Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.     

Interaction of Huntingtin-associated protein-1 with kinesin light chain: implications in intracellular trafficking in neurons

Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells.     

Reconciling Carbon-cycle Concepts, Terminology, and Methods

Recent projections of climatic change have focused a great deal of scientific and public attention on patterns of carbon (C) cycling as well as its controls, particularly the factors that determine whether an ecosystem is a net source or sink of atmospheric carbon dioxide (CO₂). Net ecosystem production (NEP), a central concept in C-cycling research, has been used by scientists to represent two different concepts. We propose that NEP be restricted to just one of its two original definitions—the imbalance between gross primary production (GPP) and ecosystem respiration (ER). We further propose that a new term—net ecosystem carbon balance (NECB)—be applied to the net rate of C accumulation in (or loss from [negative sign]) ecosystems. Net ecosystem carbon balance differs from NEP when C fluxes other than C fixation and respiration occur, or when inorganic C enters or leaves in dissolved form. These fluxes include the leaching loss or lateral transfer of C from the ecosystem; the emission of volatile organic C, methane, and carbon monoxide; and the release of soot and CO₂ from fire. Carbon fluxes in addition to NEP are particularly important determinants of NECB over long time scales. However, even over short time scales, they are important in ecosystems such as streams, estuaries, wetlands, and cities. Recent technological advances have led to a diversity of approaches to the measurement of C fluxes at different temporal and spatial scales. These approaches frequently capture different components of NEP or NECB and can therefore be compared across scales only by carefully specifying the fluxes included in the measurements. By explicitly identifying the fluxes that comprise NECB and other components of the C cycle, such as net ecosystem exchange (NEE) and net biome production (NBP), we can provide a less ambiguous framework for understanding and communicating recent changes in the global C cycle.     

A subset of Cowdria ruminantium genes important for immune recognition and protection

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.     

Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.     

CD5-Dependent CK2 Activation Pathway Regulates Threshold for T Cell Anergy

CD5 activates casein kinase 2 (CK2), a serine/threonine kinase that constitutively associates with the CK2-binding domain at the end of its cytoplasmic tail. To determine the physiological significance of CD5-dependent CK2 activation in T cells, we generated a knock-in mouse that expresses a CD5 protein containing a microdeletion with selective inability to interact with CK2 (CD5ΔCK2BD). The levels of CD5 on developing and mature T cell populations from CD5ΔCK2BD mice and CD5 wild-type (WT) mice were similar. The thymus of CD5ΔCK2BD mice contained fewer double-positive thymocytes than did that of both CD5WT and CD5 knockout (KO) mice, although the numbers of all other immature and mature T cell populations were unaltered. CD5ΔCK2BD T cells hypoproliferated and exhibited enhanced activation-induced cell death when stimulated with anti-CD3 or cognate peptide in comparison with CD5WT T cells. We also found that functional CD5-dependent CK2 signaling was necessary for efficient differentiation of naive CD4(+) T cells into Th2 and Th17 cells, but not Th1 cells. We previously showed that experimental autoimmune encephalomyelitis (EAE) in CD5KO mice was less severe and delayed in onset than in CD5WT mice. Remarkably, CD5ΔCK2BD mice recapitulated both EAE severity and disease onset of CD5KO mice. Increasing the immunization dose of myelin oligodendrocyte glycoprotein 35-55 peptide, a model that mimics high-dose tolerance, led to decreased severity of EAE in CD5WT mice but not in CD5KO or CD5ΔCK2BD mice. This property was recapitulated in in vitro restimulation assays. These results demonstrate that CD5-CK2 signaling sets the threshold for T cell responsiveness and is necessary for efficient generation of Th2 and Th17 cells.     

The resilience and functional role of moss in boreal and arctic ecosystems

CONTENTS: Summary 49 I. Mosses in the northern, high-latitude region 50 II. The role of moss in ecological resilience 51 III. Response of moss to disturbance 54 IV. Future research needs 60 V. Conclusions 62 Acknowledgements 62 References 62 SUMMARY: Mosses in northern ecosystems are ubiquitous components of plant communities, and strongly influence nutrient, carbon and water cycling. We use literature review, synthesis and model simulations to explore the role of mosses in ecological stability and resilience. Moss community responses to disturbance showed all possible responses (increases, decreases, no change) within most disturbance categories. Simulations from two process-based models suggest that northern ecosystems would need to experience extreme perturbation before mosses were eliminated. But simulations with two other models suggest that loss of moss will reduce soil carbon accumulation primarily by influencing decomposition rates and soil nitrogen availability. It seems clear that mosses need to be incorporated into models as one or more plant functional types, but more empirical work is needed to determine how to best aggregate species. We highlight several issues that have not been adequately explored in moss communities, such as functional redundancy and singularity, relationships between response and effect traits, and parameter vs conceptual uncertainty in models. Mosses play an important role in several ecosystem processes that play out over centuries - permafrost formation and thaw, peat accumulation, development of microtopography - and there is a need for studies that increase our understanding of slow, long-term dynamical processes.     

Comparison of methods to correct survival estimates and survival regression analysis on a large HIV African cohort

Objective

The evaluation of HIV treatment programs is generally based on an estimation of survival among patients receiving antiretroviral treatment (ART). In large HIV programs, loss to follow-up (LFU) rates remain high despite active patient tracing, which is likely to bias survival estimates and survival regression analyses.

Methods

We compared uncorrected survival estimates derived from routine program data with estimates obtained by applying six correction methods that use updated outcome data by a field survey targeting LFU patients in a rural HIV program in Malawi. These methods were based on double-sampling and differed according to the weights given to survival estimates in LFU and non-LFU subpopulations. We then proposed a correction of the survival regression analysis.

Results

Among 6,727 HIV-infected adults receiving ART, 9% were LFU after one year. The uncorrected survival estimates from routine data were 91% in women and 84% in men. According to increasing sophistication of the correction methods, the corrected survival estimates ranged from 89% to 85% in women and 82% to 77% in men. The estimates derived from uncorrected regression analyses were highly biased for initial tuberculosis mortality ratios (RR; 95% CI: 1.07; 0.76–1.50 vs. 2.06 to 2.28 with different correction weights), Kaposi sarcoma diagnosis (2.11; 1.61–2.76 vs. 2.64 to 3.9), and year of ART initiation (1.40; 1.17–1.66 vs. 1.29 to 1.34).

Conclusions

In HIV programs with high LFU rates, the use of correction methods based on non-exhaustive double-sampling data are necessary to minimise the bias in survival estimates and survival regressions.     

Treatment Initiation,Program Attrition and Patient Treatment Outcomes Associated with Scale-Up and Decentralization of HIV Care in Rural Malawi

Objective

To describe patient antiretroviral therapy (cART) outcomes associated with intensive decentralization of services in a rural HIV program in Malawi.

Methods

Longitudinal analysis of data from HIV-infected patients starting cART between August 2001 and December 2008 and of a cross-sectional immunovirological assessment conducted 12 (±2) months after therapy start. One-year mortality, lost to follow-up, and attrition (deaths and lost to follow-up) rates were estimated with exact Poisson 95% confidence intervals (CI) by type of care delivery and year of initiation. Association of virological suppression (<50 copies/mL) and immunological success (CD4 gain ≥100 cells/µL), with type of care was investigated using multiple logistic regression.

Results

During the study period, 4322 cART patients received centralized care and 11,090 decentralized care. At therapy start, patients treated in decentralized health facilities had higher median CD4 count levels (167 vs. 130 cell/µL, P<0.0001) than other patients. Two years after cART start, program attrition was lower in decentralized than centralized facilities (9.9 per 100 person-years, 95% CI: 9.5–10.4 vs. 20.8 per 100 person-years, 95% CI: 19.7–22.0). One year after treatment start, differences in immunological success (adjusted OR = 1.23, 95% CI: 0.83–1.83), and viral suppression (adjusted OR = 0.80, 95% CI: 0.56–1.14) between patients followed at centralized and decentralized facilities were not statistically significant.

Conclusions

In rural Malawi, 1- and 2-year program attrition was lower in decentralized than in centralized health facilities and no statistically significant differences in one-year immunovirological outcomes were observed between the two health care levels. Longer follow-up is needed to confirm these results.     

The Effects of Permafrost Thaw on Soil Hydrologic, Thermal, and Carbon Dynamics in an Alaskan Peatland

Recent warming at high-latitudes has accelerated permafrost thaw in northern peatlands, and thaw can have profound effects on local hydrology and ecosystem carbon balance. To assess the impact of permafrost thaw on soil organic carbon (OC) dynamics, we measured soil hydrologic and thermal dynamics and soil OC stocks across a collapse-scar bog chronosequence in interior Alaska. We observed dramatic changes in the distribution of soil water associated with thawing of ice-rich frozen peat. The impoundment of warm water in collapse-scar bogs initiated talik formation and the lateral expansion of bogs over time. On average, Permafrost Plateaus stored 137 ± 37 kg C m⁻², whereas OC storage in Young Bogs and Old Bogs averaged 84 ± 13 kg C m⁻². Based on our reconstructions, the accumulation of OC in near-surface bog peat continued for nearly 1,000 years following permafrost thaw, at which point accumulation rates slowed. Rapid decomposition of thawed forest peat reduced deep OC stocks by nearly half during the first 100 years following thaw. Using a simple mass-balance model, we show that accumulation rates at the bog surface were not sufficient to balance deep OC losses, resulting in a net loss of OC from the entire peat column. An uncertainty analysis also revealed that the magnitude and timing of soil OC loss from thawed forest peat depends substantially on variation in OC input rates to bog peat and variation in decay constants for shallow and deep OC stocks. These findings suggest that permafrost thaw and the subsequent release of OC from thawed peat will likely reduce the strength of northern permafrost-affected peatlands as a carbon dioxide sink, and consequently, will likely accelerate rates of atmospheric warming.