Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis.

Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4-/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4-/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4-/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.

Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium.

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

Single-nucleus RNA sequencing of plant tissues using a nanowell-based system.

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowell-based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single-nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage-specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments.

Effects of diets high in animal or plant protein on oxidative stress in individuals with type 2 diabetes: A randomized clinical trial.

High-protein diet is a promising strategy for diabetes treatment supporting body weight control, improving glycaemic status, cardiovascular risk factors and reducing liver fat. Here, we investigated effects of diets high in animal (AP) or plant (PP) protein on oxidative stress and antioxidant status in individuals with type 2 diabetes (T2DM). 37 obese individuals (age 64.3 ± 1.0 years) with T2DM were randomized to an isocaloric diet (30 energy(E)% protein, 30 E% fat and 40 E% carbohydrates) rich in AP or PP for 6 weeks. Markers of oxidative and nitrosative stress and antioxidant status in plasma and nitrate/nitrite levels in urine were assessed. Gene expression in subcutaneous adipose tissue (SAT) was analysed by RNA-Seq and real-time PCR. Both AP and PP diets similarly reduced plasma levels of malondialdehyde (PAP = 0.003, PPP = 1.6 × 10-4) and protein carbonyls (PAP = 1.2 × 10-4, PPP = 3.0 × 10-5) over 6 weeks. Nitrotyrosine (NT) increased upon both AP and PP diets (PAP = 0.005, PPP = 0.004). SAT expression of genes involved in nitric oxide (NO) and oxidative stress metabolism and urine NO metabolite (nitrate/nitrite) levels were not changed upon both diets. Plasma levels of carotenoids increased upon PP diet, whereas retinol, alpha- and gamma-tocopherol slightly decreased upon both diets. AP and PP diets similarly improve oxidative stress but increase nitrosative stress markers in individuals with T2DM. Mechanisms of the NT regulation upon high-protein diets need further investigation.

The Wnt-Driven Mll1 Epigenome Regulates Salivary Gland and Head and Neck Cancer.

We identified a regulatory system that acts downstream of Wnt/ß-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/ß-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activity and genome-wide increased H3K4 tri-methylation at promoters. Null mutations of Mll1 in tumor mice and in xenotransplanted human head and neck tumors resulted in loss of self-renewal of tumor-propagating cells and in block of tumor formation but did not alter normal tissue homeostasis. CRISPR/Cas9 mutagenesis and pharmacological interference of Mll1 at sequences that inhibit essential protein-protein interactions or the SET enzyme active site also blocked the self-renewal of mouse and human tumor-propagating cells. Our work provides strong genetic evidence for a crucial role of Mll1 in solid tumors. Moreover, inhibitors targeting specific Mll1 interactions might offer additional directions for therapies to treat these aggressive tumors.