Amyotrophic lateral sclerosis: an emerging era of collaborative gene discovery

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.     

Administration of keratinocyte growth factor down-regulates the pulmonary capacity of acetylcholine production

Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase - the acetylcholine synthesizing enzyme - was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.     

Bifunctional building blocks for glyco-architectures by TiCl4 -promoted ring opening of cyclodextrin derivatives

During our studies on the preparation of blocklike substituted 1,4-glucans by cationic ring-opening polymerization,1,2 we found that TiCl4 behaves differently from common initiators like Et3O+X- (X = PF6, SbCl6), BF3.Et2O, or methyl triflate, causing only ring opening under formation of alpha-maltooligosyl chlorides bearing one free hydroxyl group (4-OH) at the nonreducing end. These compounds are valuable building blocks for the preparation of new glyco-architectures since they are easily accessible starting materials for direct glycosylations or the preparation of a variety of oligomeric glycosyl donors like alkyl glycosides, thioglycosides, or azides. We successfully carried out and optimized the TiCl4-promoted ring opening with per-O-methylated, per-O-ethylated, and temporarily protected per-O-allylated cyclodextrins of various ring size. 1H NMR spectroscopy and high-pressure liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) were used to characterize the products.     

Soil fungal communities in a Castanea sativa (chestnut) forest producing large quantities of Boletus edulis sensu lato (porcini): where is the mycelium of porcini?

A study was conducted in a Castanea sativa forest that produces large quantities of the edible mushroom porcini (Boletus edulis sensu lato). The primary aim was to study porcini mycelia in the soil, and to determine if there were any possible ecological and functional interactions with other dominant soil fungi. Three different approaches were used: collection and morphological identification of fruiting bodies, morphological and molecular identification of ectomycorrhizae by rDNA-ITS sequence analyses and molecular identification of the soil mycelia by ITS clone libraries. Soil samples were taken directly under basidiomes of Boletus edulis, Boletus aestivalis, Boletus aereus and Boletus pinophilus. Thirty-nine ectomycorrhizal fungi were identified on root tips whereas 40 fungal species were found in the soil using the cloning technique. The overlap between above- and below-ground fungal communities was very low. Boletus mycelia, compared with other soil fungi, were rare and with scattered distribution, whereas their fruiting bodies dominated the above-ground fungal community. Only B. aestivalis ectomycorrhizae were relatively abundant and detected as mycelia in the soil. No specific fungus-fungus association was found. Factors triggering formation of mycorrhizae and fructification of porcini appear to be too complex to be simply explained on the basis of the amount of fungal mycelia in the soil.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

Urinary proteomics in chronic kidney disease: diagnosis and risk of progression beyond albuminuria

Background

The contrast between a high prevalence of chronic kidney disease (CKD) and the low incidence of end-stage renal disease highlights the need for new biomarkers of progression beyond albuminuria testing. Urinary proteomics is a promising method, but more studies focusing on progression rate and patients with hypertensive nephropathy are needed.

Results

We analyzed urine samples with capillary electrophoresis coupled to a mass-spectrometer from 18 well characterized patients with CKD stage 4–5 (of whom six with hypertensive nephropathy) and 17 healthy controls. Classification scores based on a previously developed panel of 273 urinary peptides were calculated and compared to urine albumin dipstick results. Urinary proteomics classified CKD with a sensitivity of 0.95 and specificity of 1.00. Overall diagnostic accuracy (area under ROC curve) was 0.98, which was better than for albuminuria (0.85, p = 0.02). Results for hypertensive nephropathy were similar to other CKD diagnoses. Adding the proteomic score to an albuminuria model improved detection of rapid kidney function decline (>4 ml/min/1.73 m² per year) substantially: area under ROC curve increased from 0.762 to 0.909 (p = 0.042), and 38% of rapid progressors were correctly reclassified to a higher risk and 55% of slow progressors were correctly reclassified to a lower risk category. Reduced excretion of collagen types I–III, uromodulin, and other indicators of interstitial inflammation, fibrosis and tubular dysfunction were associated with CKD diagnosis and rapid progression. Patients with hypertensive nephropathy displayed the same findings as other types of CKD.

Conclusions

Urinary proteomic analyses had a high diagnostic accuracy for CKD, including hypertensive nephropathy, and strongly improved identification of patients with rapid kidney function decline beyond albuminuria testing.     

The Cost-Effectiveness of Low-Cost Essential Antihypertensive Medicines for Hypertension Control in China: A Modelling Study

Background

Hypertension is China’s leading cardiovascular disease risk factor. Improved hypertension control in China would result in result in enormous health gains in the world’s largest population. A computer simulation model projected the cost-effectiveness of hypertension treatment in Chinese adults, assuming a range of essential medicines list drug costs.

Methods and Findings

The Cardiovascular Disease Policy Model-China, a Markov-style computer simulation model, simulated hypertension screening, essential medicines program implementation, hypertension control program administration, drug treatment and monitoring costs, disease-related costs, and quality-adjusted life years (QALYs) gained by preventing cardiovascular disease or lost because of drug side effects in untreated hypertensive adults aged 35–84 y over 2015–2025. Cost-effectiveness was assessed in cardiovascular disease patients (secondary prevention) and for two blood pressure ranges in primary prevention (stage one, 140–159/90–99 mm Hg; stage two, ≥160/≥100 mm Hg). Treatment of isolated systolic hypertension and combined systolic and diastolic hypertension were modeled as a reduction in systolic blood pressure; treatment of isolated diastolic hypertension was modeled as a reduction in diastolic blood pressure. One-way and probabilistic sensitivity analyses explored ranges of antihypertensive drug effectiveness and costs, monitoring frequency, medication adherence, side effect severity, background hypertension prevalence, antihypertensive medication treatment, case fatality, incidence and prevalence, and cardiovascular disease treatment costs. Median antihypertensive costs from Shanghai and Yunnan province were entered into the model in order to estimate the effects of very low and high drug prices. Incremental cost-effectiveness ratios less than the per capita gross domestic product of China (11,900 international dollars [Int$] in 2015) were considered cost-effective. Treating hypertensive adults with prior cardiovascular disease for secondary prevention was projected to be cost saving in the main simulation and 100% of probabilistic simulation results. Treating all hypertension for primary and secondary prevention would prevent about 800,000 cardiovascular disease events annually (95% uncertainty interval, 0.6 to 1.0 million) and was borderline cost-effective incremental to treating only cardiovascular disease and stage two patients (2015 Int$13,000 per QALY gained [95% uncertainty interval, Int$10,000 to Int$18,000]). Of all one-way sensitivity analyses, assuming adherence to taking medications as low as 25%, high Shanghai drug costs, or low medication efficacy led to the most unfavorable results (treating all hypertension, about Int$47,000, Int$37,000, and Int$27,000 per QALY were gained, respectively). The strengths of this study were the use of a recent Chinese national health survey, vital statistics, health care costs, and cohort study outcomes data as model inputs and reliance on clinical-trial-based estimates of coronary heart disease and stroke risk reduction due to antihypertensive medication treatment. The limitations of the study were the use of several sources of data, limited clinical trial evidence for medication effectiveness and harms in the youngest and oldest age groups, lack of information about geographic and ethnic subgroups, lack of specific information about indirect costs borne by patients, and uncertainty about the future epidemiology of cardiovascular diseases in China.

Conclusions

Expanded hypertension treatment has the potential to prevent about 800,000 cardiovascular disease events annually and be borderline cost-effective in China, provided low-cost essential antihypertensive medicines programs can be implemented.     

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.     

Cool,cold or colder? Spatial segregation of prions and blue petrels is explained by differences in preferred sea surface temperatures

The Southern Ocean provides one of the largest environmental gradients on Earth that lacks geographical barriers, and small but highly mobile petrels living there may offer fine models of evolution of diversity along environmental gradients. Using geolocation devices, we investigated the winter distribution of closely related petrel species breeding sympatrically in the southern Indian Ocean, and applied ecological niche models to compare environmental conditions in the habitat used. We show that thin-billed prions (Pachyptila belcheri), Antarctic prions (Pachyptila desolata) and blue petrels (Halobaena caerulea) from the Kerguelen archipelago in the southern Indian Ocean segregate latitudinally, sea surface temperature being the most important variable separating the distribution of the species. Antarctic prions spent the winter north of the Polar Front in temperate waters, whereas blue petrels were found south of the Polar Front in Antarctic waters. Thin-billed prions preferred intermediate latitudes and temperatures. Stable isotope values of feathers reflected this near complete niche separation across an ecological gradient that spans large scales, and suggest evolutionary isolation by environment. In pelagic seabirds that exploit large areas of ocean, spatial niche partitioning may not only facilitate coexistence among ecologically similar species, but may also have driven their evolution in the absence of geographical barriers.