Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

Acidification and solar drying of manure-based digestate to produce improved fertilizing products.

The increase in energy and fertilizer consumption makes it necessary to develop sustainable alternatives for agriculture. Anaerobic digestion and digestates appeared to be suitable options. However, untreated digestates still have high water content and can increase greenhouse gas emissions during storage and land application. In this study, manure-derived digestate and solid fraction of digestate after separation were treated with a novel solar drying technology to reduce their water content, combined with acidification to reduce the gaseous emissions. The acidified digestate and acidified solid fraction of digestate recovered more nitrogen and ammonia nitrogen than their respective non-acidified products (1.5-1.3 times for TN; 14 times for TAN). Ammonia and methane emissions were reduced up to 94% and 72% respectively, compared to the non-acidified ones, while N2O increased more than 3 times. Dried digestate and dried acidified digestate can be labeled as NPK organic fertilizer regarding the European regulation, and the dried solid fraction and the improved dried acidified solid fraction can be labeled as N or P organic fertilizer. Moreover, plant tests showed that N concentrations in fresh lettuce leaves were within the EU limit with all products in all the cases. However, zinc concentration appeared to be a limitation in some of the products as their concentration exceeded the European legal limits.

Precipitation of greigite and pyrite induced by Thermococcales: an advantage to live in Fe- and S-rich environments?

Thermococcales, a major order of archaea inhabiting the iron- and sulfur-rich anaerobic parts of hydrothermal deep-sea vents, have been shown to rapidly produce abundant quantities of pyrite FeS2 in iron-sulfur-rich fluids at 85°C, suggesting that they may contribute to the formation of 'low temperature' FeS2 in their ecosystem. We show that this process operates in Thermococcus kodakarensis only when zero-valent sulfur is directly available as intracellular sulfur vesicles. Whether in the presence or absence of zero-valent sulfur, significant amounts of Fe3 S4 greigite nanocrystals are formed extracellularly. We also show that mineralization of iron sulfides induces massive cell mortality but that concomitantly with the formation of greigite and/or pyrite, a new generation of cells can grow. This phenomenon is observed for Fe concentrations of 5 mM but not higher suggesting that above a threshold in the iron pulse all cells are lysed. We hypothesize that iron sulfides precipitation on former cell materials might induce the release of nutrients in the mineralization medium further used by a fraction of surviving non-mineralized cells allowing production of new alive cells. This suggests that biologically induced mineralization of iron-sulfides could be part of a survival strategy employed by Thermococcales to cope with mineralizing high-temperature hydrothermal environments.

Elucidating key plant growth-promoting (PGPR) traits in Burkholderia sp. Nafp2/4-1b (=SARCC-3049) using gnotobiotic assays and whole-genome-sequence analysis.

Burkholderia sp. Nafp2/4-1b (=SARCC-3049) is a plant growth-promoting rhizobacteria (PGPR) initially isolated from the rhizosphere of pristine grassland in South Africa, and its ability to enhance growth was previously evaluated on maize (Zea mays L.). Here, the bacterium was tested with the aim of investigating its role in improving the nodulation and growth of the forage legume lucerne (Medicago sativa L.) when it is co-inoculated with the rhizobial symbionts of this legume in the glasshouse. When the co-inoculation resulted in a statistically significant (P = 0·05) increase in the number of nodules and improved plant biomass compared with single inoculation, we sequenced and analysed its genome to gain a better understanding of the genetic determinants responsible for the observed PGPR traits. The Illumina HiSeq 2500-sequenced genome resulted in 92 scaffolds, with an N50 of 322 407 bp, a total draft genome size of 7 788 045 bp and GC content of 66·2%. Analysis of the genome sequence confirmed the presence of a number of essential genes that code for various PGPR traits. The main plant beneficial genes associated with PGPR traits in Burkholderia sp. Nafp2/4-1b include pyoverdine siderophores biosynthesis gene (PvdF); acdS that codes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase; the tryptophan synthase genes involved in auxin biosynthesis (TSA1, TSB1) and the pqqABCDE operon related to phosphate solubilization. This study generated valuable information on the potential of the PGPR Burkholderia sp. strain Nafp2/4-1b as an effective commercial inoculant, which warrants further formulation and field application studies before developing it into a low cost, environmentally safe and effective biofertilizer.

Balancing Properties with Carboxylates: A Lead Optimization Campaign for Selective and Orally Active CDK9 Inhibitors.

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Hexavalent TRAIL Fusion Protein Eftozanermin Alfa Optimally Clusters Apoptosis-Inducing TRAIL Receptors to Induce On-Target Antitumor Activity in Solid Tumors.

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621-responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. SIGNIFICANCE: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Quantification of BCL-2 Family Members by Flow Cytometry.

Flow cytometry is a powerful technique for the detection and quantification of cell surface and intracellular proteins. It enables the ability to measure the expression levels of specific proteins in a cell population of interest without the need to physically separate out the cells from within a heterogeneous population by using the appropriate cell-specific markers. It also requires fewer cells than other traditional techniques such as Western blotting. Here we describe a robust and reproducible method to measure the expression levels of the BCL-2 family members, BCL-2, BCL-XL, and MCL-1 by quantitative flow cytometry (QFCM) using validated antibodies.

Potential mechanisms of resistance to venetoclax and strategies to circumvent it.

BACKGROUND: Venetoclax (ABT-199), a first-in-class orally bioavailable BCL-2-selective inhibitor, was recently approved by the FDA for use in patients with 17p-deleted chronic lymphocytic leukemia who have received prior therapy. It is also being evaluated in numerous clinical trials for treating patients with various hematologic malignancies. As with any targeted cancer therapy, it is critically important to identify potential mechanisms of resistance, both for patient stratification and developing strategies to overcome resistance, either before it develops or as it emerges. METHODS: In order to gain a more comprehensive insight into the nature of venetoclax resistance mechanisms, we evaluated the changes in the BCL-2 family members at the genetic and expression levels in seven different venetoclax-resistant derived leukemia and lymphoma cell lines. RESULTS: Gene and protein expression analyses identified a number of different alterations in the expression of pro- and anti-apoptotic BCL-2 family members. In the resistant derived cells, an increase in either or both the anti-apoptotic proteins BCL-XL or MCL-1, which are not targeted by venetoclax was observed, and either concomitant or exclusive with a decrease in one or more pro-apoptotic proteins. In addition, mutational analysis also revealed a mutation in the BH3 binding groove (F104L) that could potentially interfere with venetoclax-binding. Not all changes may be causally related to venetoclax resistance and may only be an epiphenomenon. For resistant cell lines showing elevations in BCL-XL or MCL-1, strong synergistic cell killing was observed when venetoclax was combined with either BCL-XL- or MCL-1-selective inhibitors, respectively. This highlights the importance of BCL-XL- and MCL-1 as causally contributing to venetoclax resistance. CONCLUSIONS: Overall our study identified numerous changes in multiple resistant lines; the changes were neither mutually exclusive nor universal across the cell lines tested, thus exemplifying the complexity and heterogeneity of potential resistance mechanisms. Identifying and evaluating their contribution has important implications for both patient selection and the rational development of strategies to overcome resistance.

Development of a flow cytometric method for quantification of BCL-2 family members in chronic lymphocytic leukemia and correlation with sensitivity to BCL-2 family inhibitors.

BACKGROUND: We have developed a quantitative fluorescence cytometry (QFCM) method that can be used to measure BCL-2 family member proteins in cell lines and clinical samples. We described the validation of antibodies, methods development and application of the assay. METHOD: We characterized and validated antibodies to BCL-2, BCL-XL , and MCL-1 in cell lines to confirm specificity for flow cytometry. Each protein was measured in a panel of leukemia/lymphoma cell lines and B-cells from chronic lymphocytic leukemia (CLL) patients treated with the BCL-2/BCL-XL inhibitor navitoclax. The cellular activity of various BCL-2 family member inhibitors alone and in combination was determined to demonstrate utility of our assay to correlate protein levels with efficacy. RESULTS: We identified antibodies that were highly specific for each protein. The expression profile in cell lines as determined by molecules of equivalent soluble fluorochrome was comparable to western blot. Using our assay, BCL-2, BCL-XL , and MCL-1 protein levels were shown to correlate with response to BCL-2 family inhibitors in vitro and could be measured in clinical samples. CONCLUSIONS: This method can quantify BCL-2 family members in a specific, highly reproducible and sensitive fashion, and requires fewer cells compared to western blot. It is particularly useful for identifying BCL-2, BCL-XL , and MCL-1 protein levels in a specific cell population within a heterogeneous population like those collected from CLL patients. These data show that our QFCM method can be used to facilitate the quantification and evaluation of biomarkers predictive of response in patients treated with BCL-2 family member inhibitors. © 2016 International Clinical Cytometry Society.

Influence of carbon dioxide on numbers of Culicoides midges collected with suction light traps in South Africa.

To implement risk management against diseases transmitted by species of Culicoides Latreille, 1809 (Diptera: Ceratopogonidae), it is essential to identify all potential vectors. Light traps are the most commonly used tool for the collection of Culicoides midges. Given the indiscriminate artificial attraction of light, traps will collect all night-flying insects rather than only livestock-associated Culicoides midges. Factors that may increase the efficacy of traps, especially for livestock-associated Culicoides midges, require investigation. In the present study, results obtained with Centers for Disease Control (CDC) and Onderstepoort light traps baited with carbon dioxide (CO2 ) were compared with those of unbaited controls. Comparisons were made using two replicates of a 4 × 4 randomized Latin square design. With both trap types, the mean numbers of Culicoides midges collected in 16 baited traps were higher than those caught in 16 unbaited traps. Although exceptionally low numbers were collected with the CDC traps, the increases in the numbers and frequency of collection of Culicoides imicola Kieffer, 1913 were more pronounced in the CDC traps compared with the Onderstepoort traps. These results indicate that the addition of CO2 may increase the efficiency of these traps for the collection of C. imicola and other livestock-associated Culicoides species.

Relating DSM-5 section III personality traits to section II personality disorder diagnoses.

BACKGROUND: The DSM-5 Personality and Personality Disorders Work Group formulated a hybrid dimensional/categorical model that represented personality disorders as combinations of core impairments in personality functioning with specific configurations of problematic personality traits. Specific clusters of traits were selected to serve as indicators for six DSM categorical diagnoses to be retained in this system - antisocial, avoidant, borderline, narcissistic, obsessive-compulsive and schizotypal personality disorders. The goal of the current study was to describe the empirical relationships between the DSM-5 section III pathological traits and DSM-IV/DSM-5 section II personality disorder diagnoses. METHOD: Data were obtained from a sample of 337 clinicians, each of whom rated one of his or her patients on all aspects of the DSM-IV and DSM-5 proposed alternative model. Regression models were constructed to examine trait-disorder relationships, and the incremental validity of core personality dysfunctions (i.e. criterion A features for each disorder) was examined in combination with the specified trait clusters. RESULTS: Findings suggested that the trait assignments specified by the Work Group tended to be substantially associated with corresponding DSM-IV concepts, and the criterion A features provided additional diagnostic information in all but one instance. CONCLUSIONS: Although the DSM-5 section III alternative model provided a substantially different taxonomic structure for personality disorders, the associations between this new approach and the traditional personality disorder concepts in DSM-5 section II make it possible to render traditional personality disorder concepts using alternative model traits in combination with core impairments in personality functioning.

Clearance of systemic hematologic tumors by venetoclax (Abt-199) and navitoclax.

The Bcl-2 family inhibitors venetoclax and navitoclax demonstrated potent antitumor activity in chronic lymphocytic leukemia patients, notably in reducing marrow load and adenopathy. Subsequent trials with venetoclax have been initiated in non-Hodgkin's lymphoma and multiple myeloma patients. Traditional preclinical models fall short either in faithfully recapitulating disease progression within such compartments or in allowing the direct longitudinal analysis of systemic disease. We show that intravenous inoculation of engineered RS4;11 (acute lymphoblastic leukemia) and Granta 519 (mantle cell lymphoma) bioluminescent reporter cell lines result in tumor engraftment of bone marrow, with additional invasion of the central nervous system in the case of Granta 519. Importantly, apoptosis induction and response of these systemically engrafted tumors to Bcl-2 family inhibitors alone or in combination with standard-of-care agents could be monitored longitudinally with optical imaging, and was more accurately reflective of the observed clinical response.

Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

ZRF1 controls oncogene-induced senescence through the INK4-ARF locus.

The reactivation of the INK4-ARF locus, which is epigenetically repressed by Polycomb proteins in healthy cells, is a hallmark of senescence. One mechanism of reactivating Polycomb-silenced genes is mediated by the epigenetic factor ZRF1, which associates with ubiquitinated histone H2A. We show that cells undergoing senescence following oncogenic Ras expression have increased ZRF1 levels, and that this binds to the p15INK4b, ARF and p16INK4a promoters. Furthermore, ZRF1 depletion in oncogenic Ras-expressing cells restores proliferation by preventing Arf and p16Ink4a expression, consequently bypassing senescence. Thus, ZRF1 regulates the INK4-ARF locus during cellular proliferation and senescence, and alterations in ZRF1 may contribute to tumorigenesis.

The attraction range of the Onderstepoort 220V light trap for Culicoides biting midges as determined under South African field conditions.

Despite some limitations suction light traps are the primary tools used for the collection of Culicoides species (Diptera: Ceratopogonidae). The range of attraction of the Onderstepoort light trap is not known but an insight into the range of a trap will determine where the trap must be positioned relative to the hosts present, possible breeding sites and environmental structures in the trapping vicinity. It will therefore contribute to a more meaningful interpretation and comparison of results between trapping events. In the present study the number of Culicoides midges collected in a single trap was compared to those of traps made with an additional trap respectively 1m, 4m and 8.5m away from the first. Treatments between sites were rotated in three replicates of a 4×4 Latin square design. While interactions were found in traps 4m apart no statistically significant interactions were found when they were 8.5m apart. The range of attraction, indicated by the interaction between two traps, will be between 2m and 4m. In interpreting light trap results the limitations of this collection method needs to be taken into consideration.