The integrated stress response regulates 18S nonfunctional rRNA decay in mammals.

18S nonfunctional rRNA decay (NRD) detects and eliminates translationally nonfunctional 18S rRNA. While this process is critical for ribosome quality control, the mechanisms underlying nonfunctional 18S rRNA turnover remain elusive. NRD was originally identified and has exclusively been studied in Saccharomyces cerevisiae. Here, we show that 18S NRD is conserved in mammals. Using genome-wide CRISPR genetic interaction screens, we find that mammalian NRD acts through the integrated stress response (ISR) via GCN2 and ribosomal protein ubiquitination by RNF10. Selective ribosome profiling reveals nonfunctional 18S rRNA induces translational arrest at start sites. Indeed, biochemical analyses demonstrate that ISR activation limits translation initiation and attenuates collisions between scanning 43S preinitiation complexes and nonfunctional 80S ribosomes arrested at start sites. Overall, the ISR promotes nonfunctional 18S rRNA and 40S ribosomal protein turnover by RNF10-mediated ubiquitination. These findings establish a dynamic feedback mechanism by which the GCN2-RNF10 axis surveils ribosome functionality at translation initiation.

FANCD2 expression affects platinum response and further characteristics of high grade serous ovarian cancer in cells with different genetic backgrounds.

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

RFWD3 modulates response to platinum chemotherapy and promotes cancer associated phenotypes in high grade serous ovarian cancer.

Background: DNA damage repair is frequently dysregulated in high grade serous ovarian cancer (HGSOC), which can lead to changes in chemosensitivity and other phenotypic differences in tumours. RFWD3, a key component of multiple DNA repair and maintenance pathways, was investigated to characterise its impact in HGSOC. Methods: RFWD3 expression and association with clinical features was assessed using in silico analysis in the TCGA HGSOC dataset, and in a further cohort of HGSOC tumours stained for RFWD3 using immunohistochemistry. RFWD3 expression was modulated in cell lines using siRNA and CRISPR/cas9 gene editing, and cells were characterised using cytotoxicity and proliferation assays, flow cytometry, and live cell microscopy. Results: Expression of RFWD3 RNA and protein varied in HGSOCs. In cell lines, reduction of RFWD3 expression led to increased sensitivity to interstrand crosslinking (ICL) inducing agents mitomycin C and carboplatin. RFWD3 also demonstrated further functionality outside its role in DNA damage repair, with RFWD3 deficient cells displaying cell cycle dysregulation, reduced cellular proliferation and reduced migration. In tumours, low RFWD3 expression was associated with increased tumour mutational burden, and complete response to platinum chemotherapy. Conclusion: RFWD3 expression varies in HGSOCs, which can lead to functional effects at both the cellular and tumour levels.

Structure and biological evaluation of Caenorhabditis elegans CISD-1/mitoNEET, a KLP-17 tail domain homologue, supports attenuation of paraquat-induced oxidative stress through a p38 MAPK-mediated antioxidant defense response.

CISD-1/mitoNEET is an evolutionarily conserved outer mitochondrial membrane [2Fe-2S] protein that regulates mitochondrial function and morphology. The [2Fe-2S] clusters are redox reactive and shown to mediate oxidative stress in vitro and in vivo. However, there is limited research studying CISD-1/mitoNEET mediation of oxidative stress in response to environmental stressors. In this study, we have determined the X-ray crystal structure of Caenorhabditis elegans CISD-1/mitoNEET homologue and evaluated the mechanisms of oxidative stress resistance to the pro-oxidant paraquat in age-synchronized populations by generating C. elegans gain and loss of function CISD-1 models. The structure of the C. elegans CISD-1/mitoNEET soluble domain refined at 1.70-Å resolution uniquely shows a reversible disulfide linkage at the homo-dimeric interface and also represents the N-terminal tail domain for dimerization of the cognate kinesin motor protein KLP-17 involved in chromosome segregation dynamics and germline development of the nematode. Moreover, overexpression of CISD-1/mitoNEET in C. elegans has revealed beneficial effects on oxidative stress resistance against paraquat-induced reactive oxygen species generation, corroborated by increased activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade.

Inhibitors of the Fanconi anaemia pathway as potential antitumour agents for ovarian cancer.

The Fanconi anaemia (FA) pathway is an important mechanism for cellular DNA damage repair, which functions to remove toxic DNA interstrand crosslinks. This is particularly relevant in the context of ovarian and other cancers which rely extensively on interstrand cross-link generating platinum chemotherapy as standard of care treatment. These cancers often respond well to initial treatment, but reoccur with resistant disease and upregulation of DNA damage repair pathways. The FA pathway is therefore of great interest as a target for therapies that aim to improve the efficacy of platinum chemotherapies, and reverse tumour resistance to these. In this review, we discuss recent advances in understanding the mechanism of interstrand cross-link repair by the FA pathway, and the potential of the component parts as targets for therapeutic agents. We then focus on the current state of play of inhibitor development, covering both the characterisation of broad spectrum inhibitors and high throughput screening approaches to identify novel small molecule inhibitors. We also consider synthetic lethality between the FA pathway and other DNA damage repair pathways as a therapeutic approach.

Simulated Altitude via Re-Breathing Creates Arterial Hypoxemia but Fails to Improve Elements of Running Performance.

Acclimatization to altitude has been shown to improve elements of performance. Use of simulated altitude is popular among athletes across the sports spectrum. This work was on a handheld, re-breathing device touted to enhance performance. Seven recreationally-trained athletes used the device for 18 hours over the course of the 37-day intervention trial. The elevations simulated were progressively increased from 1,524m to 6,096m. To ascertain potential efficacy, four performance trials were included (familiarization, baseline, and 2 follow-ups). Hematological (hematocrit, hemoglobin, and lactate), physiological (respiratory exchange ratio, heart rate, and oxygen consumption), and perceptual (Borg's RPE) variables were monitored at rest, during two steady state running economy stages, and at maximal effort during each visit. The device is clearly capable of creating arterial hypoxemic conditions equating to high altitude. This fact is exemplified by average pulse oximetry values of approximately 78.5% in the final 6-day block of simulation. At the same time, there were no changes observed in any hematological (p>0.05), physiological (p>0.05), or perceptual (p>0.05) variable at either follow-up performance trial. Relative VO2 data was analyzed with a 15-breath moving average sampling frequency in accordance with our recent findings (Scheadler et al.) reported in Medicine and Science in Sports and Exercise. Effect sizes are reported within, but most were trivial (d=0.0-0.19). Overall, findings align with speculation that a more robust altitude stimulus than can be offered by short-term arterial hypoxemia is required for changes to be evidenced. The device has shown some promise in other work, but our data is not supportive.

A difficult transition.

More than 700,000 people in the UK are living with an eating disorder. They can experience physical complications, poor quality of life, disrupted relationships, emotional distress, social isolation and economic disadvantage. The risk of early death is one of the highest among patients with psychiatric disorders.

Discovery of Indazoles as Potent, Orally Active Dual Neurokinin 1 Receptor Antagonists and Serotonin Transporter Inhibitors for the Treatment of Depression.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

Integrating lifestyle approaches into osteoarthritis care.

As the lifetime risk, societal cost, and overall functional impact of osteoarthritis (OA) is imposing, it is imperative that clinicians provide an individualized care model for patients. Patients must be offered a multiplicity of care strategies and encouraged to embrace lifestyle approaches for self-managing the effects and symptoms of OA. Certainly, the attitude of the clinician and patient will directly influence receptivity and implementation of lifestyle approaches. This work proposes how the use of structured and routine assessments and cognitive therapy ideologies may complement a comprehensive treatment plan. Assessments described herein include objective and/or self-report measures of physical function, pain, attitude about social support, and sleep quality. Baseline assessments followed by systematic monitoring of the results may give patients and clinicians valuable insight into the effectiveness of the care plan. Empirical evidence from randomized trials with OA patients highlights the effectiveness of cognitive behavioral change strategies for addressing salient concerns for OA (pain control, mobility performance, and sleep quality). Cognitive restructuring can provide patients with renewed power in managing their disease. Cognitive therapy topics discussed presently include: 1) what is OA?, 2) effectiveness of exercise and FITT (frequency, intensity, time, and type) principles for OA patients, 3) goal-setting and barriers, and 4) translating to independent care. Woven within the discussion about cognitive therapy are ideas about how the results from baseline assessments and group-mediated dynamics might assist more favorable outcomes. There are a plethora of assessments and cognitive therapy topics that could be utilized in the care strategy that we are promoting, but the present topics were selected for their low clinician and patient burden and promising results in trials with OA patients. Clinicians who are comfortable and knowledgeable about a wider range of management tools may serve more effectively in the critical, central management process and help patients embrace personal care more successfully.

Biaryls as potent, tunable dual neurokinin 1 receptor antagonists and serotonin transporter inhibitors.

Depression is a serious illness that affects millions of patients. Current treatments are associated with a number of undesirable side effects. Neurokinin 1 receptor (NK1R) antagonists have recently been shown to potentiate the antidepressant effects of serotonin-selective reuptake inhibitors (SSRIs) in a number of animal models. Herein we describe the optimization of a biaryl chemotype to provide a series of potent dual NK1R antagonists/serotonin transporter (SERT) inhibitors. Through the choice of appropriate substituents, the SERT/NK1R ratio could be tuned to afford a range of target selectivity profiles. This effort culminated in the identification of an analog that demonstrated oral bioavailability, favorable brain uptake, and efficacy in the gerbil foot tap model. Ex vivo occupancy studies with compound 58 demonstrated the ability to maintain NK1 receptor saturation (>88% occupancy) while titrating the desired level of SERT occupancy (11-84%) via dose selection.

Centrally Delivered BACE1 Inhibitor Activates Microglia, and Reverses Amyloid Pathology and Cognitive Deficit in Aged Tg2576 Mice.

Multiple small-molecule inhibitors of the ß-secretase enzyme (BACE1) are under preclinical or clinical investigation for Alzheimer's disease (AD). Prior work has illustrated robust lowering of central amyloid ß (Aß) after acute administration of BACE1 inhibitors. However, very few studies have assessed the overall impact of chronically administered BACE1 inhibitors on brain amyloid burden, neuropathology, and behavioral function in aged preclinical models. We investigated the effects of a potent nonbrain-penetrant BACE1 inhibitor, delivered directly to the brain using intracerebroventricular infusion in an aged transgenic mouse model. Intracerebroventricular infusion of the BACE1 inhibitor (0.3-23.5 µg/d) for 8 weeks, initiated in 17-month-old Tg2576 mice, produced dose-dependent increases in brain inhibitor concentrations (0.2-13 µm). BACE1 inhibition significantly reversed the behavioral deficit in contextual fear conditioning, and reduced brain Aß levels, plaque burden, and associated pathology (e.g., dystrophic neurites), with maximal effects attained with ∼1 µg/d dose. Strikingly, the BACE1 inhibitor also reversed amyloid pathology below baseline levels (amyloid burden at the start of treatment), without adversely affecting cerebral amyloid angiopathy, microhemorrhages, myelination, or neuromuscular function. Inhibitor-mediated decline in brain amyloid pathology was associated with an increase in microglial ramification. This is the first demonstration of chronically administered BACE1 inhibitor to activate microglia, reverse brain amyloid pathology, and elicit functional improvement in an aged transgenic mouse model. Thus, engagement of novel glial-mediated clearance mechanisms may drive disease-modifying therapeutic benefit with BACE1 inhibition in AD.

Design, optimization, and in vivo evaluation of a series of pyridine derivatives with dual NK1 antagonism and SERT inhibition for the treatment of depression.

A series of substituted pyridines, ether linked to a phenylpiperidine core were optimized for dual NK(1)/SERT affinity. Optimization based on NK(1)/SERT binding affinities, and minimization of off-target ion channel activity lead to the discovery of compound 44. In vivo evaluation of 44 in the gerbil forced swim test (a depression model), and ex-vivo NK(1)/SERT receptor occupancy data support the potential of a dual acting compound for the treatment of depression.