Isolation and Characterization of Renal Erythropoietin-Producing Cells from Genetically Produced Anemia Mice

Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for elucidating the mechanisms involved in hypoxia and/or anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe an approach to isolate and characterize REPs. We bred mice bearing an Epo gene allele to which green fluorescent protein (GFP) reporter cDNA was knocked-in (EpoGFP) with mice bearing an Epo gene allele lacking the 3′ enhancer (EpoΔ3′E). Mice harboring the mutant EpoGFP/Δ3′E gene exhibited anemia (average Hematocrit 18% at 4 to 6 days after birth), and this perinatal anemia enabled us to identify and purify REPs based on GFP expression from the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblastic cells that reside in the peritubular interstitial space, confirming our previous observation in Epo-GFP transgenic reporter assays. Flow cytometry analyses revealed that the GFP fraction constitutes approximately 0.2% of the whole kidney cells and 63% of GFP-positive cells co-express CD73 (a marker for cortical fibroblasts and Epo-expressing cells in the kidney). Quantitative RT-PCR analyses confirmed that Epo expression was increased by approximately 100-fold in the purified population of REPs compared with that of the unsorted cells or CD73-positive fraction. Gene expression analyses showed enrichment of Hif2α and Hif3α mRNA in the purified population of REPs. The genetic approach described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression of a defined population of cells essential for Epo production in the kidney. This has provided evidence that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction. (282 words

Essential and Instructive Roles of GATA Factors in Eosinophil Development

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation

Using spin to understand the formation of LIGO's black holes

With the detection of four candidate binary black hole (BBH) mergers by the Advanced LIGO detectors thus far, it is becoming possible to constrain the properties of the BBH merger population in order to better understand the formation of these systems. Black hole (BH) spin orientations are one of the cleanest discriminators of formation history, with BHs in dynamically formed binaries in dense stellar environments expected to have spins distributed isotropically, in contrast to isolated populations where stellar evolution is expected to induce BH spins preferentially aligned with the orbital angular momentum. In this work we propose a simple, model-agnostic approach to characterizing the spin properties of LIGO's BBH population. Using measurements of the effective spin of the binaries, which is LIGO's best constrained spin parameter, we introduce a simple parameter to quantify the fraction of the population that is isotropically distributed, regardless of the spin magnitude distribution of the population. Once the orientation characteristics of the population have been determined, we show how measurements of effective spin can be used to directly constrain the underlying BH spin magnitude distribution. Although we find that the majority of the current effective spin measurements are too small to be informative, with LIGO's four BBH candidates we find a slight preference for an underlying population with aligned spins over one with isotropic spins (with an odds ratio of 1.1). We argue that it will be possible to distinguish symmetric and anti-symmetric populations at high confidence with tens of additional detections, although mixed populations may take significantly more detections to disentangle. We also derive preliminary spin magnitude distributions for LIGO's black holes, under the assumption of aligned or isotropic populations

A low-frequency IL4R locus variant in Japanese patients with intravenous immunoglobulin therapy-unresponsive Kawasaki disease

Background: Kawasaki disease (KD) is a systemic vasculitis which may be associated with coronary artery aneurysms. A notable risk factor for the development of coronary artery aneurysms is resistance to intravenous immunoglobulin (IVIG) therapy, which comprises standard treatment for the acute phase of KD. The cause of IVIG resistance in KD is largely unknown; however, the contribution of genetic factors, especially variants in immune-related genes, has been suspected. Methods: To explore genetic variants related to IVIG-unresponsiveness, we designated KD patients who did not respond to both first and second courses of IVIG therapy as IVIG-unresponsive patients. Using genomic DNA from 30 IVIG-unresponsive KD patients, we performed pooled genome sequencing targeting 39 immune-related cytokine receptor genes. Results: The single nucleotide variant (SNV), rs563535954 (located in the IL4R locus), was concentrated in IVIG-unresponsive KD patients. Individual genotyping showed that the minor allele of rs563535954 was present in 4/33 patients with IVIG-unresponsive KD, compared with 20/1063 individuals in the Japanese genome variation database (odds ratio = 7.19, 95% confidence interval 2.43-21.47). Furthermore, the minor allele of rs563535954 was absent in 42 KD patients who responded to IVIG treatment (P = 0.0337), indicating that a low-frequency variant, rs563535954, is associated with IVIG-unresponsiveness in KD patients. Although rs563535954 is located in the 3'-untranslated region of IL4R, there was no alternation in IL4R expression associated with the mior allele of rs563535954. However, IVIG-unresponsive patients that exhibited the minor allele of rs563535954 tended to be classified into the low-risk group (based on previously reported risk scores) for prediction of IVIG-resistance. Therefore, IVIG-unresponsiveness associated with the minor allele of rs563535954 might differ from IVIG-unresponsiveness associated with previous risk factors used to evaluate IVIG-unresponsiveness in KD. Conclusion: These findings suggest that the SNV rs563535954 could serve as a predictive indicator of IVIG-unresponsiveness, thereby improving the sensitivity of risk scoring systems, and may aid in prevention of coronary artery lesions in KD patients.ArticlePEDIATRIC RHEUMATOLOGY.17:34(2019)journal articl

A multi-ethnic meta-analysis identifies novel genes, including ACSL5, associated with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating progressive motor neuron disease that affects people of all ethnicities. Approximately 90% of ALS cases are sporadic and thought to have multifactorial pathogenesis. To understand the genetics of sporadic ALS, we conducted a genome-wide association study using 1,173 sporadic ALS cases and 8,925 controls in a Japanese population. A combined meta-analysis of our Japanese cohort with individuals of European ancestry revealed a significant association at the ACSL5 locus (top SNP p = 2.97 × 10−8). We validated the association with ACSL5 in a replication study with a Chinese population and an independent Japanese population (1941 ALS cases, 3821 controls; top SNP p = 1.82 × 10−4). In the combined meta-analysis, the intronic ACSL5 SNP rs3736947 showed the strongest association (p = 7.81 × 10−11). Using a gene-based analysis of the full multi-ethnic dataset, we uncovered additional genes significantly associated with ALS: ERGIC1, RAPGEF5, FNBP1, and ATXN3. These results advance our understanding of the genetic basis of sporadic ALS

東北メディカル・メガバンク事業 三世代コホート調査の進捗について

第3回日本DOHaD研究会学術集会　抄録集　【ポスター発表

Genetic Predisposition to Ischemic Stroke

Background and Purpose—The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk.Methods—We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets).Results—In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33–2.31) and 1.99 (95% confidence interval, 1.19–3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001).Conclusions—The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements