A parent-rating scale of postpartum depression: Maternity-monitoring scale by parents (MMSP).

Postpartum depression (PPD) is an illness that is difficult for the affected women themselves to recognize. Moreover, many mothers believe that mothers should not complain about the mental difficulties of taking care of their children. Therefore, in addition to self-evaluation for PPD, evaluation from others is also necessary. We aimed to develop a novel measure to screen for PPD based on a parent-rating scale that is administered to the parents of postpartum mothers. The 15-item maternity-monitoring scale by parents (MMSP) was designed and applied to the feasibility cohort (n = 61) and the emergency cohort (n = 55). The Edinburgh Postnatal Depression Scale (EPDS) (threshold score of 8/9) was used to evaluate a high risk of PPD. An egogram-based index, the over-adaptation index for depression (OAID), was performed along with the EPDS and MMSP. In the feasibility cohort, MMSP was moderately correlated with EPDS. In the emergency cohort, under the circumstance of the state of emergency declaration over the coronavirus disease 2019 in Japan, application of the MMSP was delayed, resulting in the proportion of parents who overlooked PPD symptoms in their daughters increasing from 33 % to 50 %. Our findings suggest that a novel approach of parent-rated PDD screening of postpartum women is potentially possible, and the MMSP is a potential candidate for screening. Moreover, the OAID is also helpful in identifying women with hidden PPD, along with the EPDS. The performance of the MMSP should be confirmed in the parents of patients with PPD diagnosed by psychiatrists.

Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids.

New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high- from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI-NACE-MS greatly expands the metabolome coverage of conventional aqueous-based CE-MS protocols and is a promising platform for large-scale lipidomic studies.

Metabolomic profiling of gastric cancer tissues identified potential biomarkers for predicting peritoneal recurrence.

BACKGROUND: Metabolomics is useful for analyzing the nutrients necessary for cancer progression, as the proliferation is regulated by available nutrients. We studied the metabolomic profile of gastric cancer (GC) tissue to elucidate the associations between metabolism and recurrence. METHODS: Cancer and adjacent non-cancerous tissues were obtained in a pair-wise manner from 140 patients with GC who underwent gastrectomy. Frozen tissues were homogenized and analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Metabolites were further assessed based on the presence or absence of recurrence. RESULTS: Ninety-three metabolites were quantified. In cancer tissues, the lactate level was significantly higher and the adenylate energy charge was lower than in non-cancerous tissues. The Asp, ß-Ala, GDP, and Gly levels were significantly lower in patients with recurrence than in those without. Based on ROC analyses to determine the cut-off values of the four metabolites, patients were categorized into groups at high risk and low risk of peritoneal recurrence. Logistic regression and Cox proportional hazard analyses identified ß-Ala as an independent predictor of peritoneal recurrence (hazard ratio [HR] 5.21 [95% confidence interval 1.07-35.89], p = 0.029) and an independent prognostic factor for the overall survival (HR 3.44 [95% CI 1.65-7.14], p < 0.001). CONCLUSIONS: The metabolomic profiles of cancer tissues differed from those of non-cancerous tissues. In addition, four metabolites were significantly associated with recurrence in GC. ß-Ala was both a significant predictor of peritoneal recurrence and a prognostic factor.

High Throughput Screening of Serum γ-Glutamyl Dipeptides for Risk Assessment of Nonalcoholic Steatohepatitis with Impaired Glutathione Salvage Pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m2, n = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γ-glutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves long-term migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γ-glutamyltransferease (GGT) enzyme activity (r = -0.46; p = 2.5 × 10-7), which was indicative of a low-risk (n = 64) as compared to a high-risk (n = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.

Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis.

Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 102, maintaining linearity with r2 > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for large-scale metabolome analysis of plasma for biological studies and clinical biomarker screening.

Taxonomic classification for microbiome analysis, which correlates well with the metabolite milieu of the gut.

BACKGROUND: 16S rRNA gene amplicon sequencing analysis (16S amplicon sequencing) has provided considerable information regarding the ecology of the intestinal microbiome. Recently, metabolomics has been used for investigating the crosstalk between the intestinal microbiome and the host via metabolites. In the present study, we determined the accuracy with which 16S rRNA gene data at different classification levels correspond to the metabolome data for an in-depth understanding of the intestinal environment. RESULTS: Over 200 metabolites were identified using capillary electrophoresis and time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics in the feces of antibiotic-treated and untreated mice. 16S amplicon sequencing, followed by principal component analysis (PCA) of the intestinal microbiome at each taxonomic rank, revealed differences between the antibiotic-treated and untreated groups in the first principal component in the family-, genus, and species-level analyses. These differences were similar to those observed in the PCA of the metabolome. Furthermore, a strong correlation between principal component (PC) scores of the metabolome and microbiome was observed in family-, genus-, and species-level analyses. CONCLUSIONS: Lower taxonomic ranks such as family, genus, or species are preferable for 16S amplicon sequencing to investigate the correlation between the microbiome and metabolome. The correlation of PC scores between the microbiome and metabolome at lower taxonomic levels yield a simple method of integrating different "-omics" data, which provides insights regarding crosstalk between the intestinal microbiome and the host.

Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronic intestinal inflammatory condition initiated by integrins-mediated leukocyte adhesion to the activated colonic microvascular endothelium. Calreticulin (CRT), a calcium-binding chaperone, is known as a partner in the activation of integrin α subunits (ITGAs). The relationship between their interaction and the pathogenesis of IBD is largely unknown. Here we show that a small molecule, orally active ER-464195-01, inhibits the CRT binding to ITGAs, which suppresses the adhesiveness of both T cells and neutrophils. Transcriptome analysis on colon samples from dextran sodium sulfate-induced colitis mice reveals that the increased expression of pro-inflammatory genes is downregulated by ER-464195-01. Its prophylactic and therapeutic administration to IBD mouse models ameliorates the severity of their diseases. We propose that leukocytes infiltration via the binding of CRT to ITGAs is necessary for the onset and development of the colitis and the inhibition of this interaction may be a novel therapeutic strategy for the treatment of IBD.

Metabolome analysis of esophageal cancer tissues using capillary electrophoresis-time-of-flight mass spectrometry.

Reports of the metabolomic characteristics of esophageal cancer are limited. In the present study, we thus conducted metabolome analysis of paired tumor tissues (Ts) and non-tumor esophageal tissues (NTs) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The Ts and surrounding NTs were surgically excised pair-wise from 35 patients with esophageal cancer. Following tissue homogenization and metabolite extraction, a total of 110 compounds were absolutely quantified by CE-TOFMS. We compared the concentrations of the metabolites between Ts and NTs, between pT1 or pT2 (pT1-2) and pT3 or pT4 (pT3-4) stage, and between node-negative (pN-) and node-positive (pN+) samples. Principal component analysis and hierarchical clustering analysis revealed clear metabolomic differences between Ts and NTs. Lactate and citrate levels in Ts were significantly higher (P=0.001) and lower (P<0.001), respectively, than those in NTs, which corroborated with the Warburg effect in Ts. The concentrations of most amino acids apart from glutamine were higher in Ts than in NTs, presumably due to hyperactive glutaminolysis in Ts. The concentrations of malic acid (P=0.015) and citric acid (P=0.008) were significantly lower in pT3-4 than in pT1-2, suggesting the downregulation of tricarboxylic acid (TCA) cycle activity in pT3-4. On the whole, in this study, we demonstrate significantly different metabolomic characteristics between tumor and non-tumor tissues and identified a novel set of metabolites that were strongly associated with the degree of tumor progression. A further understanding of cancer metabolomics may enable the selection of more appropriate treatment strategies, thereby contributing to individualized medicine.

[Safe Use of Recent New Drugs-Current Status and Challenges].

　In Japan and overseas, Chugai Pharmaceutical Company handles numerous biopharmaceuticals, molecular targeted therapies and other pharmaceuticals with innovative modes of action. Expert safety evaluation is essential for promoting the appropriate use of these pharmaceuticals around the world and in gaining acceptance from patients and healthcare professionals (HCPs), while speedy decision-making is crucial for the timely collection and provision of safety information and thus ensuring safety. In 2015, we collected safety information on more than 180000 cases and evaluated it from a medical standpoint. We have established a system for recording the collected information in a global database, and are conducting signal detection of adverse drug reactions using this database. With this system, we promptly disclose information to regulatory authorities in Japan, the US, Europe and Asia. We have in-house medical doctors with abundant clinical experience who conduct expert safety evaluations. Many innovative drugs, such as anticancer drugs or biopharmaceuticals, require wider-ranging, more rigorous management, including the provision of appropriate safety information to HCPs, management of distribution through wholesalers and dispensing pharmacies, and confirmation of conditions of use, in addition to all-case registration surveillance. With progress in the development of individualized medicine and drugs with new modes of action, in order for HCPs to understand the characteristics of these new drugs and use them appropriately, pharmacists and pharmaceutical companies should cooperate in promoting their appropriate use in the spirit of 'All Pharmacists for Patients'.

Plasma metabolome analysis of patients with major depressive disorder.

AIM: This study sought to characterize the plasma metabolite profiling of patients with major depressive disorder (MDD). METHODS: Psychiatric assessments were made with the Structured Clinical Interview for DSM-IV Axis I Disorders. In the exploratory cohort, plasma metabolite profiles of 34 MDD patients and 31 mentally healthy controls were compared using capillary electrophoresis-mass spectrometry. Among the candidate metabolites, we focused on a metabolite showing the largest difference. The absolute concentrations were measured in two cohorts from a psychiatric primary care clinic to characterize the accuracy of the metabolite biomarker. RESULTS: Among 23 metabolites significantly lower in the MDD group than in healthy controls, we focused on phosphoethanolamine (PEA) as a candidate. The reduction of PEA levels in MDD was checked in independent clinical sample sets. An ion-chromatography-fluorescence detection method was developed to measure plasma PEA levels. In the preliminary cohort, we examined 34 MDD and 43 non-MDD subjects. The area under the receiver-operator curve (AUC) was 0.92, with sensitivity/specificity greater than 88%, at a cut-off of 1.46 µM. In the checking cohort, with 10 MDD and 13 non-MDD subjects, AUC was 0.89, with sensitivity/specificity of 86% and 100%, respectively, at a cut-off of 1.48 µM. Plasma PEA inversely correlated with MDD severity, depressed mood, loss of interest, and psychomotor retardation. CONCLUSION: These results suggest that plasma PEA level could be a candidate biomarker of MDD in the clinical setting. Further studies comparing MDD and mentally healthy controls are needed to confirm the utility of PEA as a biomarker for depression.

Mutations of the glycine cleavage system genes possibly affect the negative symptoms of schizophrenia through metabolomic profile changes.

AIM: Hypofunction of N-methyl-D-aspartate receptors (NMDAR) may contribute to the pathophysiology of schizophrenia (SCZ). Recently, the glycine cleavage system (GCS) was shown to affect NMDAR function in the brain. GCS functional defects cause nonketotic hyperglycinemia, the atypical phenotype of which presents psychiatric symptoms similar to SCZ. Here, we examined the involvement of GCS in SCZ. METHODS: First, to identify the rare variants and the exonic deletions, we resequenced all the coding exons and the splice sites of four GCS genes (GLDC, AMT, GCSH, and DLD) in 474 patients with SCZ and 475 controls and performed multiplex ligation-dependent probe amplification analysis in SCZ. Next, we performed metabolome analysis using plasma of patients harboring GCS variants (n = 5) and controls (n = 5) by capillary electrophoresis time-of-flight mass spectrometry. The correlation between plasma metabolites and Positive and Negative Syndrome Scale score was further examined. RESULTS: Possibly damaging variants were observed in SCZ: A203V, S801N in GLDC, near the atypical nonketotic hyperglycinemia causative mutations (A202V, A802V); G825D in GLDC, a potential neural tube defect causative mutation; and R253X in AMT. Marked elevation of plasma 5-oxoproline (pyroglutamic acid), aspartate, and glutamate, which might affect NMDAR function, was observed in patients harboring GCS variants. The aspartate level inversely correlated with negative symptoms (r = -0.942, P = 0.0166). CONCLUSION: These results suggest that GCS rare variants possibly contribute to the pathophysiology of SCZ by affecting the negative symptoms through elevation of aspartate.

Metabolic profile alterations in the postmortem brains of patients with schizophrenia using capillary electrophoresis-mass spectrometry.

OBJECTIVE: We aimed to find the alterations in the profiles of low-molecular-weight metabolites in the brains of schizophrenia patients that may reflect the pathophysiology of the disorder. METHOD: Human postmortem brain tissues from the frontal cortex (15 schizophrenia patients and 15 controls) and the hippocampus (14 schizophrenia patients and 15 controls) were obtained from the Stanley Foundation Neuropathology Consortium. We analyzed ~300 metabolites, using capillary electrophoresis with time-of-flight mass spectrometry. RESULTS: In the frontal cortex, the mean levels of 29 metabolites were significantly different between the schizophrenia and control groups. In the hippocampus, only a dipeptide, glycylglycine was significantly (p≤0.001, nominal p-value) increased in schizophrenia. Glycylglycine was also significantly (p=0.007) increased in the frontal cortex of schizophrenia. The pathway analyses revealed that several metabolic pathways including KEGG "Central carbon metabolism in cancer" and "Protein digestion and absorption" were commonly affected in the frontal cortex and the hippocampus of schizophrenia patients. CONCLUSION: These findings point out alterations in glucose metabolism and proteolysis in the brains of schizophrenia.

A Novel Evaluation Method for Displacement between Carbon Beam Axis and Positioning X-ray Axis Using an Imaging Plate.

PURPOSE: We developed an evaluation method for easily calculating displacement directly between the carbon beam axis and positioning X-ray axis. METHODS: A verification image was acquired by irradiating an imaging plate with a carbon beam and X-ray. The X-ray passed through a lead plate inserted in the range compensator holder. The displacement was calculated on the verification image from the center of a wire irradiated with carbon using a multi leaf collimator (MLC) and a wire irradiated with X-ray also using MLC. The accuracy of the method was evaluated by moving the carbon beam axis, the X-ray axis, and the setup angle. The weekly changes of vertical and lateral beams in all rooms were also evaluated. RESULTS: The displacements of the carbon beam axis and the setup angle did not influence the calculation results, whereas the displacement of the X-ray axis did (R=0.999). The displacements including weekly changes were all less than 1.00 mm. CONCLUSION: An evaluation method for calculating the displacement directly and simply between the carbon beam axis and positioning X-ray axis was developed and verified. The weekly changes of displacement between axes were evaluated to be acceptable at our facility.

Reduced cerebrospinal fluid ethanolamine concentration in major depressive disorder.

Amino acids play key roles in the function of the central nervous system, and their alterations are implicated in psychiatric disorders. In the search for a biomarker for major depressive disorder (MDD), we used high-performance liquid chromatography to measure amino acids and related molecules in the cerebrospinal fluid (CSF) of 52 patients with MDD (42 depressed and 10 remitted; DSM-IV) and 54 matched controls. Significant differences were found in four amino acid concentrations between the depressed patients and controls. After Bonferroni correction, only ethanolamine (EA) levels remained significantly reduced in depressed patients (nominal P = 0.0000011). A substantial proportion of the depressed patients (40.5%) showed abnormally low CSF EA levels (<12.1 µM) (P = 0.000033; OR = 11.6, 95% CI: 3.1-43.2). When patients with low EA and those with high EA levels were compared, the former had higher scores for overall depression severity (P = 0.0033) and 'Somatic Anxiety' symptoms (P = 0.00026). In unmedicated subjects, CSF EA levels showed a significant positive correlation with levels of homovanillic acid (P = 0.0030) and 5-hydroxyindoleacetic acid (P = 0.019). To our knowledge, this is the first study showing that patients with MDD have significantly lower CSF EA concentrations compared with control subjects. CSF EA could be a state-dependent biomarker for a subtype of MDD.

Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis.

BACKGROUND: Principal component analysis (PCA) has been widely used to visualize high-dimensional metabolomic data in a two- or three-dimensional subspace. In metabolomics, some metabolites (e.g., the top 10 metabolites) have been subjectively selected when using factor loading in PCA, and biological inferences are made for these metabolites. However, this approach may lead to biased biological inferences because these metabolites are not objectively selected with statistical criteria. RESULTS: We propose a statistical procedure that selects metabolites with statistical hypothesis testing of the factor loading in PCA and makes biological inferences about these significant metabolites with a metabolite set enrichment analysis (MSEA). This procedure depends on the fact that the eigenvector in PCA for autoscaled data is proportional to the correlation coefficient between the PC score and each metabolite level. We applied this approach to two sets of metabolomic data from mouse liver samples: 136 of 282 metabolites in the first case study and 66 of 275 metabolites in the second case study were statistically significant. This result suggests that to set the number of metabolites before the analysis is inappropriate because the number of significant metabolites differs in each study when factor loading is used in PCA. Moreover, when an MSEA of these significant metabolites was performed, significant metabolic pathways were detected, which were acceptable in terms of previous biological knowledge. CONCLUSIONS: It is essential to select metabolites statistically to make unbiased biological inferences from metabolomic data when using factor loading in PCA. We propose a statistical procedure to select metabolites with statistical hypothesis testing of the factor loading in PCA, and to draw biological inferences about these significant metabolites with MSEA. We have developed an R package "mseapca" to facilitate this approach. The "mseapca" package is publicly available at the CRAN website.

[Research on the forecasting of trends in demand for pharmacists 2011-2035].

The first crop of pharmacists graduating from 6-year programs in pharmaceutical l education arrived in April 2012, and it will be important to incorporate new factors when predicting future trends in supply and demand for pharmacists. If we project supply given an exam pass rate of 75%, the supply of pharmacists will increase for the next 10 years or so if the number of exam takers is about 10000, and no decrease in the total number of pharmacists is expected until 2035. At pharmacies, a high degree of demand for the services of pharmacists can be expected to result from increases in the number of elderly patients and the number of patients receiving prescriptions, together with expanded accommodation of home health care, if the proportion of prescriptions that are actually filled up to 70%. At hospitals, demand has been projected to increase over the short term, owing to such factors as the trend toward having a resident pharmacist in each ward, advances in team medicine, and the spread of outpatient chemotherapy. Given the rising enrollment quotas for schools of pharmacy, and if the current supply and demand for pharmacists are maintained, we cannot rule out the possibility that pharmacists will come to be in excess supply within a 10-year horizon if the number of unemployed continues to decrease and the employment rate continues to improve along with changes in economic conditions and the consciousness of graduates of the 6-year programs.

Metabolomic profiling of lung and prostate tumor tissues by capillary electrophoresis time-of-flight mass spectrometry.

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.

Technical approach to individualized respiratory-gated carbon-ion therapy for mobile organs.

We propose a strategy of individualized image acquisitions and treatment planning for respiratory-gated carbon-ion therapy. We implemented it in clinical treatments for diseases of mobile organs such as lung cancers at the Gunma University Heavy Ion Medical Center in June 2010. Gated computed tomography (CT) scans were used for treatment planning, and four-dimensional (4D) CT scans were used to evaluate motion errors within the gating window to help define the internal margins (IMs) and planning target volume for each patient. The smearing technique or internal gross tumor volume (IGTV = GTV + IM), where the stopping power ratio was replaced with the tumor value, was used for range compensation of moving targets. Dose distributions were obtained using the gated CT images for the treatment plans. The influence of respiratory motion on the dose distribution was verified with the planned beam settings using 4D CT images at some phases within the gating window before the adoption of the plan. A total of 14 lung cancer patients were treated in the first year. The planned margins with the proposed method were verified with clinical X-ray set-up images by deriving setup and internal motion errors. The planned margins were considered to be reasonable compared with the errors, except for large errors observed in some cases.

Serum metabolomic profile and potential biomarkers for severity of fibrosis in nonalcoholic fatty liver disease.

BACKGROUND: Biomarker for usefulness in diagnosing advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is expected. In order to discover novel biomarkers for NAFLD and its pathogenesis, we performed matabolomics screening. METHODS: (1) The initial cohort was 44 NAFLD patients. (2) This validation cohort was 105 NAFLD patients, 26 primary biliary cirrhosis (PBC) patients, and 48 healthy controls. Using capillary electrophoresis and liquid chromatography with mass spectrometry, we analyzed low molecular weight metabolites in these groups. RESULTS: 1. In the initial cohort, we found 28 metabolites associated with advanced fibrosis. Among them, 4 sulfated steroids showed the greatest difference. A decrease of dehydroepiandrosterone sulfate (DHEA-S) and 5α-androstan-3ß ol-17-one sulfate (etiocholanolone-S) was observed with the progression of fibrosis. Furthermore, 16 hydroxydehydroepiandrosterone sulfate (16-OH-DHEA-S) increased with the progression of fibrosis. 2. In the validation cohort, the decrease of DHEA-S and etiocholanolone-S, as well as the increase of 16-OH-DHEA-S, with the progression of fibrosis was confirmed. The 16-OH-DHEA-S/DHEA-S ratio and 16-OH-DHEA-S/etiocholanolone-S ratio were even more strongly associated with the grade of fibrosis. Among PBC patients, 16-OH-DHEA-S tended to be higher in stages 3 and 4 than in stages 1 and 2. However, levels of DHEA-S, etiocholanolone-S, and the two ratios were not associated with the stage of PBC. CONCLUSION: Several metabolic products were found to be biomarkers of fibrosis in NAFLD and could also be useful for diagnosis of this condition. Our findings suggested disturbance of hormone metabolism in NAFLD and might lead to the development of new therapy.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome.

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-ß-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.