Development and validation of qPCR methods for nucleic acid biomarkers as a drug development tool: points to consider.

Nucleic acid (NA) biomarkers play critical roles in drug development. However, the global regulatory guidelines for assessing quantification methods specific to NA biomarkers are limited. The validation of analytical methods is crucial for the use of biomarkers in clinical and post-marketing evaluations of drug efficacy and adverse reactions. Given that quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) methods are the gold standards for the quantification of NA biomarkers, the Biomarker Analytical Method Validation Study Group in Japan has discussed considerations and made recommendations for the development and validation of qPCR- and RT-qPCR-based analytical methods for endogenous NA biomarkers as drug development tools. This white paper aims to contribute to the global harmonization of NA biomarker assay validation.

Prediction of Human Pharmacokinetics of Phosphorodiamidate Morpholino Oligonucleotides in Duchenne Muscular Dystrophy Patients Using Viltolarsen.

Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike other nucleic acids, are not negatively charged. Thus, PMOs differ from other ASOs in their pharmacokinetic (PK) properties. Drugs with a PMO backbone have been administered to Duchenne muscular dystrophy pediatric patients; however, appropriate methodologies are not currently available to predict their human PK from nonclinical data. In this study, we used viltolarsen as a representative PMO to investigate the applicability of the allometric scaling approach to human PK prediction. We first summarized the nonclinical and clinical PK data for viltolarsen as showing high total clearance, low serum protein binding, metabolic resistance, and urinary excretion as the unchanged drug in both animals and humans. We then investigate the PK of viltolarsen in mice, rats, cynomolgus monkeys, and dogs and used the results, with body weight, to extrapolate to humans by several methods. The estimate of human total clearance obtained from cynomolgus monkeys was the best, and body weight may be the key factor in accurately predicting human total clearance. In contrast, all of the well-known prediction methods for the volume of distribution at steady state gave underestimates. However, the human PK profiles predicted from the PK parameters in cynomolgus monkeys fit the observed human plasma concentrations well. These results are expected to contribute to the further development of PMOs. SIGNIFICANCE STATEMENT: We investigated how to predict the human PK of phosphorodiamidate morpholino oligomers from nonclinical data. The estimates of human PK parameters and profiles determined from cynomolgus monkeys by an allometric scaling approach were the most suitable, and the cynomolgus monkey body weight may be the key factor in accurately predicting human total clearance.

Emergence of second coherent regions for breathing chimera states.

Chimera states in one-dimensional nonlocally coupled phase oscillators are mostly assumed to be stationary, but breathing chimeras can occasionally appear, branching from the stationary chimeras via Hopf bifurcation. In this paper, we demonstrate two types of breathing chimeras: The type I breathing chimera looks the same as the stationary chimera at a glance, while the type II consists of multiple coherent regions with different average frequencies. Moreover, it is shown that the type I changes to the type II by increasing the breathing amplitude. Furthermore, we develop a self-consistent analysis of the local order parameter, which can be applied to breathing chimeras, and numerically demonstrate this analysis in the present system.

Cyanide Formation in Freezer Stored Foods: Freezing of a Glycine and Nitrite Mixture.

Freezing is not always the best way to keep foods safely. Some reactions are known to be accelerated in ice. Furthermore, some other reactions that are not observed in solution are also promoted in ice. We found that the formation of nitrosamines through the reaction of an amine with a nitrite is accelerated in ice. Surprisingly, cyanide is formed through the reaction of glycine with nitrite in ice but not in solution. Amines are present in many kinds of foods. Nitrite is present in vegetables and is used as a food coloring agent and to inhibit the reproduction of Clostridium botulinum. The maximum amount of cyanide formed reaches a dangerous level, and the intake of this formed cyanide in a few tens of cubic centimeters causes some people to get headaches. These facts suggest that hazardous compounds could be generated in frozen processed foods. We report here the formation of cyanide and its possible formation pathway in ice. Finally, we propose a way to prevent cyanide formation in food under frozen conditions.

Breathing multichimera states in nonlocally coupled phase oscillators.

Chimera states for the one-dimensional array of nonlocally coupled phase oscillators in the continuum limit are assumed to be stationary states in most studies, but a few studies report the existence of breathing chimera states. We focus on multichimera states with two coherent and incoherent regions and numerically demonstrate that breathing multichimera states, whose global order parameter oscillates temporally, can appear. Moreover, we show that the system exhibits a Hopf bifurcation from a stationary multichimera to a breathing one by the linear stability analysis for the stationary multichimera.

Acceleration and Reaction Mechanism of the N-Nitrosation Reaction of Dimethylamine with Nitrite in Ice.

Some reactions (e.g., oxidation of nitrite, denitrification of ammonium) are accelerated in freeze-concentrated solution (FCS) compared to those in aqueous solution. Ice is highly intolerant to impurities, and the ice excludes those that would accelerate reactions. Here we show the acceleration of the N-nitrosation reaction of dimethylamine (DMA) with nitrite to produce N-nitrosodimethylamine (NDMA) in FCS. NDMA is a carcinogenic compound, and this reaction is potentially accelerated in frozen fish/meat. The eaction rate of the N-nitrosation reaction becomes fastest at specific pH. This means that it is a third-order reaction. Theoretical pH values of the peak in the third-order reaction are higher than the experimental one. Freeze-concentration of acidic solution causes pH decrement; however, the freeze-concentration alone could not explain the difference of pH values. The theoretical value was obtained under the assumption that no solute took part in ice. However, solutes are incorporated in ice with a small distribution coefficient of solutes into ice. This small incorporation enhanced the decrement of pH values. Using the distribution coefficient of chloride and sodium ion and assuming those of nitrite and DMA to explain the enhancement, we succeeded in estimating the distribution coefficients of nitrite: 2 × 10-3 and DMA: 3 × 10-2.

Persistent chimera states in nonlocally coupled phase oscillators.

Chimera states in the systems of nonlocally coupled phase oscillators are considered stable in the continuous limit of spatially distributed oscillators. However, it is reported that in the numerical simulations without taking such limit, chimera states are chaotic transient and finally collapse into the completely synchronous solution. In this Rapid Communication, we numerically study chimera states by using the coupling function different from the previous studies and obtain the result that chimera states can be stable even without taking the continuous limit, which we call the persistent chimera state.

Cytotoxic effect of Drosophila deoxynucleoside kinase gene on replicating plasmid in HeLa cells.

To enhance the levels of transgene expression from plasmid-based nonviral vectors, replicating plasmids containing the SV40 origin and the SV40 large T antigen gene, as a model replicating unit, were constructed. The replicating luciferase plasmid DNA produced the luciferase protein more efficiently than the non-replicating luciferase plasmid DNA, as expected. Surprisingly, the introduction of the replicating plasmid DNA containing the Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) gene was highly cytotoxic and caused cell death without nucleoside analogs. Our results confirm that transgenes on a replicating plasmid represent an excellent tool for effective protein production and suggest that efficient production of the Dm-dNK protein in tumor cells could be an attractive cancer therapy.

The impact of volcanic gases from Miyake island on the chemical constituents in precipitation in the Tokyo metropolitan area.

The volcano on Miyake Island first erupted in July 2000 and continuous emission of volcanic gas from the collapsed caldera has been observed from the middle of August 2000. The large volcanic emission of SO2 had a strong influence on Tokyo metropolitan area, which is located approximately 150 km north of Miyake Island. We measured major ions in precipitation and dry deposition samples which had been collected at five sampling sites (Yokohama, Kashiwa, Fujisawa, Yokosuka, and Hachioji) in the Tokyo metropolitan area for 12 years since 1990. We have evaluated quantitatively the impact of the volcanic SO2 gas emitted from Miyake Island on the Tokyo metropolitan area by comparing depositional ionic constituents in the volcanic degassing period (from September 2000 to August 2001) with those in the normal period of the past 10 years (September 1990 to August 2000). nss-SO4(2-) concentrations in precipitation at the sampling sites in the Tokyo metropolitan area were 59.5-77.0 microeq/L during the degassing period, and 33.3-44.1 microeq/L during the normal period, respectively. The difference of nss-SO4(2-) concentrations between the two periods was statistically significant. In contrast, no significant differences were observed in the concentrations of the other major ions (NH4+, nss-Ca2+, Cl-, and NO3-) between the two periods. The impact of volcanic degassing from Miyake Island on the ionic concentrations in the precipitation of the Tokyo metropolitan area was seen only in the H+ and nss-SO4(2-) concentrations. The annual wet deposition amount of volcanic nss-SO4(2-) into the Tokyo metropolitan area has been quantitatively estimated. The annual wet deposition amounts were calculated as 701+/-277 Meq/year (22.4+/-8.9 kt SO2/year) on the total area of the Tokyo metropolitan area (14,000 km2). The wet deposition amount of nss-SO4(2-) corresponds to only 0.15% of the total annual amount of volcanic SO2 (15 Mt/year) emitted from Miyake Island from September 2000 to August 2001. This estimation is consistent with a previous study which reported that 0.3% of the atmospheric discharge of SO2 from the volcano in Mt. Etna in Sicily was deposited by precipitation to the surrounding area (1200 km2). The annual wet deposition amount of volcanic SO2 to the Tokyo metropolitan area was at the same order of the magnitude (37+/-15%) as the annual anthropogenic SO2 amount (60 kt/year) emitted from the Tokyo metropolitan area.

Long-term trend of chemical constituents in precipitation in Tokyo metropolitan area, Japan, from 1990 to 2002.

In order to understand the actual status and mechanism of acid rain, it is important to know the pH of precipitation and its chemical constituents on a continuous and regular basis over a wide area. This study examines acid rain over a wide area using an observational network in the Tokyo metropolitan area of Japan, and analyzes the major chemical constituents of every precipitation sample. Precipitation was collected continuously for a period of 12 years from June 1990 to May 2002 at several sampling sites in the Tokyo metropolitan area, and its pH and chemical constituent concentrations were measured. The average pH ranged from 4.23 to 4.62, clearly indicating acidification of precipitation over the entire Tokyo metropolitan area. A time-trend model was applied to describe temporal variations of chemical constituent concentrations, including annual change rate, seasonal variation, and precipitation effects. Seasonal and annual trends for the past 12 years were examined with the model, using the least squares method. Nonsea salt (nss)-Ca2+ shows a maximum value in early spring, a seasonality probably caused by calcium-rich particles in airborne yellow dust from Asia. Slightly decreasing annual trends of nss-SO4(2-) may correspond to the recent decreasing trend of atmospheric SO2 gas concentrations in the Tokyo metropolitan area. The annual trends of NO3-, NH4+, and nss-Ca2+ show a large site-to-site difference. The increasing NO3-, NH4+, and nss-Ca2+ concentrations at inland suburban sites may be caused by increases in their local sources such as vehicle traffic and municipal waste incineration. The annual change rate of H+ is slightly negative or almost zero at every site, so the acidification of precipitation has not become worse since 1990 over the Tokyo metropolitan area.

Daily concentrations of trace metals in aerosols in Beijing, China, determined by using inductively coupled plasma mass spectrometry equipped with laser ablation analysis, and source identification of aerosols.

This paper describes the daily concentrations of trace metals and ionic constituents in the aerosol of Beijing, China from March 2001 to August 2003. Daily PM10 concentrations were also measured from September 2001 to August 2003. The daily average PM10 concentration at Beijing, China from September 2001 to August 2003 was 171+/-117 microg m(-3) (n = 673), which is 5-fold higher than at Yokohama, Japan. Trace metal concentrations were analyzed by using inductively coupled plasma mass spectrometry equipped with a laser ablation sample introduction (LA/ICP-MS), which is a rapid and simultaneous method for multi-element analysis. The daily average metal concentrations in TSP in Beijing from March 2001 to August 2003 were: Al: 3.5+/-2.4 (n = 727), Ti: 0.47+/-0.35 (n = 720), V: 0.013+/-0.010 (n = 716), Cr: 0.019+/-0.015 (n = 618), Mn: 0.24+/-0.16 (n = 730), Fe: 5.5+/-3.9 (n = 728), Co: 0.0046+/-0.0055 (n = 629), Ni: 0.022+/-0.024 (n = 680), Cu: 0.11+/-0.11 (n = 660), Zn: 0.77+/-0.60 (n = 726), As: 0.048+/-0.047 (n = 731), Se: 0.010+/-0.010 (n = 550), Cd: 0.0068+/-0.0082 (n = 709), Sb: 0.033+/-0.036 (n = 687), and Pb: 0.43+/-0.50 (n = 728) (unit, microg m(-3)). All the metal concentrations in TSP in Beijing, China were 1.7-21.8 times higher than those in TSP in the center of Tokyo, Japan. Notably, As concentrations in TSP in Beijing were 20-fold higher than those in Tokyo. Source identification of aerosols in Beijing was carried out by using the chemical mass balance (CMB) receptor model, with the daily concentration of metals in the aerosol. The major primary sources of the aerosol of Beijing were considered to be soil dust and coal combustion. Vehicle exhaust contribution tended to increase.