TSCOT + Thymic Epithelial Cell-Mediated Sensitive CD4 Tolerance by Direct Presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells

Contributions Made by CDC25 Phosphatases to Proliferation of Intestinal Epithelial Stem and Progenitor Cells

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells

Neural Network Analysis for Microplastic Segmentation

It is necessary to locate microplastic particles mixed with beach sand to be able to separate them. This paper illustrates a kernel weight histogram-based analytical process to determine an appropriate neural network to perform tiny object segmentation on photos of sand with a few microplastic particles. U-net and MultiResUNet are explored as target networks. However, based on our observation of kernel weight histograms, visualized using TensorBoard, the initial encoder stages of U-net and MultiResUNet are useful for capturing small features, whereas the later encoder stages are not useful for capturing small features. Therefore, we derived reduced versions of U-net and MultiResUNet, such as Half U-net, Half MultiResUNet, and Quarter MultiResUNet. From the experiment, we observed that Half MultiResUNet displayed the best average recall-weighted F1 score (40%) and recall-weighted mIoU (26%) and Quarter MultiResUNet the second best average recall-weighted F1 score and recall-weighted mIoU for our microplastic dataset. They also require 1/5 or less floating point operations and 1/50 or a smaller number of parameters over U-net and MultiResUNet

MRI data-driven functional near-infrared spectroscopy mapping method for conforming functional hemodynamic neural activity to structural cortical information

Functional near-infrared spectroscopy (fNIRS) as a brain imaging technique is getting more and more interested in spite of its lower spatial resolution compared to functional magnetic resonance image (fMRI). Especially, fNIRS is gradually spread out in investigating the cortical activity of motion related tasks, because of its advantages of robustness to motion artifacts, patient acceptability, and the experimental simplicity. Since fNIRS uses internationally recognized 10-20 probe positing system, the accuracy of source localization is poor. In addition, fNIRS does not provide accurate structural information for different individual brains because its statistical parametric mapping (SPM) is a mapping method that projects the statistical change of oxygenated or deoxygenated hemoglobin in a standard brain template. This paper proposed fNIRS mapping procedure that can reflect the more accurate structural characteristics of individual brain. First, the reliability of the fNIRS localization method was verified by comparing 3D digitizer and neuro-navigator position errors. Second, the personalized fNIRS images was acquired by converting the structural information obtained from the individual MRI to the suitable form for the SPM. Two healthy subjects participated in the investigation of localization errors with carrying out two motor tasks comprising hand grasping and knee bending. From a total of 2 trials, the average of error was 4.16 mm. These results guaranteed the reliability of the fNIRS source localization method using 3D-digitizer because the error bound was within 30 mm of the measurement distance between the probes. In order to reconstruct the personalized fNIRS brain images, five stroke patients were enrolled in the body weighted support treadmill training (BWSTT) experiment. As a result of the comparison between the conventional NIRS-SPM image and the proposed personalized brain image, the proposed method clearly discovered the position of individual brain lesion and more clearly figured out the lateralization and intensity of individual brain activity. This suggests that the proposed personalized brain imaging technique is useful for observing not only the structural characteristics of brain (lateralization and localization of lesion or cortical activity) but also the functional properties (intensity of oxy- or deoxy-hemoglobin) of the brain. © Copyright 2017 American Scientific Publishers All rights reserved.1

Targeted disruption of <i>Cdc25B</i> in mice.

<p>(<b>A</b>) Structure of targeting vector and chromosomal organization of <i>Cdc25B</i> locus before and after Cre-mediated excision. The genomic organization of the mouse <i>Cdc25B</i> gene was disrupted by inserting into intron 1 the neomycin phosphotransferase cDNA driven by the phosphoglycerine kinase promoter (pGK-neo) as a selectable marker. Exons are represented by black boxes. The location of Hind III (H), Bam HI (B) and Kpn I (K) site is indicated and <i>loxP</i> sites are represented by yellow triangles. Sizes of upstream (3.3 kb) and downstream (4.5 kb) homologous arms are indicated. Position of probes used for Southern blotting are shown. Red triangles depict the locations of PCR primers used for genotyping. Abbreviations: +, wild type allele; R, recombinant allele; F, floxed allele; WT, wild type. (<b>B</b>–<b>C</b>) Southern blot analysis demonstrating homologous recombination in the <i>Cdc25B</i> locus. ES cell genomic DNA was digested with Hind III (B) and Bam HI (C), and Southern blotting was performed using the 5′ and 3′ probes shown in panel A. The genotype of each ES cell line is indicated. The location of size markers is shown on the left. (<b>D</b>) Southern blot analysis demonstrating Cre-mediated recombination in the <i>Cdc25B</i> locus. ES cell clones containing the recombinant allele were expanded and transiently transfected with a plasmid encoding Cre recombinase. Genomic DNA was digested with Kpn I (K), and Southern blotting was performed using the internal probe shown in panel A. The genotype of each ES cell line is indicated. Location of size markers is shown on left. (<b>E</b>) PCR analysis of mouse tail DNA. Mouse tail DNA was amplified with PCR primers depicted as red triangles in panel A. The wild type (+) allele produced a 383 bp PCR product and floxed allele (F) produced a 433 bp PCR product. The genotype of each mouse is indicated. The location of size markers is shown on the right.</p

Loss of homeostasis in small intestines of <i>vABKO a</i>nd <i>vTKO</i> mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Small intestines were isolated and length determinations were made. Small intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.38 (WT), 0.76 (<i>vil-Cre-ER^T2</i>), 0.003 (<i>vAKO</i>), 0.31 (<i>vBKO</i>), 0.002 (<i>vABKO</i>), 0.46 (<i>vACKO</i>) and 0.004 (<i>vTKO</i>). The small intestinal lengths of <i>vAKO</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.40 (<i>vil-Cre-ER^T2</i>), 0.03 (<i>vAKO</i>), 0.10 (<i>vBKO</i>), 0.00006 (<i>vABKO</i>), 0.36 (<i>vACKO</i>) and 0.0006 (<i>vTKO</i>). (<b>B</b>) Duodenums isolated from mice treated as described in A were photographed under a dissection microscope. Scale bar: 0.5 mm. (<b>C</b>) Significant shortening of villi in small intestines of <i>vABKO</i> and <i>vTKO</i> mice. Length of individual villi shown in panel B were measured (30 villi per mouse). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.00008 (<i>vABKO</i>) and 0.007 (<i>vTKO</i>). Villi lengths of <i>vil-Cre-ER^T2</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.001 (<i>vil-Cre-ER^T2</i>), 0.27 (<i>vAKO</i>), 0.45 (<i>vBKO</i>), 0.0005 (<i>vABKO</i>), 0.25 (<i>ACKO</i>) and 0.03 (<i>vTKO</i>).</p