Modeling the Cost and Health Impacts of Diagnostic Strategies in Patients with Suspected Transthyretin Cardiac Amyloidosis.

Background Transthyretin cardiac amyloidosis (ATTR-CMP) is an increasingly recognized and treatable cause of heart failure with preserved ejection fraction. Multimodality cardiac imaging is recommended for ATTR-CMP diagnosis, but its cost-effectiveness in current clinical practice has not been well studied. Methods and Results Using a microsimulation model, we compared the cost-effectiveness of a combination of strategies involving 99mtechnetium pyrophosphate (PYP), cardiac magnetic resonance imaging, and endomyocardial biopsy for the diagnosis of ATTR-CMP. We developed a decision analytic model to project health care costs and lifetime quality-adjusted life years for symptomatic, older patients who present with congestive heart failure, with an increased left ventricular wall thickness and a 13% prevalence of ATTR-CMP. Rates of clinical events, costs, and quality-of-life values were estimated from published literature. The analysis was conducted from a US health care system perspective with health and cost outcomes discounted annually at 3%. In the base-case scenario, using a fixed tafamidis price of $16 000 annually (previously identified cost-effective price), total health care costs per person were lowest for the PYP-only strategy ($209 415) and highest for endomyocardial biopsy strategy ($215 881). Of the 7 strategies examined, the PYP-only strategy had the highest net monetary benefit using a willingness-to-pay threshold of $100 000/quality-adjusted life year. Results were sensitive to variations in model inputs for PYP and cardiac magnetic resonance imaging specificity, cost of tafamidis, and willingness-to-pay thresholds. Conclusions Our model-based analyses showed that a PYP-only strategy to diagnose ATTR-CMP is the most cost-effective strategy, at willingness-to-pay threshold of $100 000/quality-adjusted life year. At higher threshold ($150 000/quality-adjusted life year), sequential tests involving PYP and cardiac magnetic resonance imaging may be considered cost effective.

Functional Assessment of Missense Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder.

Pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder, is caused by inactivating mutations in the ABCC6 gene. The encoded protein, ABCC6, a transmembrane transporter, has a specialized efflux function in hepatocytes by contributing to plasma levels of inorganic pyrophosphate, a potent inhibitor of mineralization in soft connective tissues. Reduced plasma inorganic pyrophosphate levels underlie the ectopic mineralization in pseudoxanthoma elasticum. In this study, we characterized the pathogenicity of three human ABCC6 missense variants using an adenovirus-mediated liver-specific ABCC6 transgene expression system in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Variants p.L420V and p.R1064W were found benign because they had abundance and plasma membrane localization in hepatocytes similar to the wild-type human ABCC6 transgene, normalized plasma inorganic pyrophosphate levels, and prevented mineralization in the dermal sheath of vibrissae in muzzle skin, a phenotypic hallmark in the Abcc6-/- mice. In contrast, p.S400F was shown to be pathogenic because it failed to normalize plasma inorganic pyrophosphate levels and had no effect on ectopic mineralization despite its normal expression and proper localization in hepatocytes. These results showed that adenovirus-mediated hepatic ABCC6 expression in Abcc6-/- mice can provide a model system to effectively elucidate the multifaceted functional consequences of human ABCC6 missense variants identified in patients with pseudoxanthoma elasticum.

Assessment of the capacity of a pyrophosphate-based mouth rinse to inhibit the formation of supragingival dental calculus. A randomized double-blind placebo-controlled clinical trial.

BACKGROUND: This study aimed to analyze the efficacy of an anti-calculus mouth rinse and its possible adverse effects on the mucosa and teeth. MATERIAL AND METHODS: This randomized double-blind placebo-controlled clinical trial included 40 patients with treated and managed periodontal disease, all with a history of rapid calculus formation. Patients used a pyrophosphate-based test mouth rinse (B) or a placebo (A). A range of parameters were measured for: saliva (saliva flow, pH and chemical composition); calculus (Volpe-Manhold [V-M] index, weight, and volume); adverse effects on mucosa and teeth; and the patients' subjective perceptive of mouth rinse efficacy. RESULTS: the test mouth rinse B produced reductions in urea, uric acid, and phosphorous, calcium, saliva flow, and increases in pH. V-M index and calculus weight decreased after using the test mouth rinse. Calculus volume decreased with both mouth rinses. No changes to the mucosa or teeth were observed. Patients perceived that the test mouth rinse was more effective. CONCLUSIONS: The test/B and placebo mouth rinses both modified certain parameters in saliva composition, particularly reductions in urea, uric acid, and phosphorous. Calcium tended to increase after using the test-B mouth rinse. The results did not demonstrate the anticalculus efficacy of the pyrophosphate-based mouth rinse or positive effects on saliva flow or composition. This field requires further research, as no product has been developed that prevents calculus formation completely.

Analogies and disparities among scintigraphic bone tracers in the diagnosis of cardiac and non-cardiac ATTR amyloidosis.

In this issue of JNC, BW Spery and Coll report a retrospective analysis of 57 patients with transthyretin-related amyloidosis (ATTR) in an advanced phase of the disease who underwent 99mTechnetium-pyrophosphate (99mTcPYP) scintigraphy. Although relatively small and "negative," the study is relevant since it broadens our knowledge on the uptake of "bone tracers" in ATTR and contributes to understand the limitations of the clinical use of scintigraphy in this disease. The paper raises, directly or indirectly, at least three questions: To what extent are the different bone tracers interchangeable for the diagnosis of ATTR cardiac amyloidosis? Are bone tracers able to image non-cardiac ATTR amyloidosis? What is the explanation for the variable performance of the different bone tracers in the diagnosis of cardiac and extracardiac ATTR amyloidosis?

A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism.

In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine-Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine-Zn(II)-pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstract A simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine-Zn(II) complex.

Full molecular trajectories of RNA polymerase at single base-pair resolution.

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.

Assessment of plant availability and environmental risk of biosolids-phosphorus in a U.S. Midwest Corn-Belt Soil.

A field experiment was conducted from 2005 to 2008 in Fulton County, Western Illinois with biosolids from conventional wastewater treatment applied as corn fertilizer in a series of P rates (0, 163, 325, 488, 650 kg P ha(-1)) along with commercial P fertilizer - triple superphosphate P (TSP) as reference to assess biosolids-P plant availability and potential loss to waterbodies through runoff. Air-dried biosolids and TSP were incorporated into surface soil at end of 2005, and corn (Zea mays) was planted for three consecutive years (2006-2008). Concentrations of soil extractable P except for Mehlich-3 P were always lower in the biosolids than TSP treatments at the same P rates. The soil potentially available P in water extractable P (WEP) and Olsen P derived from biosolids-P estimated by the exponential depletion model was 2-4% and 15-24% of total P in the applied biosolids, respectively. The residence time of biosolids-induced WEP and Olsen P in Midwest soil under annual corn cropping was 5 and 2 years, respectively. Corn tissue analysis showed lower increase in P concentration by biosolids-P than TSP. The elevation rate of soluble reactive P (SRP) concentration in simulated runoff was less by biosolids than TSP. Based on the data in this study, the plant availability and environmental risk of biosolids-P are lower than those of TSP in the Midwest soil, thus use of biosolids as P nutrient for corn would not cause a major impairment to water sources even P applied through biosolids was not completely used by annual crop.

Pig manure treatment and purification by filtration.

This study aimed to develop a new, complex pig manure treatment and filtration process. The final scheme, called the AMAK process, comprised the following successive steps: mineralization with mineral acids, alkalization with lime milk, superphosphate addition, a second alkalization, thermal treatment, and pressure filtration. The proposed method produced a filtrate with 95%, 80%, and 96% reductions in chemical oxygen demand, nitrogen content, and phosphorus content, respectively. An advantage of the proposed method was that it incorporated a crystalline phase into the solid organic part of the manure, which enabled high filtration rates (>1000 kg m(-2) h(-1)) and efficient separation. The process also eliminated odor emissions from the filtrate and sediment. The treated filtrate could be used to irrigate crops or it could be further treated in conventional biological wastewater treatment plants. The sediment could be used for producing mineral-organic fertilizer. The AMAK process is inexpensive, and it requires low investment costs.

Naphthalene carbohydrazone based dizinc(II) chemosensor for a pyrophosphate ion and its DNA assessment application in polymerase chain reaction products.

A new naphthalene carbohydrazone based dizinc(II) complex has been synthesized and investigated to act as a highly selective fluorescence and visual sensor for a pyrophosphate ion with a quite low detection limit of 155 ppb; this has also been used to detect the pyrophosphate ion released from polymerase-chain-reaction.

A two-state model for the dynamics of the pyrophosphate ion release in bacterial RNA polymerase.

The dynamics of the PPi release during the transcription elongation of bacterial RNA polymerase and its effects on the Trigger Loop (TL) opening motion are still elusive. Here, we built a Markov State Model (MSM) from extensive all-atom molecular dynamics (MD) simulations to investigate the mechanism of the PPi release. Our MSM has identified a simple two-state mechanism for the PPi release instead of a more complex four-state mechanism observed in RNA polymerase II (Pol II). We observed that the PPi release in bacterial RNA polymerase occurs at sub-microsecond timescale, which is ∼3-fold faster than that in Pol II. After escaping from the active site, the (Mg-PPi)(2-) group passes through a single elongated metastable region where several positively charged residues on the secondary channel provide favorable interactions. Surprisingly, we found that the PPi release is not coupled with the TL unfolding but correlates tightly with the side-chain rotation of the TL residue R1239. Our work sheds light on the dynamics underlying the transcription elongation of the bacterial RNA polymerase.

Studies on acedan-based mononuclear zinc complexes toward selective fluorescent probes for pyrophosphate.

We have demonstrated that mononuclear Zn(II)-dipicolylamine (DPA) complexes with an auxiliary ligand can fluorescently discriminate pyrophosphate over ATP with as high selectivity as the known fast responding dinuclear bis(ZnDPA) complexes.

A universal, fully automated high throughput screening assay for pyrophosphate and phosphate release from enzymatic reactions.

The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts ( approximately 20,000 wells per day) to ultra-high throughput environments ( approximately 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.

Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol.

To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, protein-family search, and the sequence alignment of their gene products. Predicted terpene cyclases were classified into three groups: sesquiterpene, diterpene, and other terpene cyclases. Genes of the terpene cyclase group that form an operon with a gene encoding S-adenosyl-l-methionine (SAM)-dependent methyltransferase were found in genome data of seven microorganisms belonging to actinomycetes, Streptomyces ambofaciens ISP5053, Streptomyces coelicolor A3(2), Streptomyces griseus IFO13350, Streptomyces lasaliensis NRRL3382R, Streptomyces scabies 87.22, Saccharopolyspora erythraea NRRL2338, and Micromonospora olivasterospora KY11048. Among six microorganisms tested, S. ambofaciens, S. coelicolor A3(2), S. griseus, and S. lasaliensis produced 2-MIB but M. olivasterospora produced 2-methylenebornane (2-MB) instead. The regions containing monoterpene cyclase and methyltransferase genes were amplified by PCR from S. ambofaciens, S. lasaliensis, and Saccharopolyspora erythraea, respectively, and their genes were heterologously expressed in Streptomyces avermitilis, which was naturally deficient of 2-MIB biosynthesis by insertion and deletion. All exoconjugants of S. avermitilis produced 2-MIB. Full-length recombinant proteins, monoterpene cyclase and methyltransferase of S. lasaliensis were expressed at high level in Escherichia coli. The recombinant methyltransferase catalyzed methylation at the C2 position of geranyl diphosphate (GPP) in the presence of SAM. 2-MIB was generated by incubation with GPP, SAM, recombinant methyltransferase, and terpene cyclase. We concluded that the biosynthetic pathway involves the methylation of GPP by GPP methyltransferase and its subsequent cyclization by monoterpene cyclase to 2-MIB.

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis.

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.

Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing.

A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1 M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10 microL of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis.

[The hygienic assessment of superphosphates manufactured on a base of Algerian phosphorites]. / Gigienicheskaia otsenka superfosfatov, izgotovlennykh na osnove alzhirskikh fosforitov.

Hygienic and toxicological investigations of soil, plants, and animals have shown that the superphosphates made from Algerian phosphorites little differ from those made from the apatites of the Kola Peninsula. Superphosphates A and B should be referred to as hazard class IV and the superphosphates treated by ammonium should be classified as hazard class III.