Intensive determination of storage condition effects on human plasma metabolomics

Introduction

Human plasma metabolomics offer powerful tools for understanding disease mechanisms and identifying clinical biomarkers for diagnosis, efficacy prediction and patient stratification. Although storage conditions can affect the reliability of data from metabolites, strict control of these conditions remains challenging, particularly when clinical samples are included from multiple centers. Therefore, it is necessary to consider stability profiles of each analyte.

Objectives

The purpose of this study was to extract unstable metabolites from vast metabolome data and identify factors that cause instability.

Method

Plasma samples were obtained from five healthy volunteers, were stored under ten different conditions of time and temperature and were quantified using leading-edge metabolomics. Instability was evaluated by comparing quantitation values under each storage condition with those obtained after ?80 °C storage.

Result

Stability profiling of the 992 metabolites showed time- and temperature-dependent increases in numbers of significantly changed metabolites. This large volume of data enabled comparisons of unstable metabolites with their related molecules and allowed identification of causative factors, including compound-specific enzymatic activity in plasma and chemical reactivity. Furthermore, these analyses indicated extreme instability of 1-docosahexaenoylglycerol, 1-arachidonoylglycerophosphate, cystine, cysteine and N⁶-methyladenosine.

Conclusion

A large volume of data regarding storage stability was obtained. These data are a contribution to the discovery of biomarker candidates without misselection based on unreliable values and to the establishment of suitable handling procedures for targeted biomarker quantification.

     

Cone-Beam Computed Tomography Correlates with Conventional Helical Computed Tomography in Evaluation of Lipiodol Accumulation in HCC after Chemoembolization

Background & Aims

The amount of drug-loaded lipiodol in an HCC tumor post-transarterial chemoembolization (TACE) correlates with the risk of local tumor recurrence. Lipiodol enhancement of a tumor on conventional CT, measured in Hounsfield units (HU), can predict tumor response. Here we investigate whether cone-beam CT (CBCT) can also be used to predict tumor response, providing the benefit of being able to optimize the patient’s treatment plan intra-procedurally.

Methods

A total of 82 HCC nodules (82 patients), ≤5 cm in diameter, were treated with balloon-occluded TACE using miriplatin between December 2013 and November 2014. For each patient, both CBCT and conventional CT images were obtained post-TACE. The degree of correlation between CBCT and conventional CT was determined by comparing identical regions of interest for each imaging modality using pixel values.

Results

The pixel values from conventional CT and CBCT were highly correlated, with a Pearson correlation coefficient of 0.912 (p<0.001). The location of the nodules within the liver did not affect the results; the correlation coefficient was 0.891 (p<0.001) for the left lobe and 0.926 (p<0.001) for the right lobe. The mean pixel value for conventional CT was 439 ± 279 HU, and the mean pixel value for CBCT was 416 ± 311 HU.

Conclusions

CBCT may be used as a substitute for conventional CT to quantitatively evaluate the amount of drug-loaded lipiodol within an HCC nodule and, hence, the efficacy of TACE treatment. The major benefit of using CBCT is the ability to predict the likelihood of local recurrence intra-procedurally, enabling subsequent treatment optimization.     

Summed Probability Distribution of 14C Dates Suggests Regional Divergences in the Population Dynamics of the Jomon Period in Eastern Japan

Recent advances in the use of summed probability distribution (SPD) of calibrated ¹⁴C dates have opened new possibilities for studying prehistoric demography. The degree of correlation between climate change and population dynamics can now be accurately quantified, and divergences in the demographic history of distinct geographic areas can be statistically assessed. Here we contribute to this research agenda by reconstructing the prehistoric population change of Jomon hunter-gatherers between 7,000 and 3,000 cal BP. We collected 1,433 ¹⁴C dates from three different regions in Eastern Japan (Kanto, Aomori and Hokkaido) and established that the observed fluctuations in the SPDs were statistically significant. We also introduced a new non-parametric permutation test for comparing multiple sets of SPDs that highlights point of divergences in the population history of different geographic regions. Our analyses indicate a general rise-and-fall pattern shared by the three regions but also some key regional differences during the 6^th millennium cal BP. The results confirm some of the patterns suggested by previous archaeological studies based on house and site counts but offer statistical significance and an absolute chronological framework that will enable future studies aiming to establish potential correlation with climatic changes.     

In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.     

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.     

Month of birth is associated with mortality among older people in Japan: Findings from the JAGES cohort

Month of birth (MOB) has been linked to a variety of health conditions in adulthood. This study examined the association between MOB and mortality among the healthy elderly in Japan, where a practice of traditional age reckoning was employed up until the late 1940s. The results showed male participants born in December were more likely to die earlier while those born in January had lower mortality. It is possible that social factors in early life, such as the time period when a birth is officially registered, may have implications for health that stretch across the life course.     

Evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a SARS 3CL protease inhibitor

A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL^pro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CL^pro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CL^pro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CL^pro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.