Association between Plasma Xanthine Oxidoreductase Activity and the Renal Function in a General Japanese Population: The Tohoku Medical Megabank Community-Based Cohort Study.

INTRODUCTION: Xanthine oxidoreductase (XOR) has been identified as a critical source of reactive oxygen species in various pathophysiological conditions, including hypertension, endothelial dysfunction, and atherosclerosis. This study investigated the association between XOR and renal function in a general Japanese population. METHODS: The Iwate Tohoku Medical Megabank Organization pooled individual participant data from a community-based cohort study in Iwate prefecture. Chronic kidney disease (CKD) was estimated using the estimated glomerular filtration rate of cystatin C (eGFRcys). Individuals with a history of hyperuricemia or severe renal dysfunction (eGFRcys <15 mL/min/1.73 m2 or undergoing dialysis) were excluded from the study. We performed a multinominal multivariate logistic analysis adjusted for age, blood pressure, uric acid, glycated hemoglobin A1c, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol to associate XOR activity and renal function. RESULTS: The present study included 4,248 participants (male/female: 1,373/2,875, age: 62.9 ± 11.7 years). When participants were divided according to XOR quartiles, blood pressure, body mass index, uric acid, low-density lipoprotein cholesterol, and glycated hemoglobin A1c were highest in the highest XOR quartile (all p < 0.001). The XOR activity was significantly higher in the subgroup with CKD stage G3 and G4 (G1 vs. G2 vs. G3-G4: 44.8 ± 40.5 vs. 52.0 ± 42.9 vs. 54.1 ± 43.9 pmol/h/mL, p = 0.02). The higher XOR activity was significantly associated with an increase of CKD stage: the odd ratios (95% confidence intervals) per 1 pmol/h/mL increase in XOR activity with CKD stage G1 as a reference were 1.37 (1.13-1.73) in G2 and 1.51 (1.30-1.84) in G3-G4. CONCLUSION: The present study concluded that high XOR activity was associated with the severity of CKD in a general Japanese population, suggesting that upregulated XOR activity may be involved in advanced renal dysfunction.

Study Profile of the Tohoku Medical Megabank Community-Based Cohort Study.

BACKGROUND: We established a community-based cohort study to assess the long-term impact of the Great East Japan Earthquake on disaster victims and gene-environment interactions on the incidence of major diseases, such as cancer and cardiovascular diseases. METHODS: We asked participants to join our cohort in the health check-up settings and assessment center based settings. Inclusion criteria were aged 20 years or over and living in Miyagi or Iwate Prefecture. We obtained information on lifestyle, effect of disaster, blood, and urine information (Type 1 survey), and some detailed measurements (Type 2 survey), such as carotid echography and calcaneal ultrasound bone mineral density. All participants agreed to measure genome information and to distribute their information widely. RESULTS: As a result, 87,865 gave their informed consent to join our study. Participation rate at health check-up site was about 70%. The participants in the Type 1 survey were more likely to have psychological distress than those in the Type 2 survey, and women were more likely to have psychological distress than men. Additionally, coastal residents were more likely to have higher degrees of psychological distress than inland residents, regardless of sex. CONCLUSION: This cohort comprised a large sample size and it contains information on the natural disaster, genome information, and metabolome information. This cohort also had several detailed measurements. Using this cohort enabled us to clarify the long-term effect of the disaster and also to establish personalized prevention based on genome, metabolome, and other omics information.

Association between the social isolation and depressive symptoms after the great East Japan earthquake: findings from the baseline survey of the TMM CommCohort study.

BACKGROUND: Social isolation and mental health issues have become a severe problem in disaster areas in the Great East Japan Earthquake. This study examined whether the combination of the house damage and social isolation or the combination of the death of family members and social isolation is associated with depressive symptoms among survivors using the baseline study data of the Tohoku Medical Megabank Project Community-Based Cohort Study (TMM CommCohort Study). METHODS: We used cross-sectional data from a baseline survey of 48,958 participants (18,423 males, 30,535 females; aged 60.1 ± 11.2 years) to examine the association between social isolation measured by the Lubben social network scale 6 (LSNS-6) and depressive symptoms measured by the Center for Epidemiological Studies-Depressive Scale (CES-D). The presence of social isolation and depressive symptoms was defined by an LSNS-6 score of < 12 and a CES-D score of ≥16, respectively. We performed a logistic regression analysis to determine the multivariable-adjusted odds ratio (95% confidence interval) [AOR (95% CI)] for depressive symptoms according to sex in the social isolation in comparison to without social isolation, and the associations of the combination of the house damage or the death of family members and social isolation and depressive symptoms. RESULTS: Social isolation was significantly associated with depressive symptoms (males: OR = 1.87; 95% CI = 1.72-2.04, females: OR = 2.13; 95% CI = 2.00-2.26). Both males and females respondents with severe house damage and social isolation had a greater risk of depressive symptoms in comparison to those with an undamaged house and without social isolation (males: OR = 3.40; 95% CI = 2.73-4.24, females: OR = 2.92; 95% CI = 2.46-3.46). The risk of depressive symptoms was also higher in both males and females respondents with the death of family members and social isolation in comparison to those without the death of family members and without social isolation (males: OR = 2.18; 95% CI = 1.90-2.50, females: OR = 2.60; 95% CI = 2.35-2.88). CONCLUSION: The findings suggested that a combination of social isolation and severe house damage and the death of family members caused by a large-scale natural disaster was associated with a higher risk of depressive symptoms although the interaction was not statistically significant.

Derivatization-based quadruplex LC/ESI-MS/MS method for high throughput quantification of serum dehydroepiandrosterone sulfate.

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 µg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.

Genome-wide association study identifies seven novel susceptibility loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide for which 15 disease-associated loci had been discovered. Among them, only 5 loci have been associated with POAG in Asians. We carried out a genome-wide association study and a replication study that included a total of 7378 POAG cases and 36 385 controls from a Japanese population. After combining the genome-wide association study and the two replication sets, we identified 11 POAG-associated loci, including 4 known (CDKN2B-AS1, ABCA1, SIX6 and AFAP1) and 7 novel loci (FNDC3B, ANKRD55-MAP3K1, LMX1B, LHPP, HMGA2, MEIS2 and LOXL1) at a genome-wide significance level (P < 5.0×10-8), bringing the total number of POAG-susceptibility loci to 22. The 7 novel variants were subsequently evaluated in a multiethnic population comprising non-Japanese East Asians (1008 cases, 591 controls), Europeans (5008 cases, 35 472 controls) and Africans (2341 cases, 2037 controls). The candidate genes located within the new loci were related to ocular development (LMX1B, HMGA2 and MAP3K1) and glaucoma-related phenotypes (FNDC3B, LMX1B and LOXL1). Pathway analysis suggested epidermal growth factor receptor signaling might be involved in POAG pathogenesis. Genetic correlation analysis revealed the relationships between POAG and systemic diseases, including type 2 diabetes and cardiovascular diseases. These results improve our understanding of the genetic factors that affect the risk of developing POAG and provide new insight into the genetic architecture of POAG in Asians.

Mass spectrometry-based microbiological testing for blood stream infection.

BACKGROUND: The most successful application of mass spectrometry (MS) in laboratory medicine is identification (ID) of microorganisms using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in blood stream infection. We describe MALDI-TOF MS-based bacterial ID with particular emphasis on the methods so far developed to directly identify microorganisms from positive blood culture bottles with MALDI-TOF MS including our own protocols. We touch upon the increasing roles of Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) as well. MAIN BODY: Because blood culture bottles contain a variety of nonbacterial proteins that may interfere with analysis and interpretation, appropriate pretreatments are prerequisites for successful ID. Pretreatments include purification of bacterial pellets and short-term subcultures to form microcolonies prior to MALDI-TOF MS analysis. Three commercial protocols are currently available: the Sepsityper® kit (Bruker Daltonics), the Vitek MS blood culture kit (bioMerieux, Inc.), and the rapid BACpro® II kit (Nittobo Medical Co., Tokyo). Because these commercially available kits are costly and bacterial ID rates using these kits are not satisfactory, particularly for Gram-positive bacteria, various home-brew protocols have been developed: 1. Stepwise differential sedimentation of blood cells and microorganisms, 2. Combination of centrifugation and lysis procedures, 3. Lysis-vacuum filtration, and 4. Centrifugation and membrane filtration technique (CMFT). We prospectively evaluated the performance of this CMFT protocol compared with that of Sepsityper® using 170 monomicrobial positive blood cultures. Although preliminary, the performance of the CMFT was significantly better than that of Sepsityper®, particularly for Gram-positive isolates. MALDI-TOF MS-based testing of polymicrobial blood specimens, however, is still challenging. Also, its contribution to assessment of susceptibility and resistance to antibiotics is still limited. For this purpose, liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) should be more useful because this approach can identify as many as several thousand peptide sequences. CONCLUSION: MALDI-TOF MS is now an essential tool for rapid bacterial ID of pathogens that cause blood stream infection. For the purpose of assessment of susceptibility and resistance to antibiotics of the pathogens, the roles of liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) will increase in the future.

Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes.

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.

A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses.

RATIONALE: 25-Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson-type reagent, 1,2,4-triazoline-3,5-dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson-type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. METHODS: Caged 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) analogues were prepared from 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione, DAP-PA) was used for LC/MS/MS analyses of VD metabolites. RESULTS: The solution stability of DAP-PA in ethyl acetate dramatically improved compared with that of the non-caged one. We measured the thermal retro-Diels-Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature-dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. CONCLUSIONS: DAP-PA was stable enough for mid- to long-term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

The Value of a Cystatin C-based Estimated Glomerular Filtration Rate for Cardiovascular Assessment in a General Japanese Population: Results From the Iwate Tohoku Medical Megabank Project.

BACKGROUND: Epidemiological studies have shown that high circulating cystatin C is associated with a risk of cardiovascular disease (CVD) independent of creatinine-based renal function measurements. The present study investigated the comparison between the cystatin C-based estimated glomerular filtration rate (GFRcys) and creatinine-based GFR (GFRcr) to determine whether these measurements are associated with CV biomarkers and elevated CVD risk in a general Japanese population. METHODS: The Iwate Tohoku Medical Megabank Organization pooled individual participant data from a general population-based cohort study in Iwate prefecture (n = 29,375). Chronic kidney disease (CKD) was estimated using the GFRcys, GFRcr and the urine albumin-to-creatinine ratio (UACR). RESULTS: The prevalence of CKD in the participants was found to be higher based on the GFRcr than the GFRcys. Multiple variable analyses after adjusting for baseline characteristics showed that high-sensitivity cardiac troponin T (hs-cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were associated with the GFRcys. The area under the receiver operating characteristic (AUROC) curve for identifying individuals with a high Suita score was higher for the GFRcys (AUROC = 0.68) than it was for the GFRcr (AUROC = 0.64, P < 0.001). The GFRcys provided reclassification improvement for the CVD risk prediction model by the GFRcr (net reclassification improvement = 0.341; integrated discrimination improvement = 0.018, respectively, P < 0.001). CONCLUSIONS: The GFRcys is more closely associated with CV biomarkers, including hs-cTnT and NT-proBNP levels, and a high Suita score than the GFRcr, and it provides additional value in the assessment of CVD risk using GFRcr.

An in-house centrifugation and membrane filtration technique for identifying microorganisms from positive blood culture bottles with high identification rates using matrix-assisted laser desorption ionization-Time-of-flight mass spectrometry: A preliminary report.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

Novel Risk Loci Identified in a Genome-Wide Association Study of Urolithiasis in a Japanese Population.

BACKGROUND: A family history of urolithiasis is associated with a more than doubling of urolithiasis risk, and a twin study estimating 56% heritability of the condition suggests a pivotal role for host genetic factors. However, previous genome-wide association studies (GWAS) have identified only six risk-related loci. METHODS: To identify novel urolithiasis-related loci in the Japanese population, we performed a large-scale GWAS of 11,130 cases and 187,639 controls, followed by a replication analysis of 2289 cases and 3817 controls. Diagnosis of urolithiasis was confirmed either by a clinician or using medical records or self-report. We also assessed the association of urolithiasis loci with 16 quantitative traits, including metabolic, kidney-related, and electrolyte traits (such as body mass index, lipid storage, eGFR, serum uric acid, and serum calcium), using up to 160,000 samples from BioBank Japan. RESULTS: The analysis identified 14 significant loci, including nine novel loci. Ten regions showed a significant association with at least one quantitative trait, including metabolic, kidney-related, and electrolyte traits, suggesting a common genetic basis for urolithiasis and these quantitative traits. Four novel loci were related to metabolic traits, obesity, hypertriglyceridemia, or hyperuricemia. The remaining ten loci were associated with kidney- or electrolyte-related traits; these may affect crystallization. Weighted genetic risk score analysis indicated that the highest risk group (top 20%) showed an odds ratio of 1.71 (95% confidence interval, 1.42 to 2.06) - 2.13 (95% confidence interval, 2.00 to 2.27) compared with the reference group (bottom 20%). CONCLUSIONS: Our findings provide evidence that host genetic factors related to regulation of metabolic and crystallization pathways contribute to the development of urolithiasis.

Creatine kinase muscle type specifically interacts with saturated fatty acid- and/or monounsaturated fatty acid-containing phosphatidic acids.

Diacylglycerol kinase (DGK) δ, which is a key enzyme in the pathogenesis of type 2 diabetes (T2D), preferentially generates saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs) such as 16:0/16:0-PA and 16:0/18:1-PA, but not polyunsaturated fatty acid (PUFA)-containing PAs, in glucose-stimulated myoblast cells. Here, we searched for the target proteins of 16:0/16:0-PA in the mouse skeletal muscle and identified an energy metabolizing enzyme, creatine kinase muscle type (CKM), which is correlated with T2D. CKM bound to 16:0/16:0-PA with the highest affinity (dissociation constant: 2.0 µM) among all the PA-binding proteins reported thus far. Intriguingly, CKM preferentially interacted with SFA- and/or MUFA-containing PAs, but not with PUFA-containing PAs. Notably, CKM exclusively interacted with PA, whereas the protein did not bind to other lipids such as diacylglycerol, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol (3,4,5)-trisphosphate and cardiolipin. Taken together, these results demonstrate that CKM is a very unique PA-binding protein that possesses exceedingly high affinity for PA, exceptional preference for SFA/MUFA-PA and extremely high specificity to PA and suggest that SFA/MUFA-PAs produced by DGKδ are novel regulators of CKM function.

Association between high-sensitivity cardiac troponin T and future cardiovascular incidence in a general Japanese population: results from the Tohoku medical megabank project.

Purpose: Elevation of high-sensitivity cardiac troponin T (hs-cTnT) is associated with an increased risk of cardiovascular disease (CVD). This study determined whether hs-cTnT was detectable with N-terminal pro-b-type natriuretic peptide (NT-proBNP) and related to CV risk factors in a general Japanese population. Materials and methods: The Tohoku Medical Megabank Organization pooled individual participant data for a population-based cohort study in the Iwate prefecture (n = 30,193, age = 60.2 ± 11.5 year). Results: Hs-cTnT levels were higher in participants with hypertension, diabetes mellitus than in participants without these conditions (all ps < 0.001). Logistic regression analysis demonstrated that NT-proBNP was strongly associated with elevation of hs-cTnT (OR = 3.35, 95% CI = 2.90-3.89, p < 0.001). The receiver operating characteristic curve analysis showed that hs-cTnT was one of useful biomarker for the differentiation of high risk for CVD (the Suita score ≥ 56) from a general population. Logistic regression analysis demonstrated hs-cTnT levels were related to the CVD high risk group (OR = 2.67, 95% CI = 2.28-3.14, p < 0.001). Conclusions: Hs-cTnT levels are associated with elevation of NT-proBNP and high Suita score, which suggests that elevated hs-cTnT is related to subclinical myocardial damage and indicates CV risk.

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI-MS/MS after conversion to MS/MS-fragmentable derivatives.

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D3 [25(OH)D3 ] and 24,25-dihydroxyvitamin D3 [24,25(OH)2 D3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3 -3- and -25-glucuronides, or 24,25(OH)2 D3 -3-, -24- and -25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2 D3 , respectively. The 3-glucuronide was not present for 24,25(OH)2 D3 , unlike 25(OH)D3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH)2 D3 .

Biobank Establishment and Sample Management in the Tohoku Medical Megabank Project.

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

Search for a Novel Allergen in Hen's Egg Allergy Using an IgE Immunoblotting Assay.

BACKGROUND: Food allergy is a serious health issue affecting roughly 4% of children, with a substantial effect on quality of life. Chicken egg allergy is frequently observed in infants. Therefore, some of them have to exclude hen's eggs from their daily diet to avoid allergenic symptoms. Hen's egg is composed of 2 soluble parts; one is egg white, which has been characterized as the major source of allergenicity, while the other is egg yolk, which is estimated as a miner source. Only 2 allergens from egg yolk, α-livetin (Gal d 5) and YGP42 (Gal d 6), have been described to date. METHODS: Sera from 53 patients allergic to hen's eggs and 2 patients allergic to sesame were obtained from the Department of Pediatrics, Chiba University Hospital. The study was performed using SDS-PAGE, IgE immunoblotting, and dot blotting. RESULTS: Seven bands of egg yolk were detected by IgE immunoblotting. Out of these bands, a possible new allergen was further characterized by LC-MS/MS. The 33-kDa band was identified as yolk glycoprotein (YGP40) by LC-MS/MS. A total of 21 of the 53 patients (47%) had YGP40 detected by dot blotting. CONCLUSIONS: We identified YGP40 as a new hen's egg yolk allergen and detected 4 sites of YGP40 as linear epitopes.

Capsular serotyping of Haemophilus influenzae by using matrix-associated laser desorption ionization-time of flight mass spectrometry.

Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.

Current Status of Proteomic Technologies for Discovering and Identifying Gingival Crevicular Fluid Biomarkers for Periodontal Disease.

Periodontal disease is caused by bacteria in dental biofilms. To eliminate the bacteria, immune system cells release substances that inflame and damage the gums, periodontal ligament, or alveolar bone, leading to swollen bleeding gums, which is a sign of gingivitis. Damage from periodontal disease can cause teeth to loosen also. Studies have demonstrated the proteomic approach to be a promising tool for the discovery and identification of biochemical markers of periodontal diseases. Recently, many studies have applied expression proteomics to identify proteins whose expression levels are altered by disease. As a fluid lying in close proximity to the periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for periodontal disease biomarkers because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Periodontal diseases are also promising targets for proteomics, and several groups, including ours, have applied proteomics in the search for GCF biomarkers of periodontal diseases. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize the current situation of proteomic technologies to discover and identify GCF biomarkers for periodontal diseases.

Impact of Water- and Lipid-Soluble Statins on Nonculprit Lesions in Patients with Acute Coronary Syndrome.

Statins can be differentiated into two types, based on their solubility, which have potentially differing effects on the coronary artery wall. However, suspected differences in statins' effects on plaque composition have not been systemically investigated.Sixty-seven patients with acute coronary syndrome (ACS) were randomly assigned to either atorvastatin (10 mg/day) or rosuvastatin (2.5 mg/day). Intravascular ultrasound (IVUS) and integrated backscatter (IB)-IVUS, an established tool to quantify each plaque's components, were performed immediately after emergent percutaneous coronary intervention (PCI). Follow-up IVUS was performed between 6 and 12 months after PCI. Serial changes in serum lipid profiles and plaque composition volumes were compared between the two groups.Thirty-five patients were eligible for serial IB-IVUS analyses. The mean low-density lipoprotein-cholesterol level significantly decreased in the atorvastatin and rosuvastatin groups (P < 0.001); plaque volumes were also significantly reduced from 82.0 ± 46.2 to 74.9 ± 41.3 mm3 (P = 0.01) and from 74.7 ± 35.3 to 67.7 ± 27.0 mm3 (P = 0.02), respectively. IB-IVUS revealed a significant reduction in fibrous volume from 33.8 ± 20.0 to 27.5 ± 14.9 mm3 (P < 0.01) and from 29.6 ± 13.6 to 24.8 ± 7.6 mm3 (P < 0.05), respectively; however, significant changes were not noted in the volume of the lipid pool for the atorvastatin group and the rosuvastatin group, respectively.Water- and lipid-soluble statins may be similarly effective in reducing coronary plaques in patients with ACS as judged qualitatively and quantitatively. Further study is needed to determine whether differences between water- and lipid-soluble statins affect plaque components.

Genetic Predisposition to Ischemic Stroke: A Polygenic Risk Score.

BACKGROUND AND PURPOSE: The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk. METHODS: We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets). RESULTS: In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33-2.31) and 1.99 (95% confidence interval, 1.19-3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001). CONCLUSIONS: The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors.