Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens

The autoimmune regulator (Aire)-directed ectopic expression of tissue-specific antigens (TSAs) by mature medullary thymic epithelial cells (mTECs) has been viewed as an essential mechanism in the induction of central tolerance. Recent data suggest that the survival of mTECs extends beyond the Aire+ cell population to form the post-Aire mTEC population and Hassall’s corpuscles (HCs). The nature and function of these post-Aire epithelial cells and structures, however, have remained unidentified. In this study, we characterized in detail the end-stage development of mTECs and HCs in both Aire-sufficient and Aire-deficient mice. In addition, using a transgenic mouse model in which the LacZ reporter gene is under the control of the endogenous Aire promoter, we purified and analyzed the post-Aire mTECs to characterize their function. We showed that the end-stage maturation of mTECs closely resembles that of keratinocytes and that the lack of Aire results in a marked block of mTEC differentiation, which is partially overcome by ligands for RANK and CD40. We also provide evidence that, during mTEC development, Aire is expressed only once and during a limited 1–2 day period. The following loss of Aire expression is accompanied by a quick downregulation of MHC class II and CD80, and of most of the Aire-dependent and Aire-independent TSAs, with the exception of keratinocyte-specific genes. In the final stage of maturation, the mTECs lose their nuclei to become HCs and specifically express desmogleins (DGs) 1 and 3, which, via cross-presentation by APCs, may contribute to tolerance against these pemphigus vulgaris-related TSAs

Tmprss3 loss of function impairs cochlear inner hair cell Kcnma1 channel membrane expression

Before acquiring their mature state, cochlear hair cells undergo a series of changes in expression of ion channels. How this complex mechanism is achieved is not fully understood. Tmprss3, a type II serine protease expressed in hair cells, is required for their proper functioning at the onset of hearing. To unravel the role of Tmprss3 in the acquisition of mature K+ currents, we compared their function by patch-clamp technique in wild-type Tmprss3WT and Tmprss3Y260X-mutant mice. Interestingly, only outward K+ currents were altered in Tmprss3Y260X-mutant mice. To determine by which mechanism this occurred, we compared the protein network of Tmprss3WT and Tmprss3Y260X-mutant mice using proteomic analysis. This led to the identification of a pathway related to potassium Kcnma1 channels. This pathway was validated by immunohistochemistry, focusing on the most downregulated protein that was identified as a cochlear Kcnma1-associated protein, APOA1. Finally, we show that, in contrast to Tmprss3WT, Kcnma1 channels were absent at the neck of inner hair cells (IHCs) in Tmprss3Y260X-mutant mice. In conclusion, our data suggest that lack of Tmprss3 leads to a decrease in Kcnma1 potassium channels expression in (IHCs

MicroSAGE is Highly Representative and Reproducible but Reveals Major Differences in Gene Expression Among Samples Obtained from Similar Tissues

Background: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate. Results: We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals. Conclusions: MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently

Waveguide-integrated and portable optomechanical magnetometer

Optomechanical magnetometers enable highly sensitive magnetic field sensing. However, all such magnetometers to date have been optically excited and read-out either via free space or a tapered optical fiber. This limits their scalability and integrability, and ultimately their range of applications. Here, we present an optomechanical magnetometer that is excited and read out via a suspended optical waveguide fabricated on the same silicon chip as the magnetometer. Moreover, we demonstrate that thermomechanical noise limited sensitivity is possible using portable electronics and laser. The magnetometer employs a silica microdisk resonator selectively sputtered with a magnetostrictive film of galfenol (FeGa) which induces a resonant frequency shift in response to an external magnetic field. Experimental results reveal the retention of high quality-factor optical whispering gallery mode resonances whilst also demonstrating high sensitivity and dynamic range in ambient conditions. The use of off-the-shelf portable electronics without compromising sensor performance demonstrates promise for applications.Comment: 9 pages, 4 figure

Gene Dosage–limiting Role of Aire in Thymic Expression, Clonal Deletion, and Organ-specific Autoimmunity

Inactivation of the autoimmune regulator (Aire) gene causes a rare recessive disorder, autoimmune polyendocrine syndrome 1 (APS1), but it is not known if Aire-dependent tolerance mechanisms are susceptible to the quantitative genetic changes thought to underlie more common autoimmune diseases. In mice with a targeted mutation, complete loss of Aire abolished expression of an insulin promoter transgene in thymic epithelium, but had no effect in pancreatic islets or the testes. Loss of one copy of Aire diminished thymic expression of the endogenous insulin gene and the transgene, resulting in a 300% increase in islet-reactive CD4 T cells escaping thymic deletion in T cell receptor transgenic mice, and dramatically increased progression to diabetes. Thymic deletion induced by antigen under control of the thyroglobulin promoter was abolished in Aire homozygotes and less efficient in heterozygotes, providing an explanation for thyroid autoimmunity in APS1. In contrast, Aire deficiency had no effect on thymic deletion to antigen controlled by a systemic H-2K promoter. The sensitivity of Aire-dependent thymic deletion to small reductions in function makes this pathway a prime candidate for more subtle autoimmune quantitative trait loci, and suggests that methods to increase Aire activity would be a potent strategy to lower the incidence of organ-specific autoimmunity

py4DSTEM: a software package for multimodal analysis of four-dimensional scanning transmission electron microscopy datasets

Scanning transmission electron microscopy (STEM) allows for imaging, diffraction, and spectroscopy of materials on length scales ranging from microns to atoms. By using a high-speed, direct electron detector, it is now possible to record a full 2D image of the diffracted electron beam at each probe position, typically a 2D grid of probe positions. These 4D-STEM datasets are rich in information, including signatures of the local structure, orientation, deformation, electromagnetic fields and other sample-dependent properties. However, extracting this information requires complex analysis pipelines, from data wrangling to calibration to analysis to visualization, all while maintaining robustness against imaging distortions and artifacts. In this paper, we present py4DSTEM, an analysis toolkit for measuring material properties from 4D-STEM datasets, written in the Python language and released with an open source license. We describe the algorithmic steps for dataset calibration and various 4D-STEM property measurements in detail, and present results from several experimental datasets. We have also implemented a simple and universal file format appropriate for electron microscopy data in py4DSTEM, which uses the open source HDF5 standard. We hope this tool will benefit the research community, helps to move the developing standards for data and computational methods in electron microscopy, and invite the community to contribute to this ongoing, fully open-source project

Windscapes shape seabird instantaneous energy costs but adult behavior buffers impact on offspring

Acknowledgements K. Ashbrook, M. Barrueto, K. Elner, A. Hargreaves, S. Jacobs, G. Lancton, M. LeVaillant, E. Grosbellet, A. Moody, A. Ronston, J. Provencher, P. Smith, K. Woo and P. Woodward helped in the field. J. Nakoolak kept us safe from bears. N. Sapir and two anonymous reviewers provided very useful comments on an earlier version of our manuscript. R. Armstrong at the Nunavut Research Institute, M. Mallory at the Canadian Wildlife Service Northern Research Division and C. Eberl at National Wildlife Research Centre in Ottawa provided logistical support. F. Crenner, N. Chatelain and M. Brucker customized the GPS at the IPHC-CNRS. KHE received financial support through a NSERC Vanier Canada Graduate Scholarship, ACUNS Garfield Weston Northern Studies scholarship and AINA Jennifer Robinson Scholarship and JFH received NSERC Discovery Grant funding. J. Welcker generously loaned some accelerometers. All procedures were approved under the guidelines of the Canadian Council for Animal Care.Peer reviewedPublisher PD

Investigating the Potential Role of Genetic and Epigenetic Variation of DNA Methyltransferase Genes in Hyperplastic Polyposis Syndrome

BACKGROUND: Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS. METHODS: We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters. RESULTS: No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps. CONCLUSIONS: Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.Musa Drini, Nicholas C. Wong, Hamish S. Scott, Jeffrey M. Craig, Alexander Dobrovic, Chelsee A. Hewitt, Christofer Dow, Joanne P. Young, Mark A. Jenkins, Richard Saffery and Finlay A. Macra

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program