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With the global trend towards longer life expectancies, there’s an increasing emphasis on not just living longer, but also maintaining health and wellbeing into older age. This study explores the efficacy of Ninjin’yoeito (NYT) in the early stages of frailty, a critical period for preventive interventions. Taking account of the knowledge gap regarding the association between early frailty and NYT, we use data from workplace health checkups to examine the relationship between pre-frailty severity and NYT adaption. The objective of our research is to enhance the comprehension of early treatments using NYT to prevent the progression of frailty. A total of 314 employees of the Kyoto Industrial Health Association who received workplace health checkups between November 2021 and March 2023 and consented to this study were included in the analysis. Information on gender, age, body mass index (BMI), NYT-specific symptoms assessment, the Japanese version of the General Health Questionnaire-12 (GHQ-12), and the Kihon Checklist (KCL) were obtained. The correlation analysis revealed that there was a strong positive correlation between the number of applicable NYT indications and the GHQ-12 score (r = 0.5992, p < 0.0001). Similarly, a moderate positive correlation was observed between the number of applicable NYT indications and the KCL score (r = 0.5030, p < 0.0001). In the multivariate analysis, both GHQ-12 (β = 0.49, SE = 0.06, t = 7.66, 95% CI: 0.36 to 0.62, p = 0.000) and KCL (β = 0.54, SE = 0.12, t = 4.29, 95% CI: 0.29 to 0.79, p = 0.000) showed significant positive associations with the variance in the number of applicable NYT indications, indicating that higher scores on these measures were related to a greater number of indications. NYT has the potential to be utilized not only as a therapeutic intervention for frailty, but also as a preventive measure.

Abstract Background Intrahepatic cholangiocarcinoma (ICC) is frequently associated with precursor lesions, and biliary intraepithelial neoplasia (BilIN) may play a significant role in the development of ICC. However, the exact sequence and progression of these lesions remain to be elucidated. We report a rare case of ICC that exhibited extensive longitudinal intraductal extension of high-grade BilIN in the posterior bile ducts and involved the hepatic hilum and the peripheral hepatic parenchyma. Case presentation A 70-year-old female presented with anorexia. Computed tomography (CT) revealed a 15 mm enhancing intrahepatic tumor extending to the right intrahepatic secondary confluence. This was associated with a 7 mm diameter cystic dilatation of the segment 6 bile duct (B6). Endoscopic retrograde cholangiopancreatography (ERCP) revealed stenosis at the bifurcation of the posterior bile duct branch. Bile cytology confirmed the diagnosis of adenocarcinoma cells. Therefore, the patient was diagnosed with an ICC involving the right glissonean pedicle and underwent a right hepatectomy and lymph node dissection. Histologic examination revealed the tumor consisted of moderately differentiated adenocarcinoma. In connection with this lesion, diffuse intraductal atypical epithelial cells, which were diagnosed as high-grade BilIN, was observed not only in the dilated B6 but in the entire posterior bile ducts, which measured approximately 120 mm in diameter. Furthermore, two distinct foci of adenocarcinomas were identified in the peripheral hepatic parenchyma. A lymph node metastasis was also present. The pathological diagnosis was ICC pT4N1M0 stage IVA. The patient underwent adjuvant chemotherapy and has shown no recurrence 5 years after surgery. Imaging modalities were unable to accurately assess the extent of the intraductal neoplastic lesions due to their low papillary or sessile intraductal tubular growth. No risk factors for BilIN development, which has the potential to predispose to cholangiocarcinoma, were identified in the present case. Conclusions We present a case of ICC involving the right hepatic hilum, accompanied by extensive longitudinal extensions of high-grade BilIN and multifocal microscopic invasions in peripheral hepatic parenchyma. Notably, the intraductal lesions involved the entire posterior intrahepatic bile ducts. The presence of biliary neoplasia with extensive intraductal extension, in conjunction with ICC, should be considered as a variant of BilIN.

ObjectiveCombined pulmonary fibrosis and emphysema (CPFE) is a syndrome characterized by the coexistence of emphysema and fibrotic interstitial lung disease (ILD). The aim of this study was to examine the effect of CPFE on lung cancer risk and lung cancer-related mortality in patients with rheumatoid arthritis (RA).MethodsWe conducted a multicenter retrospective cohort study of patients newly diagnosed with lung cancer at five community hospitals between June 2006 and December 2021. Patients were followed until lung cancer-related death, other-cause death, loss to follow-up, or the end of the study. We used the cumulative incidence function with Gray's test and Fine-Gray regression analysis for survival analysis.ResultsA total of 563 patients with biopsy-proven lung cancer were included (82 RA patients and 481 non-RA patients). The prevalence of CPFE was higher in RA patients than in non-RA patients (40.2% vs.10.0%) at lung cancer diagnosis. During follow-up, the crude incidence rate of lung cancer-related death was 0.29 and 0.10 per patient-year (PY) in RA and non-RA patients, and 0.32 and 0.07 per PY in patients with CPFE and patients without ILD or emphysema, respectively. The estimated death probability at 5 years differed between RA and non-RA patients (66% vs. 32%, pConclusionsRA patients with lung cancer had a higher prevalence of CPFE and increased cancer-related mortality compared with non-RA patients. Close monitoring and optimal treatment strategies tailored to RA patients with CPFE are important to improve the poor prognosis of lung cancer.

Abstract The aging of patients with tuberculosis and better therapeutic management for them are recent concerns. This study aimed to identify risk factors for adverse drug reactions (ADRs) or death in very elderly patients with pulmonary tuberculosis and to assess the association between the dosage of antituberculosis drugs and outcomes. We conducted a multicenter retrospective study at two hospitals. Hospitalized patients (≥ 80 years old) with pulmonary tuberculosis who were treated with antituberculosis drugs were enrolled. Multivariate analysis was performed to assess factors associated with ADRs or death within 60 days after treatment initiation. In total, 632 patients were included. The primary endpoint occurred in 268 patients (190 ADRs and 78 deaths). A serum albumin level

Objectives: The potential hazards of extended-spectrum antibiotic therapy for patients with community-acquired pneumonia (CAP) with low risk for drug-resistant pathogens (DRPs) remain unclear; however, risk assessment for DRPs is essential to determine the initial antibiotics to be administered. The study objective was to assess the effect of unnecessary extended-spectrum therapy on the mortality of such patients. Methods: A post hoc analysis was conducted after a prospective multicenter observational study for CAP. Multivariable logistic regression analysis was performed to assess the effect of extended-spectrum therapy on 30-day mortality. Three sensitivity analyses, including propensity score analysis to confirm the robustness of findings, were also performed. Results: Among 750 patients with CAP, 416 with CAP with a low risk for DRPs were analyzed; of these, 257 underwent standard therapy and 159 underwent extended-spectrum therapy. The 30-day mortality was 3.9% and 13.8% in the standard and extended-spectrum therapy groups, respectively. Primary analysis revealed that extended-spectrum therapy was associated with increased 30-day mortality compared with standard therapy (adjusted odds ratio 2.82; 95% confidence interval 1.20-6.66). The results of the sensitivity analyses were consistent with those of the primary analysis. Conclusion: Physicians should assess the risk for DRPs when determining the empirical antibiotic therapy and should refrain from administering unnecessary extended-spectrum antibiotics for patients with CAP with a low risk for DRPs.

Abstract The prognostic significance of absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) remains unclear in patients with postoperative pneumonia (POP). The study objectives were to investigate the prognostic effects of ALC and ANC in POP patients, and to evaluate the time courses of ALC and ANC during hospitalization. This post-hoc analysis of a single-center prospective observational study evaluated consecutive POP patients, and comparatively analyzed community-acquired pneumonia (CAP) patients to highlight features of POP. In total, 228 POP patients and 1027 CAP patients were assessed. Severe lymphopenia (ALC 7500 cells/μL) was associated with better survival, whereas CAP patients with neutrophilia tended to have a lower survival rate. Prolonged lymphopenia and delayed increase in neutrophils were characteristic time-course changes of non-survivors in POP. The time courses of ALC and ANC between survivors and non-survivors in POP trended differently from those in CAP. Our study showed that ALC and ANC at pneumonia diagnosis can serve as prognostic factors in POP patients. Differences in time-course changes of ALC and ANC between survivors and non-survivors may provide important information for future immunological research in pneumonia.

Varicella-zoster virus (VZV) infection in adults or immunocompromised patients has a more severe presentation compared to the mild disease in children. To the best of our knowledge, no reports have described the clinical course of VZV pneumonia focusing on time course of skin rash, chest computed tomography (CT) findings, and viral load. Furthermore, no reports have described the reactivation of human herpes virus 6 (HHV-6) in VZV pneumonia. Here, we report a case of severe VZV pneumonia that resulted in reactivation of HHV-6 in a patient with rheumatoid arthritis (RA). A 66-year-old female treated for RA was admitted to our hospital with papules. Her chest CT showed granular infiltrates, micronodules, and ground-glass opacities. The day after admission, because the typical skin rashes and chest CT findings were observed, she was diagnosed with VZV pneumonia and treated with acyclovir. Her skin rash then crusted over five days and entered the healing process, whereas it took approximately two weeks for her respiratory condition and chest CT findings to improve. In addition, VZV deoxyribonucleic acid (DNA) gradually decreased with treatment. On the 34th day of admission, VZV DNA was not found in the serum sample but remained in the sputum sample. Furthermore, although reactivation of HHV-6 was observed, viremia resolved without treatment. Clinicians should be able to recognize the differences in the improvement of skin rashes, respiratory status, and chest CT findings. In addition, treatment for HHV-6 reactivation should be carefully determined for each case.

Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C20:0 and C20:1 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an inherited metabolic disease caused by a defect in electron transfer flavoprotein alpha (ETFA), ETF beta (ETFB), or ETF dehydrogenase (ETFDH), and riboflavin metabolism disorders have recently been reported to present as mimicking MADD. MADD is roughly classified into neonatal (type 1 or 2) and later-onset (type 3) forms. To identify clinicogenetic characteristics in Japan, we investigated 37 Japanese patients with MADD diagnosed from 1997 to 2020. The causes of MADD were ETFDH deficiency in 26 patients, ETFA deficiency in four, ETFB deficiency in six, and riboflavin metabolism disorder in one. All 15 patients with the neonatal-onset type died by 2 years of age, while five of 22 patients with the later-onset form died by 3 years of age. Furthermore, 8 of 15 patients with the later-onset form of ETFDH deficiency treated with riboflavin were riboflavin non-responders. p.Y507D in ETFDH was identified as the most common variant (9 of 48 alleles, 18.8%). Of two patients with a homozygous p.Y507D variant, one experienced disease onset and died in the neonatal period, while the other experienced disease onset at two months of age and died at two years old, suggesting that the p.Y507D variant results in fatal outcomes. Our study concluded that more than half of Japanese patients with MADD died by three years old, and more than half of patients with the later-onset form had poor responsiveness to riboflavin, partly due to the unique Japanese p.Y507D variant in ETFDH.

For morbidly obese patients with end-stage kidney disease (ESKD), there are often difficulties in accessing, implementing, and maintaining kidney replacement therapy (KRT). Although recent weight-loss surgery has the potential to solve these problems, its therapeutic strategy and appropriate perioperative management for morbidly obese patients with ESKD have not been established. Here, we describe the case history of a 47-year-old man diagnosed with ESKD due to obesity-related glomerulopathy with an uncorrected estimated glomerular filtration rate (eGFR) of 16.1 ml/min. He hoped for kidney transplantation but was not eligible due to his high body mass index (BMI) (36.9 kg/m2). Therefore, a combination strategy for both attaining weight loss and preparing for KRT was needed. We performed modified laparoscopic sleeve gastrectomy (LSG) combined with a buried catheter for peritoneal dialysis (PD), which resulted in reduction of multiple surgical invasions while simultaneously preparing for PD. After these operations, his body mass dropped to below 30.0 kg/m2, making him a candidate for kidney transplantation, while maintaining PD. Finally, he was able to have kidney transplantation with success. Collectively, in this case, our novel therapeutic approach was able to avoid multiple surgeries, to assist catheter insertion by laparoscopy, and to provide optimal KRT for an obese patient with ESKD. Simultaneous LSG and implantation of a buried PD catheter may be a promising strategy for morbidly obese patients with ESKD.

Abstract Background Metachronous pancreatic and gallbladder cancer is a rare condition and has a dismal prognosis. Herein, we present a patient with triple metachronous primary pancreatic and gallbladder cancer associated with pancreaticobiliary maljunction who achieved long-term survival after undergoing repeat curative surgery. Case presentation A 65-year-old female patient with advanced gallbladder cancer associated with pancreaticobiliary maljunction underwent extended cholecystectomy with extrahepatic bile duct resection. The pathological diagnosis was T3N0M0 stage III A papillary adenocarcinoma with hepatic invasion. During a monthly follow-up, a diffuse hypovascular 2.0 × 1.5-cm mass was detected in the pancreatic head 6.2 years after the initial surgery. Hence, the patient underwent pancreaticoduodenectomy. Histological examination revealed T3N0M0 stage IIA well-differentiated adenocarcinoma without lymph node metastases. Marked inflammatory reaction was observed in the non-cancerous lesions of the proximal pancreatic head parenchyma containing bile pigment within ductular lumens. After 12.5 years from the initial surgery, total pancreatectomy for a 4.0 × 3.0-cm mass in the remnant pancreas was performed. Histological examination revealed T3N1M0 stage IIB moderately differentiated adenocarcinoma with lymph node metastases. Hence, surgical curative resection was achieved. Based on the pathological findings, a definitive diagnosis of triple metachronous pancreatic and gallbladder cancer was made. The pathology suggests no precursor lesions such as pancreatic intraepithelial neoplasia (PanIN) and atypical flat lesions, but marked inflammations in the non-cancerous lesions, strengthening our hypothesis that chronic inflammation induced by the pancreaticobiliary maljunction is related to carcinogenesis of the pancreas. Despite further adjuvant chemotherapy, the patient’s general condition worsened; however, she remained alive 15.2 years after the initial surgery while receiving the best supportive care. Conclusions Repeat curative surgery for triple metachronous cancer was associated with a favorable prognosis. Both the biliary tract and the pancreas should be closely monitored during follow-up among patients with pancreaticobiliary maljunction, which can be managed with curative surgery.

Recent evidence suggests that trimethylamine-N-oxide (TMAO), a metabolite of L-carnitine and choline, is linked to atherosclerosis and cardiovascular diseases. As TMAO content is very high in fish, we raised the following question: why do Japanese people, who consume lots of fish, show a low risk of atherosclerosis? To address this question, we investigated the effects of TMAO and other L-carnitine-related metabolites on carotid intima–media thickness (IMT). Participants were recruited from a small island and a mountainous region. Plasma L-carnitine, γ-butyrobetaine (γBB), TMAO, trimethyllysine (TML), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) levels were measured using liquid or gas chromatography–mass spectrometry. Plasma L-carnitine concentration was higher in men than in women. TMAO and TML were significantly higher in the residents of the island than in the mountainous people. In multiple linear regression analyses in all participants, TML showed a significant inverse association with max-IMT and plaque score (PS), whereas TMAO did not show any associations. In women, L-carnitine was positively associated with max-IMT and PS. TMAO was correlated with both EPA and DHA levels, implying that fish is a major dietary source of TMAO in Japanese people. Our study found that plasma TMAO was not an apparent risk factor for atherosclerosis in elderly Japanese people, whereas a low level of TML might be a potential risk. L-carnitine may be a marker for atherosclerosis in women.

The dispersion behavior of lithium cobalt oxide (LCO) and acetylene black (AB) particles in the preparation process of the cathode slurry of LiB is investigated from the rheological viewpoint. The cathode slurry is considered as the dispersion of coarse LCO particles in the viscoelastic AB slurry. Viscosity as well as loss modulus of the cathode slurry are estimated from those of the AB slurry using and compared the measured results. After forming the AB network structure or When AB content is high enough, LCO particles can enter and destroy the AB network structure. As a result, LCO particles and fragmented AB network structures are homogeneously mixed, exhibiting better discharge performance. Once the cathode slurry is excessively diluted, the LCO particles are excluded from the AB network structure, resulting in low discharge capacity. Over fragmentation using a high-shear device causes the AB network structure into too small segments, which also lowers the battery performance. Viscosity is helpful to understand the entrance of LCO particles into the AB network, and the storage modulus detects the destruction of the AB network structure during the preparation process of cathode slurry.

Abstract Purpose Current newborn screening (NBS) for mucopolysaccharidosis type I (MPSI) has very high false positive rates and low positive predictive values (PPVs). To improve the accuracy of presymptomatic prediction for MPSI, we propose an NBS tool based on known biomarkers, alpha‐L‐iduronidase enzyme activity (IDUA) and level of the glycosaminoglycan (GAG) heparan sulfate (HS). Methods We developed the NBS tool using measures from dried blood spots (DBS) of 5000 normal newborns from Gifu Prefecture, Japan. The tool's predictive accuracy was tested on the newborn DBS from these infants and from seven patients who were known to have early‐onset MPSI (Hurler's syndrome). Bivariate analyses of the standardized natural logarithms of IDUA and HS levels were employed to develop the tool. Results Every case of early‐onset MPSI was predicted correctly by the tool. No normal newborn was incorrectly identified as having early‐onset MPSI, whereas 12 normal newborns were so incorrectly identified by the Gifu NBS protocol. The PPV was estimated to be 99.9%. Conclusions Bivariate analysis of IDUA with HS in newborn DBS can accurately predict early MPSI symptoms, control false positive rates, and enhance presymptomatic treatment. This bivariate analysis‐based approach, which was developed for Krabbe disease, can be extended to additional screened disorders.

Abstract Background The level and profiles of blood free carnitine and acylcarnitines, obtained by acylcarnitine analysis using tandem mass spectrometry, reflect various metabolic conditions. We aimed to examine the level of free carnitine and acylcarnitines in liver cirrhosis patients by acylcarnitine analysis and determine the clinical and subjective factors associated with blood carnitine fraction levels in liver cirrhosis. Methods We compared blood carnitine fractions in 54 liver cirrhotic patients to other laboratory test results and questionnaire answers. Results In almost all patients, the blood levels of free carnitine (C0) and acetylcarnitine (C2) were within the normal reference range. However, in some patients, the levels of long-chain acylcarnitines, such as C16 and C18:1-acylcarnitine, were higher than the normal reference range. Liver function, assessed by Child-Pugh score, was significantly correlated with the blood level of each carnitine fraction measured (C0, C2, C3, C4, C6, C10, C12, C12:1, C14:1, C16, C18:1, and C18:2-acylcarnitine). Cirrhotic symptom score was significantly correlated with C0, C2, C3, C16, and C18–1-acylcarnitine blood levels. Among the 36-item short-form health survey (SF-36) items, the physical component summary was significantly associated with C0, C2, and C18–1-acylcarnitine blood levels. Conclusions Carnitine fraction levels were positively correlated with liver cirrhosis stage, particularly, long-chain acylcarnitines. Moreover, carnitine fraction levels were associated with various subjective physical symptoms in liver cirrhosis patients.

Abstract Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase deficiency (mHS deficiency) is a rare autosomal recessive inborn error of ketogenesis caused by a mutation in the HMGCS2 gene, which is characterized by non‐(hypo)‐ketotic hypoglycemia, lethargy, and hepatomegaly during acute infection and/or prolonged fasting. Clinical presentations are similar to fatty acid oxidation defects; however, diagnosis of mHS deficiency is difficult because of poor biochemical markers. We report the case of a 12‐month‐old Japanese boy with mHS deficiency who presented with a coma, and hepatomegaly, but no hypoglycemia after a febrile episode and poor oral intake. Metabolic acidosis and severe fatty liver were observed. Serum acylcarnitine analysis revealed a slightly decreased free carnitine (C0) level and an increased acetylcarnitine (C2) level. Urinary organic acid analysis revealed hypoketotic dicarboxylic aciduria, and increased excretions of glutarate, and, retrospectively, 4‐hydroxy‐6‐methyl‐2‐pyrone. Although the patient did not present with hypoglycemia, the severe fatty liver and elevated free fatty acids to total ketone bodies ratio strongly suggested an inborn error of ketogenesis. In the analysis of the HMGCS2 gene, compound heterozygous mutations of c.130_131ins C (L44PfsX29) and c.1156_1157insC (L386PfsX73) were identified, which led to the diagnosis of mHS deficiency. He had recovered without any complication by the therapy, including intravenous glucose infusion. Unlike the previously reported cases of mHS deficiency, our case did not present with hypoglycemia and the fatty liver lasted over several months. mHS deficiency should be taken into consideration when a patient has severe metabolic acidosis and fatty liver with no or subtle ketosis, even without hypoglycemia.

Flow injection analysis–tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

In Japan, carnitine palmitoyltransferase II (CPTII) deficiency has been included as one of the primary target diseases in the expanded newborn mass screening program since 2018. However, many cases of the severe infantile hepatocardiomuscular form of CPTII deficiency showed severe neurodevelopmental delay or sudden death, which indicated that management of CPTII deficiency in the acute phase remains to be studied in detail. Herein, we discuss two cases diagnosed by newborn mass screening. Patient 1 was under strict clinical management from the neonatal period, with >20 admissions in 14 months, while Patient 2 was managed using a relatively relaxed approach, with only 2 admissions in the same period. Patient 1 showed normal development; however, Patient 2 expired at the age of 1 year 2 months. To develop strategies for preventing sudden deaths in patients with CPTII deficiency, this retrospective study focused on detailed clinical management practices and biochemical findings during the acute phase. We also investigated the correlation between conventional biomarkers (such as creatine kinase) and long-chain acylcarnitines. We propose that strict monitoring and immediate medical attention, even in case of slight fever or minor abdominal symptoms, can help prevent sudden death in patients with CPTII deficiency. Considering the higher morbidity rate of such patients, strict and acute management of CPTII deficiency cannot be overemphasized.

Mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders which can lead to degenerative and irreversible skeletal, cardiovascular, pulmonary, and neurological damage. Current treatments, including hematopoietic stem cell transplantation and enzyme replacement therapy, have been found most effective if administered before clinical symptoms are present, highlighting the urgent need for the development of newborn screening.This study analyzed 18,222 dried blood spot samples from newborns for both enzyme activity and glycosaminoglycan (GAG) concentration levels. GAG levels were measured using liquid chromatography tandem mass spectrometry. Results were compared to our previously established cutoff values for three subtypes of GAGs: dermatan sulfate (DS) and heparan sulfate (HS0S and HSNS). Samples that were high for two of the three GAGs were identified and screened a second time. Samples were also measured for iduronate-2-sulfatase and alfa-L-iduronidase activity.A total of 300 samples were above the established cutoff values for at least two of the three GAGs after the first screening. One sample was determined through clinical and genetic testing to be a true positive for MPS II. The false positive rate after the first GAG screening was 1.64%. A Cochran's formula test showed that the samples available for the second screening were representative samples (p = .0000601). False positive rate after second GAG screening, extrapolated from the representative sample was 0.4%. False positive rate after enzyme activity assay by fluorimetry for IDUA and IDS enzymes was 0.21% and 0.18%. A combination of GAG and enzyme assays provided no false positive and false negative samples.Two-tier screening involving a combination of enzyme activity and multiple GAGs should be considered the gold standard for the diagnosis of MPS patients. Keywords: Newborn screening, Mucopolysaccharidosis, Fetal GAG contamination, Tandem mass spectrometry, Glycosaminoglycans

Background: Expanded newborn screening (ENBS) utilizing tandem mass spectrometry (MS/MS) for inborn metabolic diseases (IMDs), such as organic acidemias (OAs), fatty acid oxidation disorders, (FAODs), and amino acid disorders (AAs), is increasingly popular but has not yet been introduced in many Asian countries. This study aimed to determine the incidence rates of OAs, FAODs, and AAs in Asian countries and Germany using selective screening and ENBS records. Materials and methods: Selective screening for IMDs using gas chromatography–mass spectrometry and MS/MS was performed among patients suspected to be afflicted in Asian countries (including Japan, Vietnam, China, and India) between 2000 and 2015, and the results from different countries were compared. Similarly, ENBS results from Japan, South Korea, Taiwan, and Germany were compared. Additionally, the results of selective screening and ENBS in Japan were compared. Results: Among 39,270 patients who underwent selective screening, IMDs were detected in 1170. Methylmalonic acidemia was frequently identified in several countries, including Japan (81/377 diagnosed IMDs), China (94/216 IMDs), and India (72/293 IMDs). In Vietnam, however, β-ketothiolase deficiency was particularly frequent (33/250 IMDs). ENBS yielded differences in overall IMD rates by country: 1:8557 in Japan, 1:7030 in Taiwan, 1:13,205 in South Korea, and 1:2200 in Germany. Frequently discovered diseases included propionic acidemia (PPA) and phenylketonuria (PKU) in Japan, 3-methylcrotonyl-CoA carboxylase deficiency (MCCD) and PKU in Taiwan, MCCD and citrullinemia type I in South Korea, and PKU and medium-chain acyl-CoA dehydrogenase deficiency in Germany. Furthermore, in Japan, selective screening and ENBS yielded respective PPA frequencies of 14.7% and 49.4% among all organic acidemias. Conclusion: The incidence rates of IMDs vary by country. Moreover, the disease spectra of IMDs detected via selective screening differ from those detected via ENBS. Keywords: Organic acidemia, Fatty acid oxidation disorder, Amino acid disorder, Inherited metabolic disease, Expanded newborn screening, Incidence rate

Introduction: Various markers, such as C14:1 and the C14:1/C2 ratio, are used as diagnostic markers of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). However, the levels of these markers in patients with VLCADD overlap with those in heterozygous carriers and even healthy subjects. Materials and methods: In twenty-three affected patients and 15 heterozygous carriers with VLCADD, the accuracies of C14:1, C14:1/C12:1, C14:1/C2, and C14:1/C16 in dried blood spots (DBS) and serum were statistically estimated. Results: Among the serum markers, the sensitivity, specificity, positive predictive value, negative predictive value, false-positive rate, false-negative rate, and validity of C14:1/C12:1 were superior to those of C14:1, C14:1/C2, and C14:1/C16, but C14:1/C2 demonstrated a statistical advantage compared with only C14:1 and C14:1/C16. Elevation in serum C14:1/C12:1 was observed in only one heterozygous carrier, whereas almost half of the carriers displayed false positive results for the other markers. Among the DBS markers, although the accuracy of C14:1/C2 was ostensibly the best, no statistical significance was observed. Discussion: Serum C14:1/C12:1 might be useful for differentiating patients with VLCADD from heterozygous carriers. Although serum C14:1/C2 was significantly useful for the detection of VLCADD, this marker could not distinguish the affected patients from carriers. C14:1/C12:1 might be optimal compared with the other markers. Keywords: Very long-chain acyl-CoA dehydrogenase deficiency, Tetradecenoyl carnitine, Dodecenoyl carnitine, Heterozygous carrier, Newborn screening, Diagnostic markers

Abstract Background Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). Nearly 150 pathogenic mutations have been identified in FLCN. The most frequent pattern is a frameshift mutation within a coding exon. In addition, splice-site mutations have been reported, and previous studies have confirmed exon skipping in several cases. However, it is poorly understood whether there are any splice-site mutations that cause translation of intron regions in FLCN. Case presentation A 59-year-old Japanese patient with multiple pulmonary cysts and pneumothorax was hospitalized due to dyspnea. BHD was suspected and genetic testing was performed. The patient exhibited the splice-site mutation of FLCN in the 5′ end of intron 9 (c.1062 + 1G > A). Total mRNA was extracted from pulmonary cysts, and RT-PCR assessment and sequence analyses were done. Two distinct bands were generated; one was wild-type and the other was a larger-sized mutant. Sequence analysis of the latter transcript revealed the insertion of 130 base pairs of intron 9 from the beginning of the splice-site between exons 9 and 10. Conclusion To our knowledge, this is the first report of distinct intron insertion using a BHD patient’s diseased tissue-derived mRNA. The present case suggests that a splice-site mutation can lead to exon skipping as well as intron reading mRNA. The splicing process may be dependent in part on whether the donor or acceptor site is affected.

Selenium (Se) is an essential element in animals but becomes severely toxic when the amount ingested exceeds the adequate intake level. It is known that the toxicological effects of Se are highly dependent on its chemical form. In this study, we evaluated the mutagenicity of nine naturally occurring Se compounds or the so-called bioselenocompounds, including selenite, selenate, selenocyanate, selenomethionine, selenocystine, Se-methylselenocysteine, selenohomolanthionine, N-acetylgalactosamine-type selenosugar, and trimethylselenonium ion, by using the Ames test. Salmonella typhimurium TA98, TA100, and TA1535 were used for the mutagenicity evaluation in the presence or absence of S9 mix, a metabolic activator. Only selenate showed weak mutagenicity even in the absence of S9 mix. None of the bioselenocompounds except selenate exhibited mutagenicity in all the strains tested in the presence or absence of S9 mix. Selenomethionine and selenocystine reduced the number of colonies in all the strains although no other selenoamino acids exerted the same effect. These results indicate that selenate directly or indirectly injures genome. Among the bioselenocompounds tested, selenomethionine and selenocystine show antibacterial activity, but the mechanism is unclear. Keywords: Selenium, Selenoamino acid, Selenosugar, Selenocyanate, Salmonella typhimurium, Mutagenicity

The HCO3− concentration in venous serum ([HCO3−]s) is a factor commonly used for detecting the body pH and metabolic conditions. To exactly detect [HCO3−]s, the venous CO2 pressure should be kept as it is in the vein. The [HCO3−]s measurement is technically complicated to apply for huge numbers of almost heathy persons taking only basic medical examinations. The summation of [HCO3−]s and the venous serum Cl− concentration ([Cl−]s) is approximately constant; therefore, we studied if [Cl−]s could be a marker detecting metabolic conditions instead of [HCO3−]s. Venous blood was obtained from persons taking basic medical examinations (the number of persons = 107,630). Older persons showed higher values of [Cl−]s, fasting blood sugar (FBS), and glycated hemoglobin (HbA1c) than younger ones. [Cl−]s showed positive correlation to age and negative correlation to FBS and HBA1c. The negative correlation of [Cl−]s to FBS/HbA1c was obvious in persons with high FBS/HbA1c, leading us to an idea that persons with high FBS/HbA1c show high [HCO3−]s, which might be caused by low activity of carbonic anhydrase in the lung observed in persons with diabetes mellitus under acidotic conditions. Taken together, an easily measured serum electrolyte, [Cl−]s, could be a useful marker estimating metabolic conditions.

Glycosaminoglycans (GAGs) are present in proteoglycans, which play critical physiological roles in various tissues. They are known to be elevated in mucopolysaccharidoses (MPS), a group of rare inherited metabolic diseases in which the lysosomal enzyme required to break down one or more GAG is deficient. In a previous study, we found elevation of GAGs in a subset of patients without MPS. In the current study, we aim to investigate serum GAG levels in patients with conditions beyond MPS. In our investigated samples, the largest group of patients had a clinical diagnosis of viral or non-viral encephalopathy. Clinical diagnoses and conditions also included epilepsy, fatty acid metabolism disorders, respiratory and renal disorders, liver disorders, hypoglycemia, developmental disorders, hyperCKemia, myopathy, acidosis, and vomiting disorders. While there was no conclusive evidence across all ages for any disease, serum GAG levels were elevated in patients with encephalopathy and some patients with other conditions. These preliminary findings suggest that serum GAGs are potential biomarkers in MPS and other disorders. In conclusion, we propose that GAGs elevated in blood can be used as biomarkers in the diagnosis and prognosis of various diseases in childhood; however, further designed experiments with larger sample sizes are required.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Glycosaminoglycans (GAGs), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS), are the primary biomarkers in patients with mucopolysaccharidoses (MPS); however, little is known about other biomarkers. To explore potential biomarkers and their correlation with GAGs, blood samples were collected from 46 MPS II patients, 34 MPS IVA patients, and 5 MPS IVB patients. We evaluated the levels of 8 pro-inflammatory factors (EGF, IL-1β, IL-6, MIP-1α, TNF-α, MMP-1, MMP-2, and MMP-9), collagen type II, and DS, HS (HS0S, HSNS), and KS (mono-sulfated, di-sulfated) in blood.Eight biomarkers measured were significantly elevated in untreated MPS II patients, compared with those in normal controls: EGF, IL-1β, IL-6, HS0S, HSNS, DS, mono-sulfated KS, and di-sulfated KS. The same eight biomarkers remained elevated in ERT-treated patients. However, only three biomarkers remained elevated in post-HSCT MPS II patients: EGF, mono-sulfated KS, and di-sulfated KS. Post-HSCT patients with MPS II showed that IL-1β and IL-6 were normalized as HS and DS levels decreased. Eight biomarkers were significantly elevated in untreated MPS IVA patients: EGF, IL-1β, IL-6, MIP-1α, MMP-9, HSNS, mono-sulfated KS, and di-sulfated KS, and four biomarkers were elevated in MPS IVA patients under ERT: IL-6, TNF-α, mono-sulfated KS, and di-sulfated KS. There was no reduction of KS in the ERT-treated MPS IVA patient, compared with untreated patients. Two biomarkers were significantly elevated in untreated MPS IVB patients: IL-6 and TNF-α.Reversely, collagen type II level was significantly decreased in untreated and ERT-treated MPS II patients and untreated MPS IVA patients.In conclusion, selected pro-inflammatory factors can be potential biomarkers in patients with MPS II and IV as well as GAGs levels. Keywords: Morquio syndrome, Hunter syndrome, Glycosaminoglycans, Cytokines, Inflammation

Mitochondrial trifunctional protein (TFP) is a multienzyme complex that catalyzes the last three steps of the β-oxidation cycle of long-chain fatty acids. In the prenatal diagnosis of TFP deficiency, acylcarnitine (AC) analysis has been considered difficult because of limited excretion of long-chain ACs into the fetal urine and hence into the amniotic fluid. Here, we report our experience with prenatally diagnosing TFP deficiency using AC analysis of amniotic fluid. The index case was a boy born at 38 weeks gestation and weighing 2588 g. He suddenly became unconscious and hypoglycemic and died on day 6 of life. Postmortem blood AC analysis and gene sequencing revealed TFP deficiency. Therefore, the parents underwent prenatal diagnoses for their subsequent 2 pregnancies. Mutation analysis suggested that one (Case 1) was affected and the other (Case 2) was not. AC analysis also demonstrated identical results, with significantly elevated 3-hydroxy-AC levels in the amniotic fluid of the affected pregnancy compared with those of heterozygotes and normal controls (n = 2 for heterozygotes and n = 8 for normal controls). Our findings suggest that AC analysis can functionally confirm results even in families with unidentified mutations, without raising issues related to maternal cell contamination. During prenatal diagnosis, misdiagnosis has to be avoided, and combining AC analysis with gene sequencing may result in more accurate prenatal diagnosis of TFP deficiency.

Carnitine palmitoyltransferase II (CPT-2) deficiency, an autosomal recessive disorder of fatty acid oxidation, can be detected by newborn screening using tandem mass spectrometry (TMS). Our case was a boy born at 38 weeks and 6 days of gestation via normal vaginal delivery; his elder sister was affected with CPT-2 deficiency. Acylcarnitine (AC) was analyzed in both dried blood spots (DBS) and serum 2 h after birth to determine whether the boy was also affected. His C16 and C18:1 AC levels in DBS were in the normal range, while his serum long-chain AC levels were marginally increased but lower than those of his sister. After the samples were taken, he was treated with glucose infusion to prevent any catabolism for 2 days. On day 4, the long-chain AC levels in both DBS and serum obtained were higher than those on day 0 and were equivalent to those of his sister. Genetic testing confirmed the presence of the same mutation found in his sister, a homozygous F383Y mutation in the CPT2 gene, thus leading to the diagnosis of CPT-2 deficiency. The sample for TMS should be taken between days 1 and 7. If the sample is not obtained at an appropriate time, correct diagnosis may not be made, as in our case. Although early diagnosis is required, samples taken within 24 h after birth should not be used for TMS.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Fusarium fujikuroi, a member of the Fusarium fujikuroi species complex, stands out as a rice bakanae disease pathogen with a high production of gibberellic acid. Not all, but some F. fujikuroi strains are known to produce a carcinogenic mycotoxin fumonisin. Fumonisin biosynthesis is dependent on the FUM cluster composed of 16 FUM genes. The FUM cluster was detected not only from a fumonisin producing strain, but also from a fumonisin nonproducing strain that does not produce a detectable level of fumonisin. Genetic mapping indicated the causative mutation(s) of fumonisin nonproduction is present in the FUM cluster of the fumonisin nonproducing strain. Comparative analyses of FUM genes between the fumonisin producing and the nonproducing strains and gene complementation indicated that causative mutation of fumonisin nonproduction is not a single occurrence and the mutations are distributed in FUM21 and FUM7. Our research revealed a natural variation in the FUM cluster involving fumonisin production difference in F. fujikuroi.

From the perspective of safety, it is necessary to assess the risk of hydrogen-air deflagration accurately. Especially, flame propagation velocity is one of the most important factors. Propagation velocity of outwardly expanding flame has been estimated from burning velocity of a flat flame considering influence of thermal expansion at a flame front; however, this conventional method is not enough to estimate an actual propagation velocity because flame propagation is accelerated owing to cellular flame front caused by intrinsic instability in hydrogen-air deflagration. Therefore, the dynamic propagation characteristics of hydrogen-air deflagration need to be understood. We performed explosion tests in a closed chamber which has 300mm diameter windows at atmospheric pressure and room temperature and in the range of equivalence ratio from 0.2 to 1.0. In the explosion tests, dynamic behaviors of flame propagation were observed by using high speed Schlieren photography. Analyzing the obtained Schlieren images, flame radius and flame propagation velocity were measured. As the result, cellular flame fronts formed and flame propagations of hydrogen-air mixture were accelerated in the range of equivalence ratio from 0.3 to 1.0. At the equivalence ratio of 0.2, a flame floated up and could not propagate downward because the influence of buoyancy exceeded a laminar burning velocity. In the range of smooth flame at small radius, considering the correlation of flame propagation velocity with flame stretch, flame propagation velocity of flat flame and Markstein length were obtained and critical flame radius was found. In the range of cellular flame at large radius, flame propagation velocity was fitted well by using a regression equation which including a logarithm of flame radius and characteristics of flame acceleration was discussed based upon the regression equation. Based upon these considerations, we propose this regression equation as simple model of flame propagation velocity.

Bovine mastitis is an inflammation of the mammary gland caused by bacterial infection in dairy cattle. It is the most costly disease in the dairy industry because of the high use of antibiotics. Staphylococcus aureus is one of the major causative agents of bovine mastitis and antimicrobial resistance. Therefore, new strategies to control bacterial infection are required in the dairy industry. One potential strategy is bacteriophage (phage) therapy. In the present study, we examined the host range of previously isolated S. aureus phages ΦSA012 and ΦSA039 against S. aureus strains isolated from mastitic cows. These phages could kill all S. aureus (93 strains from 40 genotypes) and methicillin-resistant S. aureus (six strains from six genotypes) strains tested. Using a mouse mastitis model, we demonstrated that ΦSA012 reduced proliferation of S. aureus and inflammation in the mammary gland. Furthermore, intravenous or intraperitoneal phage administration reduced proliferation of S. aureus in the mammary glands. These results suggest that broad host range phages ΦSA012 is potential antibacterial agents for dairy production medicine.

We report here that pink grape berries were obtained by a short insertion in the intron of the MybA1 gene, a gene that regulates grape berry color. Genetic variation was detected among the MybA1 genes from grapes cultivated worldwide. PCR analysis of the MybA1 gene demonstrated that the size of the MybA1 gene in the red allele differs among grapes. Oriental V. vinifera bearing pink berries has the longest MybA1 gene among grapes, whereas the shortest MybA1 gene was detected in occidental V. vinifera grapes. The nucleotide sequences of the MybA1 genes demonstrated that oriental V. vinifera has two additional gene fragments (44 bp and 111 bp) in the promoter region of the MybA1 gene in the red allele and another 33 bp fragment in the second intron of the MybA1 gene in the red allele. The short insertion in the intron decreased the transcription activity in the model system and retained MybA1 transcripts with unspliced intron in the total RNA. From the experiments using deletion mutants of the 33 bp short insertion, 16 bp of the 3' end in the insertion is a key structure for a defect in splicing of MybA1 transcripts. Thus, a weakly colored grape berry might be a result of the short insertion in the intron of a color regulatory gene. This is new evidence concerning the molecular mechanism of the fate of grape berry color. These findings are expected to contribute to the further understanding of the color variation in grape berries, which is correlated with the evolutional events occurring in the MybA1 gene of grapes.

We report a 1-year-7-month-old boy with West syndrome who had associated secondary adrenal insufficiency as a side effect of synthetic ACTH therapy. Serial investigation using corticotropin-releasing hormone (CRH) stimulation tests revealed the time course of his hypothalamic-pituitary-adrenal (HPA) axis recovery after the secondary adrenal insufficiency. Three days after completion of the ACTH therapy, the basal cortisol, peak cortisol, and peak ACTH levels were all low. One month after ACTH therapy, the basal cortisol level exceeded the cutoff level for intact adrenocortical function, and the peak ACTH level had improved. Five months after ACTH therapy, the peak cortisol level exceeded the cutoff level for intact adrenocortical function. The secondary adrenal insufficiency after ACTH therapy and the four months' time lag between the recovery timing of the basal and peak cortisol levels on CRH stimulation tests were notable findings. This follow-up data is valuable information for understanding the timeline for the process of recovery of the HPA axis from secondary adrenal insufficiency, that should lead to appropriate protocols for adrenal testing and adrenocorticosteroid replacement for patients who have undergone ACTH therapy. We also reviewed previous studies on secondary adrenal insufficiency after ACTH therapy in terms of incidence rate, onset risk factors, and recovery from it. Based on our own experience and previous reports, we suggest secondary adrenal insufficiency after ACTH therapy as follows: regarding the total synthetic ACTH dose administered, approximately 0.2 mg/kg of ACTH could cause secondary adrenal insufficiency. As for the required period for convalescence from secondary adrenal insufficiency, it would take from two to five months. [ABSTRACT FROM AUTHOR]