IAN Family Critically Regulates Survival and Development of T Lymphocytes

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell–receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins

A pristine record of outer Solar System materials from asteroid Ryugu’s returned sample

Volatile and organic-rich C-type asteroids may have been one of the main sources of Earth’s water. Our best insight into their chemistry is currently provided by carbonaceous chondritic meteorites, but the meteorite record is biased: only the strongest types survive atmospheric entry and are then modified by interaction with the terrestrial environment. Here we present the results of a detailed bulk and microanalytical study of pristine Ryugu particles, brought to Earth by the Hayabusa2 spacecraft. Ryugu particles display a close compositional match with the chemically unfractionated, but aqueously altered, CI (Ivuna-type) chondrites, which are widely used as a proxy for the bulk Solar System composition. The sample shows an intricate spatial relationship between aliphatic-rich organics and phyllosilicates and indicates maximum temperatures of ~30 °C during aqueous alteration. We find that heavy hydrogen and nitrogen abundances are consistent with an outer Solar System origin. Ryugu particles are the most uncontaminated and unfractionated extraterrestrial materials studied so far, and provide the best available match to the bulk Solar System composition

Quantitative Distribution of DNA, RNA, Histone and Proteins Other than Histone in Mammalian Cells, Nuclei and a Chromosome at High Resolution Observed by Scanning Transmission Soft X-Ray Microscopy (STXM)

Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s⁻π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution

High lymphocyte counts before antithymocyte globulin administration predict acute graft-versus-host disease

Antithymocyte globulin (ATG) reduces severe acute and chronic graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (PBSCT). However, risk factors for severe acute GVHD in PBSCT using ATG remain to be determined. We conducted a single-center, retrospective study to analyze the association of acute GVHD requiring systemic corticosteroid (SC-aGVHD) with absolute lymphocyte counts (ALC) before the administration of ATG or conditioning in 53 patients with HLA-matched PBSCT using low-dose thymoglobulin (2 mg/kg) after myeloablative conditioning. The cumulative incidence of SC-aGVHD was 17.0% and ALC before ATG were significantly higher in patients with SC-aGVHD compared to that in patients without it (median, 0.15 x 10(9)/L vs 0.06 x 10(9)/L, P = 0.047). The cumulative incidence of SC-aGVHD was significantly higher in patients with high ALC before ATG (>= 0.15 x 10(9)/L) than in those with low ALC (38.5% vs 10.0%, P = 0.016). Non-relapse mortality (NRM) was also significantly higher in the high ALC before ATG group than the low ALC before ATG group (2-year NRM: 23.9% vs 6.0%, P = 0.048), leading to worse survival (2-year overall survival: 69.2% vs 83.5%, P = 0.039). Our study suggested that high ALC before ATG is a risk factor for SC-aGVHD

Aleukemic Extramedullary Blast Crisis as an Initial Presentation of Chronic Myeloid Leukemia with E1A3 BCR-ABL1 Fusion Transcript

Right neck swelling and pain occurred in a 49-year-old man. A Blood count showed a slight increase in platelet count without leukemoid reaction. After a biopsy of the cervical mass and bone marrow aspiration, a diagnosis of extramedullary blast crisis (EBC) of chronic myeloid leukemia (CML) was made. Fluorescence in situ hybridization (FISH) analysis showed a BCR-ABL1 fusion signal, but results of real-time polymerase chain reaction (RT-PCR) for major and minor BCR-ABL1 transcripts were negative. We identified a rare e1a 3 BCR-ABL1 fusion transcript. Administration of dasatinib resulted in disappearance of the extramedullary tumor. This is the first reported case of CML-EBC with e1a3 transcript. An aleukemic extramedullary tumor can be the initial presentation of CML

Knockdown of IAN5 in 23–1–8 T Lymphocytes

<div><p>(A) 23–1–8 T lymphocyte clones expressing shRNAs were analyzed for <i>IAN5</i> mRNA expression and cultured in the presence or absence of IL-2. Cell viability was quantified by PI staining and flow cytometry analysis. </p> <p>(B and C) Cells in 48-h culture were analyzed for apoptosis induction. Frequencies of Annexin-V-positive cells (B) or mitochondrial membrane potential (Δψm)-negative cells (C) are shown.</p> <p>(D) 23–1–8 T cells expressing shRNAs with or without human <i>Bcl-xL</i> were analyzed for <i>IAN5</i> expression by quantitative RT-PCR and for human <i>Bcl-xL</i> expression by conventional RT-PCR (left panel). Cells were cultured in the presence or absence of IL-2 for 72 h or in the presence of IL-2 and 5 μM helenalin for 48 h, and cell viability was quantified by PI staining (right panel). </p> <p>Graphs show means ± standard errors.</p> <p>NS, not significant ( <i>p</i> ≥ 0.05);^**<i>p</i> < 0.01. </p></div

Interaction of IAN Family Proteins with Bcl-2 Family Proteins

<div><p>(A) 293T cells were co-transfected with FLAG-tagged IAN molecules together with Bcl-2, Bcl-xL, HA-tagged Bax, HA-tagged Bak, HA-tagged Bad, BimEL, HA-tagged IκBα, or EGFP. Cell lysates were IP with anti-FLAG M2 antibody and IB with indicated antibodies.</p> <p>(B) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were IP with anti-FLAG M2 antibody and IB with anti-Bcl-2 or anti-Bcl-xL antibody.</p> <p>(C) 23–1–8 T cells expressing FLAG-tagged IAN4 or FLAG-tagged IAN5 were IP with normal IgG or anti-Bcl-2 or anti-Bcl-xL antibody and IB with anti-FLAG M2 antibody. Arrows indicate FLAG-tagged IAN4 or FLAG-tagged IAN5.</p> <p>(D) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were cultured in the presence or absence of IL-2 for 36 h. Cell lysates were IP with anti-FLAG M2 antibody and IB with anti-Bax antibody.</p> <p>(E) Nuclear and heavy membrane fractions prepared from 23–1–8 T cells were lysed in buffer containing 1% CHAPS. The lysates were IP with normal rabbit IgG or anti-IAN4 antibody and IB with anti-Bcl-2 antibody. Means and standard errors ( <i>n</i> = 4) of relative intensities of the bands were analyzed by using NIH Image software. </p> <p>^*<i>p</i> < 0.05;^**<i>p</i> < 0.01. </p></div