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Takuma Yuri,1,2 Nariyuki Mura,1,3,4 Kyosuke Hoshikawa,4 Hugo Giambini,2 Hiromi Fujii,1 Yoshiro Kiyoshige1,4 1Graduate School of Health Sciences, Yamagata Prefectural University of Health Sciences, Yamagata, Japan; 2Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, TX, USA; 3Department of Orthopaedic Surgery, Yoshioka Hospital, Tendo, Japan; 4Department of Physical Therapy, Yamagata Prefectural University of Health Sciences, Yamagata, JapanCorrespondence: Takuma YuriGraduate School of Health Sciences, Yamagata Prefectural University of Health Sciences, 260 Kamiyanagi, Yamagata, 990-2212, JapanTel +81-23-686-6741Email d2167013@yachts.ac.jpPurpose: Ultrasound elastography has been used to evaluate the skeletal muscle stiffness as a biomarker for sarcopenia assessment. However, there is no consensus with respect to the size and location of the region of interest in assessing such fat infiltrated muscle. The objective of this study was to determine which cross-sectional area should be measured in torn disuse muscle with fat infiltration to accurately measure muscle activity using real-time tissue elastography (RTE).Methods: Twenty-seven patients, whose rotator cuff muscle with torn tendon was successfully repaired, were followed by programmed rehabilitation. RTE measurements of the supraspinatus muscle were obtained during muscle contraction before and one-year after surgery so that the activity value was defined as the difference between elastography measurements at rest and elastography measurements during contraction. Given that the patients with successfully repaired and completed rehabilitation showed an increased activity value, the sensitivity for three regions of interest; posterior portion of the anterior-middle subregion (AM-p), anterior region (AR), and whole cross-sectional area of the supraspinatus (whole) were compared with the number of patients showing an increase in activity values as sensitivity analysis.Results: The sensitivity showing an increase in activity values was 74.1% for the AM-p area, 70.4% for the AR area, and 81.5% for the whole area. Intraclass correlation coefficient1,3 was 0.87– 0.97 for the AM-p area, 0.88– 0.98 for the AR area and 0.92– 0.99 for the whole area.Conclusion: The whole cross-sectional area is suitable to measure muscle activity in muscle with fat infiltration. The results in this study will provide some beneficial information when ultrasound elastography is used for the assessment of sarcopenia muscle with fat infiltration.Keywords: ultrasound elastography, region of interest, sarcopenia, fat infiltration, supraspinatus muscle

Background: Multiple factors are involved in the pathogenesis of primary biliary cholangitis (PBC), a chronic cholestatic liver disease, characterized by intrahepatic cholangiopathy. In particular, studies have suggested that environmental factors such as the presence of granulomas in the portal vein region are important for the development of PBC. This study aimed to comprehensively analyze and identify foreign-derived antigens in PBC liver tissue to confirm their involvement in PBC pathogenesis., Methods: Portal areas and hepatocyte regions were selectively dissected from formalin-fixed paraffin-embedded PBC liver tissue samples using the microlaser method, followed by total DNA extraction. We then validated whether the bacterial strains identified through 16S rRNA metagenomic analysis were detected in PBC liver tissues., Results: The most frequently detected bacterial genera in the PBC liver tissue samples were Sphingomonas panacis, Providencia, and Cutibacterium. These bacterial genera were also detected in the other PBC samples. Validation for the detection of S. panacis, the most abundant genus, revealed polymerase chain reaction bands extracted from the portal areas of all samples. They were also more highly expressed than bands detected in the hepatocyte region., Conclusion: S. panacis antigen was specifically detected in the portal areas of PBC liver tissues. The introduction of foreign-derived antigens into the liver as an environmental factor could be a possible mechanism for the development of PBC., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Katsumi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Micro-Tom is a cultivar of tomato (Solanum lycopersicum), which is known as a major crop and model plant in Solanaceae. Micro-Tom has phenotypic traits such as dwarfism, and substantial EMS-mutagenized lines have been reported. After Micro-Tom was generated in Florida, USA, it was distributed to research institutes worldwide and used as a genetic resource. In Japan, the Micro-Tom lines have been genetically fixed; currently three lines have been re-distributed from three institutes, but many phenotypes among the lines have been observed. We have determined the genome sequence de novo of the Micro-Tom KDRI line, one of the Micro-Tom lines distributed from Kazusa DNA Research Institute (KDRI) in Japan, and have built chromosome-scale pseudomolecules. Genotypes among six Micro-Tom lines, including three in Japan, one in the United States, one in France, and one in Brazil showed phenotypic alternation. Here, we unveiled the swift emergence of genetic diversity in both phenotypes and genotypes within the Micro-Tom genome sequence during its propagation. These findings offer valuable insights crucial for the management of bioresources., (© The Author(s) 2024. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Background: A torn rotator cuff muscle deteriorates over time leading with an increase in muscle atrophy and fatty infiltration. There are several clinical assessments for evaluating the atrophy of the torn supraspinatus muscle. However, it is unclear which approach can more accurately estimate the activity of the torn supraspinatus muscle. The purpose of this study was to determine which magnetic resonance imaging-based muscle atrophy imaging assessment currently implemented in the clinical setting accurately estimates the activity of the torn supraspinatus muscle., Methods: Forty patients who were diagnosed with a rotator cuff tear and were candidates for repairs were selected for this study. Cross-sectional area, occupation ratio, and tangent sign were analyzed on T1-weighted oblique sagittal plane magnetic resonance images in which the scapular spine leads to the Y-section. Muscle belly ratio of the supraspinatus muscle was analyzed by calculating the ratio of the width of the muscle belly to the distance from the greater tubercle to the proximal end of the muscle on T1-weighted coronal plane magnetic resonance imaging images. Fatty infiltration was evaluated using the Goutallier classification system. Tear size was obtained intraoperatively by measuring the width and length of the tear and classified based on the Cofield's classification. To assess activity of the torn supraspinatus muscle, participants were first instructed to sit on a chair with the affected arm resting on a table and the shoulder abducted to 60° in the scapular plane with neutral rotation. Elasticity of the supraspinatus muscle belly was then obtained at rest and during isometric contraction using with real-time tissue elastography. Muscle activity, a surrogate for contractility, was defined as the difference between the elasticities measured at rest and during isometric contraction. A stepwise multiple regression analysis was used to investigate independent factors, such as sex, tear width, cross-sectional area, occupation ratio, tangent sign, and muscle belly ratio, related to muscle activity., Results: Stepwise multiple regression analysis ( R 2  = 0.522, P  < .001) revealed that supraspinatus muscle activity was significantly correlated with muscle belly ratio ( β  = 0.306, P  = .044) and Goutallier stage ( β  = -0.490, P  = .002)., Conclusion: Estimations of muscle belly ratio are most suitable for assessing the activity of a torn supraspinatus muscle compared to other clinical measurements., (© 2023 The Authors.)

Crosstalk among organs/tissues is important for regulating systemic metabolism. Here, we demonstrate inter-organ crosstalk between hepatic insulin and hypothalamic leptin actions, which maintains survival during food shortages. In inducible liver insulin receptor knockout mice, body weight is increased with hyperphagia and decreased energy expenditure, accompanied by increased circulating leptin receptor (LepR) and decreased hypothalamic leptin actions. Additional hepatic LepR deficiency reverses these metabolic phenotypes. Thus, decreased hepatic insulin action suppresses hypothalamic leptin action with increased liver-derived soluble LepR. Human hepatic and circulating LepR levels also correlate negatively with hepatic insulin action indices. In mice, food restriction decreases hepatic insulin action and energy expenditure with increased circulating LepR. Hepatic LepR deficiency increases mortality with enhanced energy expenditure during food restriction. The liver translates metabolic cues regarding energy-deficient status, which is reflected by decreased hepatic insulin action, into soluble LepR, thereby suppressing energy dissipation and assuring survival during food shortages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although liver regeneration has been extensively studied, the effects of bile-derived extracellular vesicles (bile EVs) on hepatocytes has not been elucidated. We examined the influence of bile EVs, collected from a rat model of 70% partial hepatectomy (PH), on hepatocytes. We produced bile-duct-cannulated rats. Bile was collected over time through an extracorporeal bile duct cannulation tube. Bile EVs were extracted via size exclusion chromatography. The number of EVs released into the bile per liver weight 12 h after PH significantly increased. Bile EVs collected 12 and 24 h post-PH, and after sham surgery (PH12-EVs, PH24-EVs, sham-EVs) were added to the rat hepatocyte cell line, and 24 h later, RNA was extracted and transcriptome analysis performed. The analysis revealed that more upregulated/downregulated genes were observed in the group with PH24-EVs. Moreover, the gene ontology (GO) analysis focusing on the cell cycle revealed an upregulation of 28 types of genes in the PH-24 group, including genes that promote cell cycle progression, compared to the sham group. PH24-EVs induced hepatocyte proliferation in a dose-dependent manner in vitro, whereas sham-Evs showed no significant difference compared to the controls. This study revealed that post-PH bile Evs promote the proliferation of the hepatocytes, and genes promoting cell cycles are upregulated in hepatocytes.

A newly developed semi-automatic synthetic luminescence substrate (SALS) method for measuring endotoxin was compared with the existing turbidimetric kinetic assay (TKA) using leukocyte-rich plasma to verify its usefulness. As a result, the endotoxin levels by this method were higher than that by the existing assay in most specimens, and the time required for measurement was much shorter. In addition, the leukocyte-rich plasma endotoxin level minus the plasma endotoxin levels were named leukocyte-associated endotoxin, and these levels per one leukocyte were compared. As a result, those levels were highly correlated with the endotoxin measurement levels of leukocyte-rich plasma. The correlation coefficient of SALS method was superior to the existing TKA method, the endotoxin level by this method may be close to true endotoxin levels., (© 2023. The Author(s).)

Objective We investigated the clinical characteristics of patients with reversible cerebral vasoconstrictor syndrome who had a history of migraine before the onset and considered the relationship between these two pathologies. Methods We investigated 98 patients who underwent magnetic resonance angiography within 14 days of the onset of reversible cerebral vasoconstriction syndrome at our hospital. Of these, 11 cases involved recurrences, so data from 87 patients were analyzed. Materials All consecutive patients diagnosed with reversible cerebral vasoconstrictor syndrome at our institution between October 2010 and July 2021. Results Fifty of the 87 patients (57%) had a history of migraine. A multivariate analysis revealed that the following clinical factors were significantly more frequent in patients with a history of migraine than in those without such a history: female sex; emotional situations as a trigger of the onset; presence of deep and subcortical white matter hyperintensity, absence of vasoconstriction in the M1 portion of the middle cerebral artery, and absence of other cerebral lesions on initial magnetic resonance imaging; absence of vasoconstriction of the basilar artery on follow-up magnetic resonance imaging; and progression of deep and subcortical white matter hyperintensity in the chronic stage. Conclusion Reversible cerebral vasoconstrictor syndrome patients with a history of migraine showed clinical features of migraine, including one aspect of cerebral small-vessel disease due to endothelial dysfunction, as a common causative condition.

Presepsin (sCD14-ST) is used as a marker for sepsis diagnosis. The production mechanism of presepsin is unique in that it is produced through phagocytosis of microorganisms. However, some studies have demonstrated that non-infected patients had increased presepsin levels and that presepsin is related to the risk or severity of diseases. This study was designed to describe a sensitive sandwich enzyme-linked immunosorbent assay for mouse presepsin developed to investigate the association of presepsin with diseases. Polyclonal antibodies were generated from peptide-immunized rabbit antiserum. Mouse presepsin standard was prepared using the recombinant method as an Fc-fusion protein. The linear detection range of the method was 4.7-300 pg/mL with a detection limit of 1.4 pg/mL. The assay detected mouse presepsin where mouse soluble CD14 (sCD14) was digested by cathepsin D proteinase and the cross-reactivity of sCD14 was not observed. The normal levels of mouse presepsin and sCD14 were compared; 65.9 ± 21.4 pg/mL and 43.2 ± 7.2 ng/mL were determined, respectively. Moreover, the levels of presepsin and sCD14 were compared with a lipopolysaccharide (LPS)-injected sepsis mouse model. The newly developed analytical method had high specificity to presepsin and is an efficient tool for studying the association between presepsin and diseases., (© 2022. The Author(s).)

Background: Knowledge of the morphological and functional differences in the anatomic subregions of the supraspinatus (SSP) and infraspinatus (ISP) muscles during forward flexion will provide useful information in the management of shoulder joint disorders. The purpose of this study was to investigate whether the SSP and ISP muscle subregions exhibit independent roles during forward flexion of the shoulder joint., Methods: Eight healthy male volunteers without any restriction in their shoulder joints were recruited for this study. Participants were instructed to sit on a chair with their back against the backrest. Shear modulus (kPa) was measured as a surrogate for muscle stiffness using shear wave elastography on the SSP and ISP muscle subregions. Active measurements of the nondominant arm were obtained during isometric contraction at a neutral position and every 15° intervals from 30° to 150° during forward flexion. Friedman test and Dunn's post hoc test were used to evaluate differences in measurement outcomes among angles during forward flexion in each muscle subregion., Results: Active stiffness outcomes of the anterior-middle subregion of the SSP muscle during forward flexion increased from 30° up to 45°, reaching a value of 182.4 ± 32.1 kPa ( P  < .001). Stiffness of the anterior-superficial subregion of the SSP muscle was highest at 30° (125.0 ± 20.6 kPa; P  < .019) and linearly decreased up to 105° with increasing shoulder angle position. Stiffness of the superior, middle, and inferior subregions of ISP muscle presented a mountain-shaped trend, with peaks of 99.9 ± 23.5 kPa at 90° ( P  < .013), 144.2 ± 11.2 kPa at 90° ( P  < .013), and 122.9 ± 27.9 kPa at 105° ( P  < .007), respectively. Finally, the stiffness outcomes of the pectoralis major and anterior region of the deltoid muscles showed a mountain-shaped trend with peaks of 89.4 ± 23.5 kPa at 60° ( P  < .007) and 176.7 ± 22.9 kPa at 90° ( P  < .026), respectively., Conclusions: The SSP and ISP muscle subregions play a significant role during active forward flexion motion. While closely overlapped, the activity of the muscle subregions changed during the forward flexion motion range, starting with an active anterior-superficial subregion of the SSP muscle at the initial range of motion and an active inferior subregion of the ISP muscle toward midrange of motion. The SSP and ISP subregions did not demonstrate independent functional behavior during forward flexion., (© 2022 The Authors.)

The effects of the climate change including an increase in the average global temperatures, and abnormal weather events such as frequent and severe heatwaves are emerging as a worldwide ecological concern due to their impacts on plant vegetation and crop productivity. In this review, the molecular processes of plants in response to heat stress-from the sensing of heat stress, the subsequent molecular cascades associated with the activation of heat shock factors and their primary targets (heat shock proteins), to the cellular responses-have been summarized with an emphasis on the classification and functions of heat shock proteins. Vegetables contain many essential vitamins, minerals, antioxidants, and fibers that provide many critical health benefits to humans. The adverse effects of heat stress on vegetable growth can be alleviated by developing vegetable crops with enhanced thermotolerance with the aid of various genetic tools. To achieve this goal, a solid understanding of the molecular and/or cellular mechanisms underlying various responses of vegetables to high temperature is imperative. Therefore, efforts to identify heat stress-responsive genes including those that code for heat shock factors and heat shock proteins, their functional roles in vegetable crops, and also their application to developing vegetables tolerant to heat stress are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kang, Lee, Hoshikawa, Kang and Jang.)

Climate change is a major threat to global food security. Changes in climate can directly impact food systems by reducing the production and genetic diversity of crops and their wild relatives, thereby restricting future options for breeding improved varieties and reducing the ability to adapt crops to future challenges. The global surface temperature is predicted to rise by an average of 0.3°C during the next decade, and the Paris Agreement (Paris Climate Accords) aims to limit global warming to below an average of 2°C, preferably to 1.5°C compared to pre-industrial levels. Even if the goal of the Paris Agreement can be met, the predicted rise in temperatures will increase the likelihood of extreme weather events, including heatwaves, making heat stress (HS) a major global abiotic stress factor for many crops. HS can have adverse effects on plant morphology, physiology, and biochemistry during all stages of vegetative and reproductive development. In fruiting vegetables, even moderate HS reduces fruit set and yields, and high temperatures may result in poor fruit quality. In this review, we emphasize the effects of abiotic stress, especially at high temperatures, on crop plants, such as tomatoes, touching upon key processes determining plant growth and yield. Specifically, we investigated the molecular mechanisms involved in HS tolerance and the challenges of developing heat-tolerant tomato varieties. Finally, we discuss a strategy for effectively improving the heat tolerance of vegetable crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hoshikawa, Pham, Ezura, Schafleitner and Nakashima.)

Background: This study investigated hyperintense vessel signs (HVS) on fluid-attenuated inversion recovery imaging in the P1-2 portions of posterior cerebral arteries (PCAs) as a "hyperintense PCA sign" and HVS of cortical arteries. We retrospectively examined whether these signs would be useful in diagnosing reversible cerebral vasoconstriction syndrome (RCVS) in the acute phase., Methods: Eighty patients with RCVS who underwent initial magnetic resonance imaging (MRI) within 7 days of onset were included in this study. HVS and related clinical factors were examined., Results: On initial MRI of RCVS patients, hyperintense PCA sign and HVS of cortical arteries were seen in 21 cases (26%) and 38 cases (48%), respectively. In patients showing hyperintense PCA sign, vasoconstriction of the A2-3 portion was a significant clinical factor. Conversely, vasoconstriction of the M1 and P1 portions and the presence of white matter hyperintensity on initial and chronic-stage MRI were significantly associated with the presence of HVS in cortical arteries., Conclusion: Because rich collateral flow exists around PCAs, the frequency of hyperintense PCA sign is not high. However, hyperintense PCA sign findings in patients with suspected RCVS offer credible evidence of extreme flow decreases due to vasoconstriction in peripheral PCAs and other arteries associated with the collateral circulation of PCAs. Conversely, HVS in cortical arteries tend to reflect slow antegrade circulation due to vasoconstriction of peripheral vessel and major trunks. Both signs appear useful for auxiliary diagnosis of acute-phase RCVS., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Surgical Neurology International.)

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools., (© 2021 Japanese Society for Plant Biotechnology.)

Autonomic nerve fibers in the liver are distributed along the portal tract, being involved in the regulation of blood flow, bile secretion and hepatic metabolism, thus contributing to systemic homeostasis. The present study investigated changes in hepatic nerve fibers in liver biopsy specimens from patients with normal liver, viral hepatitis and non-alcoholic steatohepatitis, in relation to clinical background. The areal ratio of nerve fibers to the total portal area was automatically calculated for each sample. The nerve fiber areal ratios (NFAR) for total nerve fibers and sympathetic nerve fibers were significantly lower in liver affected by chronic hepatitis, particularly viral hepatitis, and this was also the case for advanced liver fibrosis. However, the degree of inflammatory activity did not affect NFAR for either whole nerves or sympathetic nerves. Comparison of samples obtained before and after antiviral treatment for HCV demonstrated recovery of NFAR along with improvement of liver fibrosis., Competing Interests: The authors have declared that no competing interests exist.

In recent years, the development of diagnostics using artificial intelligence (AI) has been remarkable. AI algorithms can go beyond human reasoning and build diagnostic models from a number of complex combinations. Using next-generation sequencing technology, we identified hepatitis C virus (HCV) variants resistant to directing-acting antivirals (DAA) by whole genome sequencing of full-length HCV genomes, and applied these variants to various machine-learning algorithms to evaluate a preliminary predictive model. HCV genomic RNA was extracted from serum from 173 patients (109 with subsequent sustained virological response [SVR] and 64 without) before DAA treatment. HCV genomes from the 109 SVR and 64 non-SVR patients were randomly divided into a training data set (57 SVR and 29 non-SVR) and a validation-data set (52 SVR and 35 non-SVR). The training data set was subject to nine machine-learning algorithms selected to identify the optimized combination of functional variants in relation to SVR status following DAA therapy. Subsequently, the prediction model was tested by the validation-data set. The most accurate learning method was the support vector machine (SVM) algorithm (validation accuracy, 0.95; kappa statistic, 0.90; F-value, 0.94). The second-most accurate learning algorithm was Multi-layer perceptron. Unfortunately, Decision Tree, and Naive Bayes algorithms could not be fitted with our data set due to low accuracy (< 0.8). Conclusively, with an accuracy rate of 95.4% in the generalization performance evaluation, SVM was identified as the best algorithm. Analytical methods based on genomic analysis and the construction of a predictive model by machine-learning may be applicable to the selection of the optimal treatment for other viral infections and cancer., Competing Interests: NO authors have competing interests

Aim: Strict endotoxin limits are enforced for implants and catheters inserted into the body. However, no standard limit has been set for single-use sterile surgical gloves. Materials & methods: Four types of gloves sold in Japan were dipped in saline and that endotoxin levels were measured. Cytokine producing activity of gloves in blood was also measured. Results: Three of the four types of gloves showed endotoxin contamination. We also confirmed an increase in cytokine production in these gloves except one glove in which anionic surfactants was found. Conclusion: The extent to which detected endotoxins enter the body during surgery is controversial, but strict endotoxin limits need to be established.

Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0-400 μM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6β-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 μM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 μM) and 5-MeMBI (≥100 μM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms., Competing Interests: The authors report no declarations of interest., (© 2020 The Author(s).)

While the 'Micro-Tom' TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing coupled with 96-plex exome capture resulted in the acquisition of an exome sequence dataset with an average read count of 5.6 million for the 95 mutants. Over 98% of the capture target region could be covered by the short reads with an averaged read depth of 12.8, which enabled us to identify single nucleotide polymorphisms and Indels in a genome-wide manner. By subtracting intra-cultivar DNA variations that are present between wild-type 'Micro-Tom' lines, we identified 241,391 mutation candidates in 95 mutant individuals. Of these, 64,319 and 6,480 mutations were expected to cause protein amino acid substitutions or premature stop codon, respectively. Based on the exome mutation dataset, a mutant line designated 'TOMJPW601' was found to carry a premature stop codon mutation (W261*) in a putative auxin influx carrier gene SlLAX1 ( Solyc09G014380 ), consistent with our previous report of its curly leaf phenotype. Our results suggested that a population-scale mutation database developed by multiplexed exome sequencing could be used for in silico mutant screening, which in turn could contribute to both gene function research and breeding programs., (© 2019 The Japanese Society for Plant Cell and Molecular Biology.)

A number of plant microRNAs have been demonstrated to regulate developmental processes by integrating internal and environmental cues. Recently, the Arabidopsis thaliana F-box protein HAWAIIAN SKIRT ( HWS ) gene has been described for its role in miRNA biogenesis. We have isolated in a forward genetic screen a tomato ( Solanum lycopersicum ) line mutated in the putative ortholog of HWS . We show that the tomato hws-1 mutant exhibits reduction in leaflet serration, leaflet fusion, some degree of floral organ fusion, and alteration in miRNA levels, similarly to the original A. thaliana hws-1 mutant. We also describe novel phenotypes for hws such as facultative parthenocarpy, reduction in fertility and flowering delay. In slhws-1 , the parthenocarpy trait is influenced by temperature, with higher parthenocarpy rate in warmer environmental conditions. Conversely, slhws-1 is able to produce seeds when grown in cooler environment. We show that the reduction in seed production in the mutant is mainly due to a defective male function and that the levels of several miRNAs are increased, in accordance with previous HWS studies, accounting for the abnormal leaf and floral phenotypes as well as the altered flowering and fruit development processes. This is the first study of HWS in fleshy fruit plant, providing new insights in the function of this gene in fruit development., (Copyright © 2019 Damayanti, Lombardo, Masuda, Shinozaki, Ichino, Hoshikawa, Okabe, Wang, Fukuda, Ariizumi and Ezura.)

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.

High-density lipoprotein (HDL) particles that are formed in vivo adopt a disk-shaped structure, in which the periphery of the discoidal phospholipid bilayer is surrounded by apolipoprotein. Such discoidal nanoparticles can be reconstituted with certain apolipoproteins and phospholipids and are commonly called lipid nanodisks. Apolipoprotein E (apoE), one of the HDL constituent proteins, serves as a ligand for the low-density lipoprotein (LDL) receptor. Thus, it is considered that biocompatible delivery vehicles targeting LDL receptors could be prepared by incorporating apoE as the protein component of lipid nanodisks. To enhance targeting efficiency, we designed lipid nanodisks with a large number of ligands using a peptide with the LDL receptor-binding region of apoE combined with a high lipid affinity sequence (LpA peptide). In our study, the LpA peptide spontaneously formed discoidal complexes (LpA nanodisks) of approximately 10 nm in size, equivalent to native HDL. LpA peptides on nanodisks adopted highly α-helical structures, a competent conformation capable of interacting with LDL receptors. As anticipated, the uptake of LpA nanodisks into LDL receptor-expressing cells (HepG2) was higher than that of apoE nanodisks, suggesting an enhanced targeting efficiency via the enrichment of LDL receptor-binding regions on the particle. Biodistribution studies using 111 In-labeled LpA nanodisks showed little splenic accumulation and prolonged retention in blood circulation, reflecting the biocompatibility of LpA nanodisks. High accumulation of 111 In-labeled LpA nanodisks was observed in the liver as well as in implanted tumors, which abundantly express LDL receptors. Thus, LpA nanodisks are potential biocompatible delivery vehicles targeting LDL receptors.

Severe neuroinflammation is associated with blood brain barrier (BBB) disruption in CNS diseases. Although microglial activation and the subsequent changes in cytokine/chemokine (C/C) concentrations are thought to be key steps in the development of neuroinflammation, little data are available concerning the interaction of microglia with BBB cells. In this study, we investigated this interaction by adding LPS-activated microglia (LPS-MG) to the abluminal side of a BBB model composed of endothelial cells (EC), pericytes (Peri) and astrocytes (Ast). We then examined the abluminal concentrations of 27 C/Cs and the interactions between the LPS-MG and BBB cells. LPS-MG caused collapse of the BBB, revealed by decreases in the trans-endothelial electrical resistance (TEER) and by changes in the expression levels of tight junction (TJ) proteins. Under these conditions, 19 C/Cs were markedly increased on the abluminal side. Unexpectedly, although LPS-MG alone released 10 of the 19 C/Cs, their concentrations were much lower than those detected on the abluminal side of the BBB model supplemented with LPS-MG. Co-culture of LPS-MG with Ast caused marked increases in 12 of the 19 C/Cs, while co-culture of LPS-MG with EC and Peri resulted in a significant increase in only 1 of the 19 C/Cs (fractalkine). These results suggest that C/C dynamics in this system are not only caused by activated microglia but also are due to the interaction between activated microglia and astrocytes.

Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a , SlYABBY1a , FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED , GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp., (© 2018 The Japanese Society for Plant Cell and Molecular Biology.)

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells.

Background and Purpose: Reversible cerebral vasoconstriction syndrome is characterized by thunderclap headache and diffuse segmental vasoconstriction that resolves spontaneously within 3 months. Previous reports have proposed that vasoconstriction first involves small distal arteries and then progresses toward major vessels at the time of thunderclap headache remission. The purpose of this study was to confirm centripetal propagation of vasoconstriction on MRA at the time of thunderclap headache remission compared with MRA at the time of reversible cerebral vasoconstriction syndrome onset., Materials and Methods: Of the 39 patients diagnosed with reversible cerebral vasoconstriction syndrome at our hospital during the study period, participants comprised the 16 patients who underwent MR imaging, including MRA, within 72 hours of reversible cerebral vasoconstriction syndrome onset (initial MRA) and within 48 hours of thunderclap headache remission., Results: In 14 of the 16 patients (87.5%), centripetal propagation of vasoconstriction occurred from the initial MRA to remission of thunderclap headache, with typical segmental vasoconstriction of major vessels. These mainly involved the M1 portion of the MCA (10 cases), P1 portion of the posterior cerebral artery (10 cases), and A1 portion of the anterior cerebral artery (5 cases)., Conclusions: This study found evidence of centripetal propagation of vasoconstriction on MRA obtained at the time of thunderclap headache remission, compared with MRA obtained at the time of reversible cerebral vasoconstriction syndrome onset. If clinicians remain unsure of the diagnosis during early-stage reversible cerebral vasoconstriction syndrome, this time point represents the best opportunity to diagnose reversible cerebral vasoconstriction syndrome with confidence., (© 2016 by American Journal of Neuroradiology.)

Although carbon nanotubes (CNTs) are used in many fields, including energy, healthcare, environmental technology, materials, and electronics, the adverse effects of CNTs in the brain are poorly understood. In this study, we investigated the effects of CNTs on cultured microglia, as microglia are the first responders to foreign materials. We compared the effects of sonicated suspensions of 5 kinds of CNTs and their flow-through filtered with a 0.22 µm membrane filter on microglial viability. We found that sonicated suspensions caused microglial cell damage, but their flow-through did not. The number of microglial aggregates was well correlated with the extent of the damage. We also determined that the CNT agglomerates consisted of two groups: one was phagocytosed by microglia and caused microglial cell damage, and the other caused cell damage without phagocytosis. These results suggest that phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by CNT agglomerates and it is important to conduct studies about the relationships between physical properties of nanomaterial-agglomerates and cell damage.

TOMATOMA (http://tomatoma.nbrp.jp/) is a tomato mutant database providing visible phenotypic data of tomato mutant lines generated by ethylmethane sulfonate (EMS) treatment or γ-ray irradiation in the genetic background of Micro-Tom, a small and rapidly growing variety. To increase mutation efficiency further, mutagenized M3 seeds were subjected to a second round of EMS treatment; M3M1 populations were generated. These plants were self-pollinated, and 4,952 lines of M3M2 mutagenized seeds were generated. We checked for visible phenotypes in the M3M2 plants, and 618 mutant lines with 1,194 phenotypic categories were identified. In addition to the phenotypic information, we investigated Brix values and carotenoid contents in the fruits of individual mutants. Of 466 samples from 171 mutant lines, Brix values and carotenoid contents were between 3.2% and 11.6% and 6.9 and 37.3 µg g(-1) FW, respectively. This metabolite information concerning the mutant fruits would be useful in breeding programs as well as for the elucidation of metabolic regulation. Researchers are able to browse and search this phenotypic and metabolite information and order seeds of individual mutants via TOMATOMA. Our new Micro-Tom double-mutagenized populations and the metabolic information could provide a valuable genetic toolkit to accelerate tomato research and potential breeding programs., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A 45-year-old woman presented with extensive deep cerebral venous thrombosis (DCVT) identified by typical direct thrombosed sign in the internal cerebral vein, vein of Galen, and straight sinus on both computed tomography and T(2)*-weighted magnetic resonance imaging. Moreover, rare direct sign, probably of the direct lateral vein, was also obtained. Direct sign of the thrombosed vein or sinus is important for the diagnosis of DCVT. The present case indicates the usefulness of attenuated vein sign and T(2)*-weighted imaging for the diagnosis of direct thrombosed vein in patients with DCVT.

Patients with non-traumatic, non-aneurysmal, and non-perimesencephalic subarachnoid hemorrhage (SAH) tend to have clots circumscribed along the cortical convexity, a condition referred to as acute cortical SAH. Cerebral venous thrombosis (CVT) is a potential cause of cortical SAH. The study tried to establish the diagnosis and management of cortical SAH caused by CVT. Retrospective review of 145 patients with non-traumatic SAH identified 15 patients with no ruptured aneurysm. Clinical features were investigated with a specific focus on patients with SAH caused by CVT. Eight of the 15 patients had perimesencephalic SAH, and 7 had cortical SAH. SAH caused by CVT was diagnosed in 4 of the 7 patients with cortical SAH. The cortical SAH involved the unilateral convexity or sylvian cistern and spared the basal cistern on computed tomography in all 4 patients. CVT occurred in the transverse sinus and cortical vein (1 patient), insular vein (1 patient), and cortical vein (2 patients). Identification of thrombosed veins or sinuses was established directly by T(2)*-weighted and diffusion-weighted magnetic resonance (MR) imaging in the acute stage and diffusion-weighted and T(1)-weighted MR imaging in the subacute stage. All patients had cortical swelling without findings of venous hemorrhagic infarction on T(2)*-weighted MR imaging. None of the 4 patients received active treatment, and all had favorable outcomes. CVT in patients with non-traumatic cortical SAH should be first excluded as a potential hemorrhagic cause by MR imaging for thrombosed veins or sinuses before initiating antifibrinolytic therapy.

The diagnostic efficacy of fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging and computed tomography (CT) for acute subarachnoid hemorrhage (SAH) were compared and the problems with diagnosis were investigated in 81 patients with aneurysmal SAH within 24 hours after onset who underwent FLAIR imaging and CT on admission. The number of hematomas in the cisterns and ventricles were evaluated by clot scores. In addition, the frequency of undetected hematomas was calculated for the cisterns and ventricles. Clot scores were significantly higher for FLAIR imaging than for CT in the lateral sylvian, quadrigeminal, and convexity cisterns. On the other hand, clot scores were significantly higher for CT than for FLAIR imaging in the interhemispheric and medial sylvian cisterns. The overall frequency of undetected SAH was 2% for FLAIR imaging and 14% for CT. With the exception of the interhemispheric and medial sylvian cisterns, the frequency of undetected SAH was higher for CT than for FLAIR imaging. In this study, FLAIR imaging was more sensitive than CT for the detection of acute SAH within 24 hours after onset. However, the diagnostic efficacy of FLAIR imaging was reduced in comparatively tight cisterns.

Diffusion-weighted magnetic resonance (MR) imaging findings of 72 patients with subarachnoid hemorrhage (SAH) in the acute stage were investigated to determine the clinical implications of subarachnoid clots detected by diffusion-weighted MR imaging on admission. Correlations between SAH detected by diffusion-weighted MR imaging and clinical factors were analyzed retrospectively. Diffusion-weighted MR imaging detected SAH in 34 of the 72 patients on admission. Thirty-three of 46 patients classified as Fisher group 3 had SAH detected by diffusion-weighted MR imaging. SAH detected by diffusion-weighted MR imaging on admission was significantly associated with both SAH imaging score and chronic hydrocephalus. SAH was detected by diffusion-weighted MR imaging on admission in patients who had dense clots. Detection of SAH by diffusion-weighted MR imaging was correlated with pathological conditions associated with the volume of subarachnoid clot, such as chronic hydrocephalus.

The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

A 69-year-old woman had acute cholecystitis that mimicked right bundle branch block with coved-type ST-segment elevation in the precordial electrocardiogram leads (Brugada-type ST shift). The patient did not have obvious heart disease, syncope, or a family history of sudden death. The coved-type ST-segment elevation disappeared as the acute inflammation subsided. Intravenous administration of pilsicainide, a pure sodium channel blocker, could reproduce the Brugada-type ST shift. This is the first report of the Brugada-type ST shift occurring in association with acute cholecystitis.