Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNAseq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat with histone demethylase inhibitor GSKJ4 revealed synergy. Together, these data suggest a promising therapeutic strategy for DIPG.

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34 hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

Summary Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric cancer with limited therapeutic options. The majority of cases of DIPG exhibit a mutation in histone-3 (H3K27M) that results in oncogenic transcriptional aberrancies. We show here that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. Identification of super-enhancers in DIPG provides insights toward the cell of origin, highlighting oligodendroglial lineage genes, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. The findings presented demonstrate transcriptional vulnerabilities and elucidate previously unknown mechanisms of DIPG pathobiology.

Climate warming is substantially shifting the leaf phenological events of plants, and thereby impacting on their individual fitness and also on the structure and functioning of ecosystems. Previous studies have largely focused on the climate impact on spring phenology, and to date the processes underlying leaf senescence and their associated environmental drivers remain poorly understood. In this study, experiments with temperature gradients imposed during the summer and autumn were conducted on saplings of European beech to explore the temperature responses of leaf senescence. An additional warming experiment during winter enabled us to assess the differences in temperature responses of spring leaf-out and autumn leaf senescence. We found that warming significantly delayed the dates of leaf senescence both during summer and autumn warming, with similar temperature sensitivities (6-8 days delay per °C warming), suggesting that, in the absence of water and nutrient limitation, temperature may be a dominant factor controlling the leaf senescence in European beech. Interestingly, we found a significantly larger temperature response of autumn leaf senescence than of spring leaf-out. This suggests a possible larger contribution of delays in autumn senescence, than of the advancement in spring leaf-out, to extending the growing season under future warmer conditions.

BACKGROUND AND PURPOSEGroup III pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronanmediated mechanisms promoting PH associated with IPF are not fully understood. EXPERIMENTAL APPROACHExplanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis. KEY RESULTSIn patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA-dependent manner. CONCLUSIONS AND IMPLICATIONSTaken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis. Abbreviations4MU, 4-methylumbelliferone; GEFT, guanine nucleotide exchange factor 25; HABP2, hyaluronan-binding protein 2; HAS, hyaluronan synthase; HYAL, hyaluronidase; IF, immunofluorescence; IHC, immunohistochemistry; IPF, idiopathic pulmonary fibrosis; LVSP, left ventricle systolic pressure; mPAP, mean pulmonary arterial pressure; PASMC, pulmonary artery smooth muscle cell; PH, pulmonary hypertension; RVH, right ventricle hypertrophy; RVSP, right ventricle systolic pressure; αSMA, α-smooth muscle actin

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Abstract. Rainfall-runoff models are common tools for river discharge estimation in the field of hydrology. In ungauged basins, the dependence on observed river discharge data for calibration restricts applications of rainfall-runoff models. The strong correlation between quantities of river cross-sectional water surface width obtained from remote sensing and corresponding in situ gauged river discharge has been verified by many researchers. In this study, a calibration scheme of rainfall-runoff models based on satellite observations of river width at basin outlet is illustrated. One distinct advantage is that this calibration is independent of river discharge information. The at-a-station hydraulic geometry is implemented to facilitate shifting the calibration objective from river discharge to river width. The generalized likelihood uncertainty estimation (GLUE) is applied to model calibration and uncertainty analysis. The calibration scheme is demonstrated through a case study for simulating river discharge at Pakse in the Mekong Basin. The effectiveness of the calibration scheme and uncertainties associated with utilization of river width observations from space are examined from model input-state-output behaviour, capability of reproducing river discharge and posterior parameter distribution. The results indicate that the satellite observation of the river width is a competent surrogate of observed discharge for the calibration of rainfall-runoff model at Pakse and the proposed method has the potential for improving reliability of river discharge estimation in basins without any discharge gauging.

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