Triggering Deep Convection with a Probabilistic Plume Model

A model unifying the representation of the planetary boundary layer and dry, shallow and deep convection, the Probabilistic Plume Model (PPM), is presented. Its capacity to reproduce the triggering of deep convection over land is analysed in detail. The model accurately reproduces the timing of shallow convection and of deep convection onset over land, which is a major issue in many current general climate models. The PPM is based on a distribution of plumes with varying thermodynamic states (potential temperature and specific humidity) induced by surface layer turbulence. Precipitation is computed by a simple ice microphysics, and with the onset of precipitation, downdrafts are initiated and lateral entrainment of environmental air into updrafts is reduced. The most buoyant updrafts are responsible for the triggering of moist convection, causing the rapid growth of clouds and precipitation. Organization of turbulence in the subcloud layer is induced by unsaturated downdrafts, and the effect of density currents is modeled through a reduction of the lateral entrainment. The reduction of entrainment induces further development from the precipitating congestus phase to full deep cumulonimbus. Model validation is performed by comparing cloud base, cloud top heights, timing of precipitation and environmental profiles against cloud resolving models and large-eddy simulations for two test cases. These comparisons demonstrate that PPM triggers deep convection at the proper time in the diurnal cycle, and produces reasonable precipitation. On the other hand, PPM underestimates cloud top height

CHK1 inhibition as a strategy for targeting fanconi anemia (FA) DNA repair pathway deficient tumors

<p>Abstract</p> <p>Background</p> <p>DNA repair deficient tumor cells have been shown to accumulate high levels of DNA damage. Consequently, these cells become hyper-dependent on DNA damage response pathways, including the CHK1-kinase-mediated response. These observations suggest that DNA repair deficient tumors should exhibit increased sensitivity to CHK1 inhibition. Here we offer experimental evidence in support of this hypothesis.</p> <p>Results</p> <p>Using isogenic pairs of cell lines differing only in the Fanconi Anemia (FA) DNA repair pathway, we showed that FA deficient cell lines were hypersensitive to <it>CHK1 </it>silencing by independent siRNAs as well as CHK1 pharmacologic inhibition by Gö6976 and UCN-01. In parallel, an siRNA screen designed to identify gene silencings synthetically lethal with CHK1 inhibition identified genes required for FA pathway function. To confirm these findings <it>in vivo</it>, we demonstrated that whole zebrafish embryos, depleted for <it>FANCD2 </it>by a morpholino approach, were hypersensitive to Gö6976. Silencing of FA genes led to hyper-activation of CHK1 and vice versa. Furthermore, inactivation of CHK1 in FA deficient cell lines caused increased accumulation of DNA strand and chromosomal breakages. These results suggest that the functions subserved by CHK1 and the FA pathway mutually compensate in maintaining genome integrity. As CHK1 inhibition has been under clinical trial in combination with cisplatin, we showed that the FA specific tumoricidal effect of CHK1 inhibition and cisplatin was synergistic.</p> <p>Conclusion</p> <p>Taken together, these results suggest CHK1 inhibition as a strategy for targeting FA deficient tumors.</p

MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability

Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression. DOI: http://dx.doi.org/10.7554/eLife.02445.00

PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity

A Probabilistic Bulk Model of Coupled Mixed Layer and Convection. Part I: Clear-Sky Case

A new bulk model of the convective boundary layer, the probabilistic bulk convection model (PBCM), is presented. Unlike prior bulk approaches that have modeled the mixed-layer-top buoyancy flux as a constant fraction of the surface buoyancy flux, PBCM implements a new mixed-layer-top entrainment closure based on the mass flux of updrafts overshooting the inversion. This mass flux is related to the variability of the surface state (potential temperature θ and specific humidity q) of an ensemble of updraft plumes. The authors evaluate the model against observed clear-sky weak and strong inversion cases and show that PBCM performs well. The height, state, and timing of the boundary layer growth are accurately reproduced. Sensitivity studies are performed highlighting the role of the main parameters (surface variances, lateral entrainment). The model is weakly sensitive to the exact specification of the variability at the surface and is most sensitive to the lateral entrainment of environmental air into the rising plumes. Apart from allowing time-dependent top-of-the-boundary-layer entrainment rates expressed in terms of surface properties, which can be observed in situ, PBCM naturally takes into account the transition to the shallow convection regime, as described in a companion paper. Thus, PBCM represents an important step toward a unified framework bridging parameterizations of mixed-layer entrainment velocity in both clear-sky and moist convective boundary layers

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are &#945;2&#946;2 tetramers, in which the &#946; subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the &lt;i&gt;Caenorhabditis&lt;/i&gt; &lt;i&gt;elegans&lt;/i&gt; catalytic &#945; subunit isoforms PHY-1 and PHY-2 and the &#946; subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer is the major form, while PHY-1/PDI- 2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode &lt;i&gt;Caenorhabditis&lt;/i&gt; &lt;i&gt;briggsae&lt;/i&gt; revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the &lt;i&gt;C. briggsae&lt;/i&gt; and &lt;i&gt;C. elegans&lt;/i&gt; subunits. In addition to a PHY-1/PHY-2(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer and a PHY-1/PDI-2 dimer, an active (PHY- 2)&lt;sub&gt;2&lt;/sub&gt;(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer was formed in &lt;i&gt;C. briggsae&lt;/i&gt; instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the &lt;i&gt;Caenorhabditis&lt;/i&gt; PHY-2 polypeptides determine their assembly properties. Genetic disruption of &lt;i&gt;C. briggsae phy-1&lt;/i&gt; (&lt;i&gt;Cb-dpy-18&lt;/i&gt;) via a &lt;i&gt;Mos1&lt;/i&gt; insertion resulted a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding &lt;i&gt;C. elegans&lt;/i&gt; mutants (&lt;i&gt;Ce-dpy-18&lt;/i&gt;). &lt;i&gt;C. briggsae&lt;/i&gt; &lt;i&gt;phy-2&lt;/i&gt; RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in &lt;i&gt;phy-1&lt;/i&gt; mutants. Genetic complementation of the &lt;i&gt;C. briggsae&lt;/i&gt; and &lt;i&gt;C. elegans&lt;/i&gt; &lt;i&gt;phy-1&lt;/i&gt; mutants was achieved by injection of a wild type &lt;i&gt;phy-1&lt;/i&gt; gene from either species

Small-Molecule Inhibitors of USP1 Target ID1 Degradation in Leukemic Cells

Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine–based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Mol Cancer Ther; 12(12); 2651–62. ©2013 AACR

Heterogeneity and Clonal Evolution of Acquired PARP Inhibitor Resistance in TP53- and BRCA1-Deficient Cells

Homologous recombination (HR)-deficient cancers are sensitive to poly- ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53(-/-) and BRCA1(-/-) epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. Significance: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.Peer reviewe

Inhibition of TGF beta 1 and TGF beta 3 promotes hematopoiesis in Fanconi anemia

Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor beta (TGF beta) pathway, regulated by the TGF beta 1, TGF beta 2, and TGF beta 3 ligands. Accordingly, the TGF beta pathway is an attractive therapeutic target for FA. While inhibition of TGF beta 1 and TGF beta 3 promotes blood cell expansion, inhibition of TGF beta 2 is known to suppress hematopoiesis. Here, we report the effects of AVID200, a potent TGF beta 1- and TGF beta 3-specific inhibitor, on FA hematopoiesis. AVID200 promoted the survival of murine FA HSPCs in vitro. AVID200 also promoted in vitro the survival of human HSPCs from patients with FA, with the strongest effect in patients progressing to severe aplastic anemia or myelodysplastic syndrome (MDS). Previous studies have indicated that the toxic upregulation of the nonhomologous end-joining (NHEJ) pathway accounts, at least in part, for the poor growth of FA HSPCs. AVID200 downregulated the expression of NHEJ-related genes and reduced DNA damage in primary FA HSPC in vitro and in in vivo models. Collectively, AVID200 exhibits activity in FA mouse and human preclinical models. AVID200 may therefore provide a therapeutic approach to improving BMF in FA. (c) 2020 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.Peer reviewe

Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8+T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8+T-cells and PD-L1+macrophages and PD-L1+tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8+T-cells with PD-L1+macrophages in the first, and exhausted CD8+T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second. A Phase I/II trial previously revealed variable anti-tumor efficacy of the PARP inhibitor niraparib in combination with the PD-1 inhibitor pembrolizumab in platinum-resistant ovarian cancer patients. Here, the authors perform an integrated genomic and immunomics analysis of tumor samples from the same patients and find potential predictive biomarkers of response to such combination therapy.Peer reviewe