The Roles of Cullin RING Ligases and the Anaphase Promoting Complex/Cyclosome in the Regulation of DNA Double Strand Break Repair

Historically, genome maintenance has been viewed as the largely independent activities of (1) ubiquitin ligases driving unidirectional cell cycle progression and, (2) the activity of cellular checkpoints that monitor DNA integrity and DNA replication. It is well established that the DNA damage response (DDR) checkpoint machinery promotes the activation of repair mechanisms in addition to opening a window for repair. Emerging evidence demonstrates an integrated network of the central cell cycle driving E3 ubiquitin ligases and the checkpoint machinery, as well as deubiquitinating enzymes, which intermittently cooperate and antagonize one another to define windows of checkpoint and repair activities to optimize genome stability and cellular health. A growing number of components of the ubiquitin machinery are involved in the DDR. Herein, we focus on the regulation of cell cycle checkpoints and the DNA repair mechanisms for double strand breaks (DSBs) by the coordinated activities of Cullin RING ligases (CRLs) and the anaphase promoting complex/cyclosome (APC/C)

Cyclin E overexpression impairs progression through mitosis by inhibiting APCCdh1

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APCCdh1) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APCCdh1 substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E–mediated mitotic phenotype

Limitations of Self-Assembly at Temperature One (extended abstract)

We prove that if a subset X of the integer Cartesian plane weakly self-assembles at temperature 1 in a deterministic (Winfree) tile assembly system satisfying a natural condition known as *pumpability*, then X is a finite union of doubly periodic sets. This shows that only the most simple of infinite shapes and patterns can be constructed using pumpable temperature 1 tile assembly systems, and gives strong evidence for the thesis that temperature 2 or higher is required to carry out general-purpose computation in a tile assembly system. Finally, we show that general-purpose computation is possible at temperature 1 if negative glue strengths are allowed in the tile assembly model

Glutathione-triggered disassembly of isothermally responsive polymer nanoparticles obtained by nanoprecipitation of hydrophilic polymers

The encapsulation and selective delivery of therapeutic compounds within polymeric nanoparticles offers hope for the treatment of a variety of diseases. Traditional approaches to trigger selective cargo release typically rely on polymer degradation which is not always sensitive to the biological location of a material. In this report, we prepare nanoparticles from thermoresponsive polymers with a ‘solubility release catch’ at the chain-end. This release catch is exclusively activated in the presence of intracellular glutathione, triggering an ‘isothermal’ response and promoting a change in polymer solubility. This solubility switch leads to specific and rapid nanoparticle disassembly, release of encapsulated cargo and produces completely soluble polymeric side-products

Degree of egg-taking by humans determines the fate of maleo (Macrocephalon maleo) nesting grounds across Sulawesi

The version of record of this article, first published in Biodiversity and Conservation, is available online at Publisher’s website: http://dx.doi.org/10.1007/s10531-022-02527-1The maleo (Macrocephalon maleo) of Sulawesi, Indonesia, is culturally iconic and Critically Endangered, but the causes of its decline have never been systematically analyzed nor its nesting grounds comprehensively surveyed. We visited 122 previously known and 58 previously unrecorded sites, collecting data and interviewing local people at each site. We used ordinal logistic regression to fit models with combinations of 18 different predation, habitat, and nesting ground variables to determine the strongest predictors of nesting ground success, as represented by maleo numbers. At least 56% of known nesting grounds are now inactive (abandoned), and 63% of remaining active sites host ≤ 2 pairs/day at peak season. Egg-taking by humans is the single biggest driver of maleo decline. Protecting eggs in situ predicts higher numbers than protecting eggs through hatchery methods. After egg-taking, quality (not length) of the travel corridor connecting nesting ground to primary forest best predicts nesting ground success. Being inside a federally protected area is not a primary driver of success, and does not ensure persistence: 28% of federally protected nesting grounds have become inactive. Local conservation efforts protected nesting grounds 2‒3 times better than federal protection. We update the methodology for assessing nesting ground status, and recommend five measures for maleo conservation, the foremost being to protect nesting grounds from egg-taking by humans at all remaining active sites

A Helminth Immunomodulator Exploits Host Signaling Events to Regulate Cytokine Production in Macrophages

Parasitic worms alter their host's immune system to diminish the inflammatory responses directed against them, using very efficient immunomodulating molecules. We have previously shown that the helminth immunomodulator cystatin (AvCystatin) profoundly reduces the progression of inflammatory diseases via modulation of macrophages. Here we elucidate the signaling events in macrophages triggered by AvCystatin. Labeled AvCystatin was predominantly taken up by macrophages and subsequently induced the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38. IL-10 expression induced by AvCystatin in macrophages was tyrosine kinase sensitive and dependent on activation of both MAP kinases, in clear contrast to expression of IL-12/23p40. In addition, phosphorylation of the transcription factors CREB and STAT3 was induced by AvCystatin and regulated by phospho-ERK. Chemical inhibition of phosphoinositide 3-kinase (PI3K) reduced AvCystatin-induced cytokine release; however, AKT, the downstream target of PI3K, was not activated following AvCystatin exposure. To characterize signaling elements involved in alteration of the macrophage phenotype we applied mathematical modeling. Experimental testing of the in silico generated hypotheses identified dual specificity phosphatase (DUSP) 1 and 2, as regulators in AvCystatin triggered macrophages in vitro and in vivo. In particular, DUSP1 was subsequently found to be responsible for regulation of ERK- and p38-phosphorylation and controlled the IL-10 expression in macrophages by AvCystatin. Thus, we show that AvCystatin exploits activation and deactivation pathways of MAP kinases to induce regulatory macrophages. This study provides insights into molecular mechanisms of macrophage manipulation by parasites and highlights the utility of mathematical modeling for the elucidation of regulatory circuits of immune cells

Whipworm genome and dual-species transcriptome analyses provide molecular insights into an intimate host-parasite interaction.

Whipworms are common soil-transmitted helminths that cause debilitating chronic infections in man. These nematodes are only distantly related to Caenorhabditis elegans and have evolved to occupy an unusual niche, tunneling through epithelial cells of the large intestine. We report here the whole-genome sequences of the human-infective Trichuris trichiura and the mouse laboratory model Trichuris muris. On the basis of whole-transcriptome analyses, we identify many genes that are expressed in a sex- or life stage-specific manner and characterize the transcriptional landscape of a morphological region with unique biological adaptations, namely, bacillary band and stichosome, found only in whipworms and related parasites. Using RNA sequencing data from whipworm-infected mice, we describe the regulated T helper 1 (TH1)-like immune response of the chronically infected cecum in unprecedented detail. In silico screening identified numerous new potential drug targets against trichuriasis. Together, these genomes and associated functional data elucidate key aspects of the molecular host-parasite interactions that define chronic whipworm infection

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1∆) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription

TRIB1 confers therapeutic resistance in GBM cells by activating the ERK and Akt pathways

GBM (Glioblastoma) is the most lethal CNS (Central nervous system) tumor in adults, which inevitably develops resistance to standard treatments leading to recurrence and mortality. TRIB1 is a serine/threonine pseudokinase which functions as a scaffold platform that initiates degradation of its substrates like C/EBPα through the ubiquitin proteasome system and also activates MEK and Akt signaling. We found that increased TRIB1 gene expression associated with worse overall survival of GBM patients across multiple cohorts. Importantly, overexpression of TRIB1 decreased RT/TMZ (radiation therapy/temozolomide)-induced apoptosis in patient derived GBM cell lines in vitro. TRIB1 directly bound to MEK and Akt and increased ERK and Akt phosphorylation/activation. We also found that TRIB1 protein expression was maximal during G2/M transition of cell cycle in GBM cells. Furthermore, TRIB1 bound directly to HDAC1 and p53. Importantly, mice bearing TRIB1 overexpressing tumors had worse overall survival. Collectively, these data suggest that TRIB1 induces resistance of GBM cells to RT/TMZ treatments by activating the cell proliferation and survival pathways thus providing an opportunity for developing new targeted therapeutics

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation