Testing for an unusual distribution of rare variants

Technological advances make it possible to use high-throughput sequencing as a primary discovery tool of medical genetics, specifically for assaying rare variation. Still this approach faces the analytic challenge that the influence of very rare variants can only be evaluated effectively as a group. A further complication is that any given rare variant could have no effect, could increase risk, or could be protective. We propose here the C-alpha test statistic as a novel approach for testing for the presence of this mixture of effects across a set of rare variants. Unlike existing burden tests, C-alpha, by testing the variance rather than the mean, maintains consistent power when the target set contains both risk and protective variants. Through simulations and analysis of case/control data, we demonstrate good power relative to existing methods that assess the burden of rare variants in individuals.     

Stream Nitrate Responds Rapidly to Decreasing Nitrate Deposition

Ecosystem acidification and eutrophication resulting from increased deposition of dissolved inorganic nitrogen (DIN) are issues of increasing global concern. Consequently, costly policy decisions are being implemented to decrease nitrogen oxide (NO _x ) emissions. Although declining DIN deposition along with rapid declines of DIN in surface waters have been reported in parts of Europe, the same observation is just emerging in North America. Here we find a significant decline in bulk deposition NO₃ ⁻ during the later part of a 28-year record in southcentral Ontario, Canada. Despite high N retention and substantial inter-annual variability in the long-term record due to periods of drought, we find significant declines in annual NO₃ ⁻ concentrations and export at six out of 11 streams that drain upland-dominated catchments. In contrast, five streams draining primarily wetland-dominated catchments with lower levels of NO₃ ⁻ show no decreasing trend in NO₃ ⁻ concentration or export. The rapid response in stream NO₃ ⁻ to declining atmospheric inputs was observed at sites with historically moderate inputs of DIN (~870 mg m⁻² y⁻¹) in bulk deposition. Topographic features such as slope, and related catchment features including wetland cover, appear to influence which catchments will respond positively to declining DIN deposition. These findings force us to revise our original conceptualization of the N saturation status of these catchments.     

Capturing the context of maternal deaths from verbal autopsies: a reliability study of the maternal data extraction tool (M-DET)

Background

The availability of quality data to inform policy is essential to reduce maternal deaths. To characterize maternal deaths in settings without complete vital registration systems, we designed and assessed the inter-rater reliability of a tool to systematically extract data and characterize the events that precede a nationally representative sample of maternal deaths in India.

Method/Principal Findings

Of 1017 nationally representative pregnancy-related deaths, which occurred between 2001 and 2003, we randomly selected 105 reports. Two independent coders used the maternal data extraction tool (questions with coding guidelines) to collect information on antenatal care access, final pregnancy outcome; planned place of birth and care provider; community consultation, transport, admission, hospital referral; and verification of cause of death assignment. Kappa estimated inter-rater agreement was calculated and classified as poor (K≤0.4), moderate (K = 0.4-≤0.6), substantial (K = 0.6-≤0.8) and high (K>0.8) using the criteria from Landis & Koch. The data extraction tool had high agreement for gestational age, pregnancy outcome, transport, death en route and admission to hospital; substantial agreement for receipt of antenatal care, planned place of birth, readmission and referral to higher level hospital, and whether or not death occurred in the intrapartum period; moderate to substantial agreement for classification of deaths as direct or indirect obstetric deaths or incidental deaths; moderate agreement for classification of community healthcare consultation and total number of healthcare contacts; and poor agreement for the classification of deaths as sudden deaths and other/unknown cause of death. The ability of the tool to identify the most-responsible-person in labour varied from moderate agreement to high agreement.

Conclusions

This data extraction tool achieved good inter-rater reliability and can be used to collect data on events surrounding maternal deaths and for verification/improvement of underlying cause of death.     

Production of D-amino acids from D,L-5-substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity

Conversion of D,L-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine by Agrobacterium tumefaciens RU-OR involved a racemase, an hydantoinase and an unusual D-selective N-carbamylamino acid amidohydrolase which was active at alkaline pH and was not inhibited by N-carbamyl-L-amino acids. Enzyme activity was induced by growth in media containing 2-thiouracil. A mutant strain (RU-ORL5) was isolated, which expressed both the hydantoinase and N-carbamylamino acid amidohydrolase enzymes in the absence of inducer. © Rapid Science Ltd. 1998     

Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: A potential mechanism for loss of mitochondrial transmembrane potential and apoptosis

This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (ΔΨ_m), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C₂-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of ΔΨ_m. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis. (Mol Cell Biochem 278: 39–51, 2005)     

Crystal Structure of AhpE from Mycobacterium tuberculosis, a 1-Cys peroxiredoxin

All living systems require protection against the damaging effects of reactive oxygen species. The genome of Mycobacterium tuberculosis, the cause of TB, encodes a number of peroxidases that are thought to be active against organic and inorganic peroxides, and are likely to play a key role in the ability of this organism to survive within the phagosomes of macrophages. The open reading frame Rv2238c in M.tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family. The crystal structure of AhpE, determined at 1.87 A resolution (R(cryst)=0.179, R(free)=0.210), reveals a compact single-domain protein with a thioredoxin fold. AhpE forms both dimers and octamers; a tightly-associated dimer and a ring-like octamer, generated by crystallographic 4-fold symmetry. In this native structure, the active site Cys45 is in its oxidized, sulfenic acid (S-O-H) state. A second crystal form of AhpE, obtained after soaking in sodium bromide and refined at 1.90 A resolution (R(cryst)=0.242, R(free)=0.286), reveals the reduced structure. In this structure, a conformational change in an external loop, in two of the four molecules in the asymmetric unit, allows Arg116 to stabilise the Cys45 thiolate ion, and concomitantly closes a surface channel. This channel is identified as the likely binding site for a physiological reductant, and the conformational change is inferred to be important for the reaction cycle of AhpE.     

Involvement of phycobilisome diffusion in energy quenching in cyanobacteria

Nonphotochemical quenching (NPQ) of excitation energy is a well-established phenomenon in green plants, where it serves to protect the photosynthetic apparatus from photodamage under excess illumination. The induction of NPQ involves a change in the function of the light-harvesting apparatus, with the formation of quenching centers that convert excitation energy into heat. Recently, a comparable phenomenon was demonstrated in cyanobacteria grown under iron-starvation. Under these conditions, an additional integral membrane chlorophyll-protein, IsiA, is synthesized, and it is therefore likely that IsiA is required for NPQ in cyanobacteria. We have previously used fluorescence recovery after photobleaching to show that phycobilisomes diffuse rapidly on the membrane surface, but are immobilized when cells are immersed in high-osmotic strength buffers, apparently because the interaction between phycobilisomes and reaction centers is stabilized. Here, we show that when cells of the cyanobacterium Synechocystis sp. PCC 6803 subjected to prolonged iron-deprivation are immersed in 1 m phosphate buffer, NPQ can still be induced as normal by high light. However, the formation of the quenched state is irreversible under these conditions, suggesting that it involves the coupling of free phycobilisomes to an integral-membrane complex, an interaction that is stabilized by 1 m phosphate. Fluorescence spectra are consistent with this idea. Fluorescence recovery after photobleaching measurements confirm that the induction of NPQ in the presence of 1 m phosphate is accompanied by immobilization of the phycobilisomes. We propose as a working hypothesis that a major component of the fluorescence quenching observed in iron-starved cyanobacteria arises from the coupling of free phycobilisomes to IsiA.