A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

A new composite indicator consisting of left ventricular extracellular volume, N-terminal fragment of B-type natriuretic peptide, and left ventricular end-diastolic volume is useful for predicting reverse remodeling after catheter ablation for atrial fibrillation.

Recently, myocardial extracellular volume (ECV) analysis has been measurable on computed tomography (CT) using new software. We evaluated the use of cardiac CT to estimate the myocardial ECV of left ventricular (LV) myocardium (LVM) to predict reverse remodeling (RR) in cases of atrial fibrillation (AF) after catheter ablation (CA). Four hundred and seven patients underwent CA for AF in our institution from April 2014 to Feb 2021. Of these, 33 patients (8%) with an LVEF ≤ 50% and who had undergone CT were included in our study. We estimated the LVM ECV using commercial software to analyze the CT data. RR was defined as an improvement in LVEF to > 50% after CA. LVEF increased to > 50% in 24 patients (73%) after CA. In all 24 patients, LVM ECV, LV end-diastolic and end-systolic volumes (LVEDV and LVESV), and the n-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) were significantly lower than in the other nine patients (P = 0.0037, 0.0273, 0.0443, and < 0.0001). On receiver operating characteristic curve analysis, the best cut-off of ECV, LVEDV, LVESV and NT-proBNP for the prediction of RR were 37.73%, 120 mL, 82 mL, and 1267 pg/mL, respectively. We newly defined the ENL (ECV, NT-proBNP, and LVEDV) score as the summed score for the presence or absence (1 or 0; maximum score = 3) of ECV, NT-proBNP, and LVEDV values less than or equal to each best cut-off value, and found that this score gave the highest area under the curve for the prediction of RR (0.9583, P < 0.0001). The ENL score may be useful for predicting RR in patients with AF undergoing CA.

Evaluation of extracellular volume by computed tomography is useful for prediction of prognosis in dilated cardiomyopathy.

Cardiac computed tomography (CT) is useful for the screening of coronary artery stenosis, and extracellular volume fraction (ECV) analysis by CT using new dedicated software is now available. Here, we evaluated the utility of ECV analysis using cardiac CT to predict patient prognosis in cases with dilated cardiomyopathy (DCM). We analyzed 70 cases with DCM and cardiac computed tomography (CT) with available late-phase images. We evaluated the ECV of the left ventricular myocardium (LVM) using commercially available software (Ziostation 2, Ziosoft Inc, Japan). ECV on LVM was 33.96 ± 5.04%. Major adverse cardiac events (MACE) occurred in 21 cases (30%). ECV of the LVM on CT, endo-systolic volume, and rate of significant valvular disease were significantly higher in cases with MACE than in those without (37.16 ± 5.91% vs. 32.59 ± 3.95%, 194 ± 109 vs. 138 ± 78 ml and 57% vs. 20%, all P values < 0.05). LVEF was significantly lower in cases with MACE than in those without (23 ± 8 vs. 31 ± 11%, P = 0.0024). The best cut-off value of ECV on LVM for prediction of MACE was 32.26% based on receiver operating characteristics analysis. Cases with ECV ≥ 32.26% had significantly higher MACE based on Kaplan-Meier analysis (P = 0.0032). Only ECV on LVM was an independent predictor of MACE based on a multivariate Cox proportional hazards model (P = 0.0354). Evaluation of ECV on LVM by CT is useful for predicting MACE in patients with DCM.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates ß-Glucanase Expression in Tannerella forsythia in Response to Fusobacterium nucleatum Sensing.

The periodontal pathogen Tannerella forsythia expresses a ß-glucanase (TfGlcA) whose expression is induced in response to Fusobacterium nucleatum, a bridge bacterium of the oral cavity. TfGlcA cleaves ß-glucans to release glucose, which can serve as a carbon source for F. nucleatum and other cohabiting organisms. A two-gene cluster encoding a putative extracytoplasmic function (ECF) sigma factor and a FecR-like anti-sigma factor has been recognized upstream of a TfGlcA operon. We characterized and analyzed the role of these putative ECF sigma and anti-sigma factors in the regulation of TfGlcA expression. For this purpose, deletion mutants were constructed and analyzed for ß-glucanase expression. In addition, an Escherichia coli-produced ECF sigma factor recombinant protein was evaluated for transcriptional and DNA binding activities. The results showed that the recombinant protein promoted transcription by the RNA polymerase core enzyme from the glcA promoter. Furthermore, in comparison to those in the parental strain, the ß-glucanase expression levels were significantly reduced in the ECF sigma-factor deletion mutant and increased significantly in the FecR anti-sigma factor deletion mutant. The levels did not change in the mutants following coincubation with the F. nucleatum whole cells or cell extracts. Finally, the levels of ß-glucanase produced by T. forsythia strains paralleled F. nucleatum biomass in cobiofilms. In conclusion, we identified a ß-glucanase operon regulatory system in T. forsythia comprising an ECF sigma factor (TfSigG) and a cognate FecR-like anti-sigma factor responsive to F. nucleatum and potentially other stimuli. IMPORTANCE Previous studies have shown that F. nucleatum forms robust biofilms with T. forsythia utilizing glucose from the hydrolysis of ß-glucans by T. forsythia ß-glucanase, induced by F. nucleatum. In this study, we showed that a regulatory system comprising of an ECF sigma factor, TfSigG, and a FecR-like anti-sigma factor, TfFecR, is responsible for the ß-glucanase induction in response to F. nucleatum, suggesting that this system plays roles in the mutualistic interactions of T. forsythia and F. nucleatum. The findings suggest the development and potential utility of small-molecule inhibitors targeting the ß-glucanase activity or the TfSigG/TfFecR system as therapeutic drugs against dental plaque formation and periodontitis.

Melatonin MT2 receptor is expressed and potentiates contraction in human airway smooth muscle.

Nocturnal asthma is characterized by heightened bronchial reactivity at night, and plasma melatonin concentrations are higher in patients with nocturnal asthma symptoms. Numerous physiological effects of melatonin are mediated via its specific G protein-coupled receptors (GPCRs) named the MT1 receptor, which couples to both Gq and Gi proteins, and the MT2 receptor, which couples to Gi. We investigated whether melatonin receptors are expressed on airway smooth muscle; whether they regulate intracellular cyclic AMP (cAMP) and calcium concentrations ([Ca2+]i), which modulate airway smooth muscle tone; and whether they promote airway smooth muscle cell proliferation. We detected the mRNA and protein expression of the melatonin MT2 but not the MT1 receptor in native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by RT-PCR, immunoblotting, and immunohistochemistry. Activation of melatonin MT2 receptors with either pharmacological concentrations of melatonin (10-100 µM) or the nonselective MT1/MT2 agonist ramelteon (10 µM) significantly inhibited forskolin-stimulated cAMP accumulation in HASM cells, which was reversed by the Gαi protein inhibitor pertussis toxin or knockdown of the MT2 receptor by its specific siRNA. Although melatonin by itself did not induce an initial [Ca2+]i increase and airway contraction, melatonin significantly potentiated acetylcholine-stimulated [Ca2+]i increases, stress fiber formation through the MT2 receptor in HASM cells, and attenuated the relaxant effect of isoproterenol in guinea pig trachea. These findings suggest that the melatonin MT2 receptor is expressed in ASM, and modulates airway smooth muscle tone via reduced cAMP production and increased [Ca2+]i.

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice.

Dendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8- LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8- and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8- LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.

Expression of seed storage proteins responsible for maintaining kernel traits and wheat flour quality in common wheat under heat stress conditions.

Heat stress during grain filling has been documented to decrease wheat grain yield and quality in arid regions worldwide. We studied the effect of heat stress on wheat flour quality in heat tolerant cultivars to define the effects of heat stress on flour quality and to identify germplasm combining traits for heat tolerance and good flour quality. We studied the kernel phenotypic traits, the expression of seed storage proteins (SSPs), and the resulting flour quality under heat and normal conditions. Under heat stress, all cultivars yielded narrow-shaped seeds, and increased protein contents as compared to the control plants grown under normal conditions. The specific sedimentation values used to estimate the gluten quality varied between cultivars. We identified cultivars that could maintain good flour quality under heat stress conditions: 'Imam', which possessed the Glu-D1d allele responsible for the suitable bread-making; 'Bohaine', which displayed high expression level of SSPs; and 'Condor', which possessed slight variations in the ratio of each SSP under heat stress conditions. Combining the desirable traits from these cultivars could yield a wheat cultivar with heat tolerance and good flour quality.

Short Coaptation Length is a Predictor of Recurrent Mitral Regurgitation After Mitral Valve Plasty.

BACKGROUND: Long-term predictors of recurrent mitral regurgitation (MR) after mitral valve plasty (MVP) remain to be elucidated. This study sought to determine the prognostic factors of recurrent MR during long-term follow-up after MVP, by analysing findings of three-dimensional transoesophageal echocardiography (TEE) conducted after MVP. METHODS: This study analysed 207 patients who underwent MVP for A2 and/or P2 prolapse and received TEE before discharge. Recurrent MR was defined as moderate or worse regurgitation detected by annual transthoracic echocardiography. RESULTS: During a median follow-up period of 49 months after MVP, 18 patients experienced recurrent MR and six patients needed reoperation. In the recurrent group, 16 of 18 patients showed less than moderate MR before discharge. Patients in the recurrent group underwent repair for worse MR (effective orifice area, 54±19 vs 44±16 mm2; p=0.01) and had shorter A2-P2 coaptation length (5.3±1.4 vs 7.3±1.5 mm; p<0.001) after MVP compared with the non-recurrent group. Cox proportional hazards regression analysis identified the A2-P2 coaptation length as significant risk of recurrent MR (coaptation length increase: HR, 0.44; 95% CI, 0.32-0.59; p<0.0001). The receiver operator characteristics curve demonstrated that a coaptation length of <5.6 mm had 78% sensitivity and 89% specificity for predicting recurrent MR. CONCLUSION: Coaptation length measured by post-MVP TEE predicted the tendency of recurrent MR. Patients with short coaptation length should be carefully monitored, even when residual MR is less than moderate after MVP.

The short-chain free fatty acid receptor FFAR3 is expressed and potentiates contraction in human airway smooth muscle.

Emerging evidence suggests that gut microbiota-derived short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) are important modulators of the inflammatory state in diseases such as asthma. However, the functional expression of the Gi protein-coupled free fatty acid receptors (FFAR2/GPR43 and FFAR3/GPR41) has not been identified on airway smooth muscle (ASM). Classically, acute activation of Gi-coupled receptors inhibits cyclic AMP (cAMP) synthesis, which impairs ASM relaxation and can also induce crosstalk between Gi- and Gq-signaling pathways, potentiating increases in intracellular Ca2+ concentration ([Ca2+]i), favoring ASM contraction. In contrast, chronic activation of Gi-coupled receptors can sensitize adenylyl cyclase resulting in increased cAMP synthesis favoring relaxation. We questioned whether the Gi-coupled FFAR2 or FFAR3 is expressed in human ASM, whether they modulate cAMP and [Ca2+]i, and whether SCFAs modulate human ASM tone. We detected the protein expression of FFAR3 but not FFAR2 in native human ASM and primary cultured human airway smooth muscle (HASM) cells. In HASM cells, acute activation of FFAR3 with SCFAs inhibited forskolin-stimulated cAMP accumulation, but chronic activation did not sensitize cAMP synthesis. SCFAs induced [Ca2+]i increases that were attenuated by pertussis toxin, gallein, U73122, or xestospongin C. Acute treatment with SCFAs potentiated acetylcholine-stimulated [Ca2+]i increases and stress fiber formation in cells and contraction of ex vivo human airway tissues. In contrast, chronic pretreatment of human ASM with propionate did not potentiate airway relaxation. Together, these findings demonstrate that FFAR3 is expressed in human ASM and contributes to ASM contraction via reduced cAMP and increased [Ca2+]i.

Transcriptional control of monocyte and macrophage development.

Monocytes and macrophages play critical roles in immune responses, tissue homeostasis and disease progression. There are a number of functionally and phenotypically distinct subpopulations throughout the body. However, the mechanisms by which macrophage and monocyte heterogeneity is established remain unclear. Recent studies have suggested that most tissue-resident macrophages originate from fetal progenitors but not from hematopoietic stem cells, whereas some subpopulations are derived from adult monocytes. In addition, transcription factors specifically required for the development of each subpopulation have been identified. Interestingly, local environmental factors such as heme, retinoic acid and RANKL induce the expression and/or activation of tissue-specific transcription factors, thereby controlling transcriptional programs specific for the subpopulations. Thus, distinct differentiation pathways and local microenvironments appear to contribute to the determination of macrophage transcriptional identities. In this review, we highlight recent advances in our knowledge of the transcriptional control of macrophage and monocyte development.

Definitive diagnosis of mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome caused by a recurrent de novo mutation in the POLD1 gene.

Segmental progeroid syndromes with lipodystrophy are extremely rare, heterogeneous, and complex multi-system disorders that are characterized by phenotypic features of premature aging affecting various tissues and organs. In this study, we present a "sporadic/isolated" Japanese woman who was ultimately diagnosed with mandibular hypoplasia, deafness, progeroid features, and progressive lipodystrophy (MDPL) syndrome (MIM #615381) using whole exome sequencing analysis. She had been suspected as having atypical Werner syndrome and/or progeroid syndrome based on observations spanning a 30-year period; however, repeated genetic testing by Sanger sequencing did not identify any causative mutation related to various subtypes of congenital partial lipodystrophy (CPLD) and/or mandibular dysplasia with lipodystrophy (MAD). Recently, MDPL syndrome has been described as a new entity showing progressive lipodystrophy. Furthermore, polymerase delta 1 (POLD1) gene mutations on chromosome 19 have been identified in patients with MDPL syndrome. To date, 21 cases with POLD1-related MDPL syndrome have been reported worldwide, albeit almost entirely of European origin. Here, we identified a de novo mutation in exon 15 (p.Ser605del) of the POLD1 gene in a Japanese case by whole exome sequencing. To the best of our knowledge, this is the first identified case of MDPL syndrome in Japan. Our results provide further evidence that mutations in POLD1 are responsible for MDPL syndrome and serve as a common genetic determinant across different ethnicities.

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells.

Basophils and mast cells play critical roles in host defense against pathogens and allergic disorders. However, the molecular mechanism by which these cells are generated is not completely understood. Here we demonstrate that interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of several myeloid lineages, also regulates basophil and mast cell development. Irf8(-/-) mice displayed a severe reduction in basophil counts, which was accounted for by the absence of pre-basophil and mast cell progenitors (pre-BMPs). Although Irf8(-/-) mice retained peripheral tissue mast cells, remaining progenitors from Irf8(-/-) mice including granulocyte progenitors (GPs) were unable to efficiently generate either basophils or mast cells, indicating that IRF8 also contributes to the development of mast cells. IRF8 appeared to function at the GP stage, because IRF8 was expressed in GPs, but not in basophils, mast cells, and basophil/mast cell-restricted progenitor cells. Furthermore, we demonstrate that GATA2, a transcription factor known to promote basophil and mast cell differentiation, acts downstream of IRF8. These results shed light on the pathways and mechanism underlying the development of basophils and mast cells.