Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus.

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

Development of native mass spectrometry with nanoelectrospray ionization coupled to size exclusion chromatography for proteins.

RATIONALE: Native mass spectrometry (MS) is an analytical technique used to determine the molecular mass of protein complexes without cross-linking. Size exclusion chromatography (SEC) coupled with native MS using conventional electrospray ionization (ESI) has been reported to allow online buffer exchange. To detect a wide variety of protein complexes without a collapse in the ionization process, it is important to build an online system that enables robust analysis with a low flow rate. METHODS: We created an online native MS system equipped with nanoESI connected to the SEC component (online SEC/nanoESI system) and optimized several parameters for SEC separation and ionization. The constructed system was used to measure a solution consisting of a protein mixture of various molecular masses (10-300 kDa) to verify characteristics such as the measurable molecular mass range, reproducibility, and online buffer exchange. RESULTS: The optimal flow rates for SEC separation and nanoESI analysis using this system were 200 and 1 µL/min, respectively. This system was able to analyze proteins in the ranges of 10-300 and 20-300 kDa for protein samples in ammonium acetate and nonvolatile buffer, respectively. Furthermore, the results of consecutive measurements showed that the relative standard deviations of the retention times and observed masses for each protein were sufficiently small. CONCLUSIONS: We created an online SEC/nanoESI system and evaluated its utility for the analysis of various proteins in conventional measurement solvent and nonvolatile buffer. As a result, the structural stability and resolution of the proteins were found to be sufficient when using online buffer exchange. Therefore, this online SEC/nanoESI system would be a useful technique for obtaining mass spectra of various proteins automatically with good resolution, simply by loading samples into an autosampler.

Chronic magnesium deficiency causes reversible mitochondrial permeability transition pore opening and impairs hypoxia tolerance in the rat heart.

Chronic magnesium (Mg) deficiency induces and exacerbates various cardiovascular diseases. We previously investigated the mechanisms underlying decline in cardiac function caused by chronic Mg deficiency and the effectiveness of Mg supplementation on this decline using the Langendorff-perfused isolated mouse heart model. Herein, we used the Langendorff-perfused isolated rat heart model to demonstrate the chronic Mg-deficient rats (Mg-deficient group) had lower the heart rate (HR) and left ventricular pressure (LVDP) than rats with normal Mg levels (normal group). Furthermore, decline in cardiac function due to hypoxia/reoxygenation injury was significantly greater in the Mg-deficient group than in the normal group. Experiments on mitochondrial permeability transition pore (mPTP) using isolated mitochondria revealed that mitochondrial membrane was fragile in the Mg-deficient group, implying that cardiac function decline through hypoxia/reoxygenation injury is associated with mitochondrial function. Mg supplementation for chronic Mg-deficient rats not only improved hypomagnesemia but also almost completely restored cardiac and mitochondrial functions. Therefore, proactive Mg supplementation in pathological conditions induced by Mg deficiency or for those at risk of developing hypomagnesemia may suppress the development and exacerbation of certain disease states.

International co-validation on absolute quantification of single nucleotide variants of KRAS by digital PCR.

The precise quantification of KRAS single nucleotide variant (SNV) is critical for the treatment and prognosis of lung and colorectal cancer. Validation of digital PCR (dPCR) as a method for accurate quantification of KRAS SNV has great clinical importance. An international co-validation on absolute quantification of KRAS SNV by dPCR was conducted among three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). A candidate reference material (RM) was provided by NIM and three measurands were reported: copy number concentration (Tc) of KRAS G12A mutation and wild type and KRAS G12A fractional abundance (FA). Homogeneity and stability assessment showed that the study materials provided by NIM were sufficiently homogeneous and stable during the study period. En number performance statistics was used to evaluate equivalence of the study among the three NMIs. All En values for both Tc and KRAS G12A FA≤1 showed good agreement and consistency with the reference value within the expanded uncertainty. This indicates that dPCR with full uncertainty evaluation can serve as a candidate primary reference measurement procedure (PRMP) for the KRAS SNV measurement and value assignment of reference materials.

Effect of bat pinna on sensing using acoustic finite difference time domain simulation.

The practicality of the finite-difference time-domain (FDTD) method was confirmed by comparing head-related transfer functions obtained from a three-dimensional (3D) digital model of a bat (Rhinolophus ferrumequinum nippon) head with acoustic experiments using a 3D printed physical model. Furthermore, we simulated the auditory directionality using a 3D digital model that was modified based on the pinna movement of a bat during echolocation and found that the alternating movements of the left and right pinna result in a binaural sound pressure difference for vertical sources. Using the FDTD method, suitable for simulating acoustics in large spaces, we could analyze in detail the binaural echoes that bats receive and the acoustic cues they use for echolocation.

Effects of a single dose of tablets containing lactononadecapeptide on cognitive function in healthy adults: a randomized, double-blind, cross-over, placebo-controlled trial.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE) is a memory-improving peptide. The current study aimed to determine the effects of a single dose of tablets containing LNDP on cognitive function in healthy Japanese men aged 30-59 years. A randomized, double-blind, cross-over, placebo-controlled trial was conducted in participants randomly assigned to receive LNDP or placebo tablets. The Uchida-Kraepelin test was used to induce cognitive load in participants as a model of work load. Cognitive function was evaluated using the Japanese version of the CNS Vital Signs. Composite memory and verbal memory were significantly higher following consumption of LNDP than placebo tablets. Carryover effects were observed in attention and concentration domains so that period 1 data was analyzed. LNDP consumption led to higher processing speed, executive function, and cognitive flexibility than placebo. Thus, supplementation with a single dose of LNDP tablets may improve cognitive functions including memory, attention, concentration, and information processing in daily life.

Enhancing the accuracy of measurement of small molecule organic biomarkers.

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.

Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis.

To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the expanded uncertainty (k = 2) of CRM 6201-b are (40.0 ± 1.6) µmol kg(-1).

Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification.

Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a ß-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

Optimizing virus inactivation methods for molecular detection techniques: Implications for viral protein and RNA measurements.

To facilitate the development of effective viral detection techniques, a positive control material is required for validating their quantitative performance. Inactivated viruses serve as viable control materials, as they can be handled without the constraints of biohazard safety facilities. However, inactivation alters the structure of viral component molecules, necessitating the selection of inactivation methods that have minimal effects on the target molecules relevant to molecular detection techniques. Only a limited number of studies have investigated inactivation methods to produce viral control materials. Therefore, the aim of this study was to investigate various virus inactivation methods and evaluate their impact on molecular detection techniques, with a specific focus on viral proteins and RNA. We evaluated the effects of ultraviolet (UV) irradiation, heat, beta-propiolactone (BPL), hydrogen peroxide (H2O2), and perchloric acid (HClO4) inactivation methods to identify the most effective technique and its optimal conditions. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-digital polymerase chain reaction (RT-dPCR) were employed as model assays to assess the effects of these treatments on protein and RNA measurements. Among the evaluated methods, UV and heat treatments demonstrated minimal interference with ELISA, while heat treatment had the least impact on RT-dPCR measurements. Consequently, our findings revealed that heat inactivation holds the potential for producing inactivated viruses that can be effectively used in molecular detection techniques targeting both viral protein and RNA.

Emerin deficiency does not exacerbate cardiomyopathy in a murine model of Emery-Dreifuss muscular dystrophy caused by an LMNA gene mutation.

Emery-Dreifuss muscular dystrophy (EDMD), caused by mutations in genes encoding nuclear envelope proteins, is clinically characterized by muscular dystrophy, early joint contracture, and life-threatening cardiac abnormalities. To elucidate the pathophysiological mechanisms underlying striated muscle involvement in EDMD, we previously established a murine model with mutations in Emd and Lmna (Emd-/-/LmnaH222P/H222P; EH), and reported exacerbated skeletal muscle phenotypes and no notable cardiac phenotypes at 12 weeks of age. We predicted that lack of emerin in LmnaH222P/H222P mice causes an earlier onset and more pronounced cardiac dysfunction at later stages. In this study, cardiac abnormalities of EDMD mice were compared at 18 and 30 weeks of age. Contrary to our expectations, physiological and histological analyses indicated that emerin deficiency causes no prominent differences of cardiac involvement in LmnaH222P/H222P mice. These results suggest that emerin does not contribute to cardiomyopathy progression in LmnaH222P/H222P mice.

The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments.

Salinity stress can greatly reduce seed production because plants are especially sensitive to salt during their reproductive stage. Here, we show that the sodium ion transporter AtHKT1;1 is specifically expressed around the phloem and xylem of the stamen in Arabidopsis thaliana to prevent a marked decrease in seed production caused by salt stress. The stamens of AtHKT1;1 mutant under salt stress overaccumulate Na+, limiting their elongation and resulting in male sterility. Specifically restricting AtHKT1;1 expression to the phloem leads to a 1.5-fold increase in the seed yield upon sodium ion stress. Expanding phloem expression of AtHKT1;1 throughout the entire plant is a promising strategy for increasing plant productivity under salinity stress.

Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of ß-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

Characterization and Value Assignment of a Monoclonal Antibody Reference Material, NMIJ RM 6208a, AIST-MAB.

Monoclonal antibodies have been established as the largest product class of biopharmaceuticals. Since extensive characterization is required for development and quality control of monoclonal antibody, a widely available reference material (RM) is needed. Herein, a humanized IgG1κ monoclonal antibody reference material, RM 6208-a, AIST-MAB, was established by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The monoclonal antibody solution was produced as a pharmaceutical grade using a Chinese hamster ovary-derived cell line. The assigned indicative value represents the concentration of the antibody with a heterotetrameric structure including oligomeric forms, determined by an amino acid analysis using isotope dilution mass spectrometry, and their homogeneity and stability were assessed. In addition to antibody concentration, various physicochemical properties, including peptide mapping data, charge variants, and aggregates, were examined. This RM is intended for use in validation of analytical procedures and instruments such as a system suitability test for quantification of antibody. It is also intended for comparing and evaluating the results of antibody analyses across analytical methods and analytical laboratories such as inter-laboratory comparison. Both the material and the set of data from our study provide a tool for an accurate and reliable characterization of product quality attributes of monoclonal antibodies in biopharmaceutical and metrology communities.

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors of ABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA-induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (-)-catechin gallate (CG) and (-)-gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage-dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA-induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (ICa ) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co-treatment with CG/GCG and ABA suppressed ABA-induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark-induced stomatal closure or light- and fusicoccin-induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+ -related signaling pathways. This study further supports the crucial role of ICa channels of the plasma membrane in ABA-induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress-induced signal transduction pathways.

Elevation of the Plasma Levels of TNF Receptor 2 in Association with Those of CD25, OX40, and IL-10 and HTLV-1 Proviral Load in Acute Adult T-Cell Leukemia.

Adult T-cell leukemia/lymphoma (ATL) cells express TNF receptor type-2 (TNFR2) on their surface and shed its soluble form (sTNFR2). We previously reported that sTNFR2 levels were highly elevated in the plasma of patients with acute ATL. To investigate whether its quantitation would be helpful for the diagnosis or prediction of the onset of acute ATL, we examined the plasma levels of sTNFR2 in a large number of specimens obtained from a cohort of ATL patients and asymptomatic human T-cell leukemia virus type 1 (HTLV-1) carriers (ACs) and compared them to those of other candidate ATL biomarkers (sCD25, sOX40, and IL-10) by enzyme-linked immunosorbent assays (ELISA) and HTLV-1 proviral loads. We observed that sTNFR2 levels were significantly elevated in acute ATL patients compared to ACs and patients with other types of ATL (chronic, smoldering, and lymphoma). Importantly, sTNFR2 levels were significantly correlated with those of sCD25, sOX40, and IL-10, as well as proviral loads. Thus, the present study confirmed that an increase in plasma sTNFR2 levels is a biomarker for the diagnosis of acute ATL. Examination of plasma sTNFR2 alone or in combination with other ATL biomarkers may be helpful for the diagnosis of acute ATL.

A missense mutation of the gene encoding voltage-dependent sodium channel (Nav1.1) confers susceptibility to febrile seizures in rats.

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Na(v)1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic-clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Na(v)1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

Expanding role of the jumonji C domain as an RNA hydroxylase.

JmjC (Jumonji C) domain-containing proteins are known to be an extensive family of Fe(II)/2-oxoglutarate-dependent oxygenases involved in epigenetic regulation of gene expression by catalyzing oxidative demethylation of methylated histones. We report here that a human JmjC protein named Tyw5p (TYW5) unexpectedly acts in the biosynthesis of a hypermodified nucleoside, hydroxywybutosine, in tRNA(Phe) by catalyzing hydroxylation. The finding provides an insight into the expanding role of JmjC protein as an RNA hydroxylase.

Impaired active demethylation of the paternal genome in pronuclear-stage rat zygotes produced by in vitro fertilization or intracytoplasmic sperm injection.

This study was designed to investigate the effect of in vitro production systems of rat zygotes such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) on demethylation dynamics of the paternal genome. Immunostaining with anti-5-methylcytosine indicated that in vivo derived zygotes harvested at 20 hr post-hCG injection had no active demethylation of paternal genomes as a mean relative methylation (RM) value, total fluorescence in the paternal genome divided by that in maternal one, at 1.17. Leaving zygotes in vivo or in culture for an additional 4 or 8 hr resulted in significant decreases of RM values to 0.14-0.31. Since in vitro-derived zygotes were produced using oocytes harvested at 14 hr post-hCG injection, zygotes at 6 hr after IVF or ICSI were considered developmentally comparable to the in vivo derived zygotes harvested at 20 hr post-hCG injection, with RM values at 1.04 and 0.92, respectively. At 10 hr post-IVF and ICSI, the RM values of the in vitro derived zygotes decreased significantly, but to a lesser extent compared with in vivo derived zygotes, to 0.53 and 0.62, respectively, without further decreases at 14 hr. Treatment of IVF-derived zygotes with 5-aza-2'-deoxycytidine (5-azadC; an inhibitor for methylation) or trichostatin A (TSA; an inhibitor for deacetylation) resulted in the decreased RM values at 14 hr post-IVF. However, developmental potential of the 5-azadC- or TSA-treated IVF zygotes up to the blastocyst stage was not improved. Thus, the demethylation dynamics of the paternal genome in pronuclear-stage rat zygotes was impaired by routine protocols for in vitro embryo production such as IVF and ICSI.