results generated by laboratory technicians using the BD FACSPresto in a high functioning laboratory.

Conclusions

The BD FACSPresto performed favorably in the laboratory setting compared to the conventional reference standard technologies; however, the lower sensitivities indicated that up to 20% of patients tested in the field in need of treatment would be missed. The BD FACSPresto is a technology that can allow for greater decentralization and wider access to CD4+ T cell testing and ART.     

Heterogeneous pattern of retinal nerve fiber layer in multiple sclerosis. High resolution optical coherence tomography: potential and limitations

Background

Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique.

Methodology

Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP).

Principal Findings

All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9–540 months).

Conclusions

RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).     

Ultrastructure and Development of the Ooecial Walls in Some Calloporid Bryozoans (Gymnolaemata: Cheilostomata)

In the brood chambers (ovicells) of six calloporid cheilostomes studied each skeletal wall consists of four calcified layers: (1) a very thin superficial layer of planar spherulitic crystallites, (2) an upper (outer) layer with wall-perpendicular prismatic ultrastructure, (3) an intermediate lamellar layer, and (4) a lower (inner) wall-perpendicular prismatic layer. Comparative studies of both the ovicell wall ultrastructure and early ovicell formation showed a hypothetical opportunity for evolving complex (multilayered) skeletal walls by fusion of the initially separated gymnocystal and cryptocystal calcifications in Cheilostomata. In two species studied, a bilobate pattern in the final stage of the formation of the ooecial roof was encountered in specimens with the cuticle preserved. A possible explanation to this finding is discussed – the bilobate pattern is suggestive of the hypothetical origin of the brood chamber from (1) two flattened spines, or (2) reduction in spine number of an originally multispinous ovicell.     

Mechanisms of liver steatosis in rats with systemic carnitine deficiency due to treatment with trimethylhydraziniumpropionate

Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of beta-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points. A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation.     

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.