Development of an experimental method of systematically estimating protein expression limits in HEK293 cells

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was similar to 5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression

High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease

<p>Abstract</p> <p>Background</p> <p>The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.</p> <p>Methods</p> <p>CJD patients (n = 114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma.</p> <p>Results</p> <p>The cut-off value of the 14-3-3 gamma ELISA was > 1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI).</p> <p>Conclusion</p> <p>The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease.</p

Neural regulation in tooth regeneration of Ambystoma mexicanum

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration

Transcriptome Analyses of Immune System Behaviors in Primary Polyp of Coral Acropora digitifera Exposed to the Bacterial Pathogen Vibrio coralliilyticus under Thermal Loading

Elevated sea surface temperature associated with global warming is a serious threat to coral reefs. Elevated temperatures directly or indirectly alter the distribution of coral-pathogen interactions and thereby exacerbate infectious coral diseases. The pathogenic bacterium Vibrio coralliilyticus is well-known as a causative agent of infectious coral disease. Rising sea surface temperature promotes the infection of corals by this bacterium, which causes several coral pathologies, such as bacterial bleaching, tissue lysis, and white syndrome. However, the effects of thermal stress on coral immune responses to the pathogen are poorly understood. To delineate the effects of thermal stress on coral immunity, we performed transcriptome analysis of aposymbiotic primary polyps of the reef-building coral Acropora digitifera exposed to V. coralliilyticus under thermal stress conditions. V. coralliilyticus infection of coral that was under thermal stress had negative effects on various molecular processes, including suppression of gene expression related to the innate immune response. In response to the pathogen, the coral mounted various responses including changes in protein metabolism, exosome release delivering signal molecules, extracellular matrix remodeling, and mitochondrial metabolism changes. Based on these results, we provide new insights into innate immunity of A. digitifera against pathogen infection under thermal stress conditions

Computed tomography findings of intersigmoid hernia

Purpose: To evaluate the computed tomography findings of intersigmoid hernias. Material and methods: Between April 2010 and March 2018, 7 patients who were surgically diagnosed with intersigmoid hernia in 3 institutions were enrolled in this study. Two radiologists evaluated imaging findings for the herniated small bowel, the distance between the occlusion point and bifurcation of the left common iliac artery, and the anatomic relationship with adjacent organs. Results: All patients were male, and their mean age (standard deviation, range) was 61.0 (13.5, 36-85) years. The mean size of the bowel loops was 5.2 (1.3, 4.0-8.3) cm in the caudal direction, 3.6 (0.8, 2.5-5.1) cm in the lateral, and 3.4 (0.6, 2.5-4.7) cm in the anterior-posterior direction. The volume was 37.9 (27.8, 15.6-103.0) cm3 approximated by an ellipse, and 24.0 (17.7, 9.9-65.6) cm3 approximated by a truncated cone. The obstruction point was located 3.6 (0.6, 2.8-4.7) cm inferior to the bifurcation of the left common iliac artery. In all cases, the small bowel ran under the point at which the inferior mesenteric vessels bifurcated to the superior rectal vessels and the sigmoid vessels and formed a sac-like appearance between the left psoas muscle and the sigmoid colon. The ureter ran dorsal to the point of the bowel stenosis, and the left gonadal vein ran outside the small bowel loops. Conclusions: All cases showed common imaging findings, which may be characteristic of men's intersigmoid hernia. In addition, the fossa's position was lower, and the size was larger than in the previous study, which may be a risk factor

MADGAN: unsupervised medical anomaly detection GAN using multiple adjacent brain MRI slice reconstruction.

BACKGROUND: Unsupervised learning can discover various unseen abnormalities, relying on large-scale unannotated medical images of healthy subjects. Towards this, unsupervised methods reconstruct a 2D/3D single medical image to detect outliers either in the learned feature space or from high reconstruction loss. However, without considering continuity between multiple adjacent slices, they cannot directly discriminate diseases composed of the accumulation of subtle anatomical anomalies, such as Alzheimer's disease (AD). Moreover, no study has shown how unsupervised anomaly detection is associated with either disease stages, various (i.e., more than two types of) diseases, or multi-sequence magnetic resonance imaging (MRI) scans. RESULTS: We propose unsupervised medical anomaly detection generative adversarial network (MADGAN), a novel two-step method using GAN-based multiple adjacent brain MRI slice reconstruction to detect brain anomalies at different stages on multi-sequence structural MRI: (Reconstruction) Wasserstein loss with Gradient Penalty + 100 [Formula: see text] loss-trained on 3 healthy brain axial MRI slices to reconstruct the next 3 ones-reconstructs unseen healthy/abnormal scans; (Diagnosis) Average [Formula: see text] loss per scan discriminates them, comparing the ground truth/reconstructed slices. For training, we use two different datasets composed of 1133 healthy T1-weighted (T1) and 135 healthy contrast-enhanced T1 (T1c) brain MRI scans for detecting AD and brain metastases/various diseases, respectively. Our self-attention MADGAN can detect AD on T1 scans at a very early stage, mild cognitive impairment (MCI), with area under the curve (AUC) 0.727, and AD at a late stage with AUC 0.894, while detecting brain metastases on T1c scans with AUC 0.921. CONCLUSIONS: Similar to physicians' way of performing a diagnosis, using massive healthy training data, our first multiple MRI slice reconstruction approach, MADGAN, can reliably predict the next 3 slices from the previous 3 ones only for unseen healthy images. As the first unsupervised various disease diagnosis, MADGAN can reliably detect the accumulation of subtle anatomical anomalies and hyper-intense enhancing lesions, such as (especially late-stage) AD and brain metastases on multi-sequence MRI scans

Correlation between Organelle Genetic Variation and RNA Editing in Dinoflagellates Associated with the Coral Acropora digitifera

In order to develop successful strategies for coral reef preservation, it is critical that the biology of both host corals and symbiotic algae are investigated. In the Ryukyu Archipelago, which encompasses many islands spread over ∼500 km of the Pacific Ocean, four major populations of the coral Acropora digitifera have been studied using whole-genome shotgun (WGS) sequence analysis (Shinzato C, Mungpakdee S, Arakaki N, Satoh N. 2015. Genome-wide single-nucleotide polymorphism (SNP) analysis explains coral diversity and recovery in the Ryukyu Archipelago. Sci Rep. 5:18211.). In contrast, the diversity of the symbiotic dinoflagellates associated with these A. digitifera populations is unknown. It is therefore unclear if these two core components of the coral holobiont share a common evolutionary history. This issue can be addressed for the symbiotic algal populations by studying the organelle genomes of their mitochondria and plastids. Here, we analyzed WGS data from ∼150 adult A. digitifera, and by mapping reads to the available reference genome sequences, we extracted 2,250 sequences representing 15 organelle genes of Symbiodiniaceae. Molecular phylogenetic analyses of these mitochondrial and plastid gene sets revealed that A. digitifera from the southern Yaeyama islands harbor a different Symbiodiniaceae population than the islands of Okinawa and Kerama in the north, indicating that the distribution of symbiont populations partially matches that of the four host populations. Interestingly, we found that numerous SNPs correspond to known RNA-edited sites in 14 of the Symbiodiniaceae organelle genes, with mitochondrial genes showing a stronger correspondence than plastid genes. These results suggest a possible correlation between RNA editing and SNPs in the two organelle genomes of symbiotic dinoflagellates