G-quadruplex DNA structure is a positive regulator of MYC transcription.

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.

A library-derived peptide inhibitor of the BZLF1 transcription factor.

Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.

DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody.

G-quadruplexes (G4s) are four-stranded DNA secondary structures that occur in the human genome and play key roles in transcription, replication, and genome stability. G4-specific molecular probes are of vital importance to elucidate the structure and function of G4s. The scFv antibody BG4 has been a widely used G4 probe but has various limitations, including relatively poor in vitro expression and the inability to be expressed intracellularly to interrogate G4s in live cells. To address these considerations, we describe herein the development of SG4, a camelid heavy-chain-only derived nanobody that was selected against the human Myc DNA G4 structure. SG4 exhibits low nanomolar affinity for a wide range of folded G4 structures in vitro. We employed AlphaFold combined with molecular dynamics simulations to construct a molecular model for the G4-nanobody interaction. The structural model accurately explains the role of key amino acids and Kd measurements of SG4 mutants, including arginine-to-alanine point mutations that dramatically diminish G4 binding affinity. Importantly, predicted amino acid-G4 interactions were subsequently confirmed experimentally by biophysical measurements. We demonstrate that the nanobody can be expressed intracellularly and used to image endogenous G4 structures in live cells. We also use the SG4 protein to positionally map G4s in situ and also on fixed chromatin. SG4 is a valuable, new tool for G4 detection and mapping in cells.

Exploring the binding of rationally engineered tandem-repeat proteins to E3 ubiquitin ligase Keap1.

The process of displaying functional peptides by 'grafting' them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display.

Existing validated clinical prediction rules for predicting response to physiotherapy interventions for musculoskeletal conditions have limited clinical value: A systematic review.

OBJECTIVE: To systematically review clinical prediction rules (CPRs) that have undergone validation testing for predicting response to physiotherapy-related interventions for musculoskeletal conditions. STUDY DESIGN AND SETTING: PubMed, EMBASE, CINAHL and Cochrane Library were systematically searched to September 2020. Search terms included musculoskeletal (MSK) conditions, physiotherapy interventions and clinical prediction rules. Controlled studies that validated a prescriptive CPR for physiotherapy treatment response in musculoskeletal conditions were included. Two independent reviewers assessed eligibility. Original derivation studies of each CPR were identified. Risk of bias was assessed with the PROBAST tool (derivation studies) and the Cochrane Effective Practice and Organisation of Care group criteria (validation studies). RESULTS: Nine studies aimed to validate seven prescriptive CPRs for treatment response for MSK conditions including back pain, neck pain, shoulder pain and carpal tunnel syndrome. Treatments included manipulation, traction and exercise. Seven studies failed to demonstrate an association between CPR prediction and outcome. Methodological quality of derivation studies was poor and for validation studies was good overall. CONCLUSION: Results do not support the use of any CPRs identified to aid physiotherapy treatment selection for common musculoskeletal conditions, due to methodological shortcomings in the derivation studies and lack of association between CPR and outcome in validation studies.

Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc.

c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy. Here we reassess the challenges of directly targeting c-Myc, evaluate lessons learned from current inhibitors, and explore how future strategies such as miniaturisation of Omomyc and targeting E-box binding could facilitate translation of c-Myc inhibitors into the clinic.

Structural and mechanistic insights into the Keap1-Nrf2 system as a route to drug discovery.

The proteins Keap1 and Nrf2 together act as a cytoprotective mechanism that enables cells to overcome electrophilic and oxidative stress. Research has shown that manipulating this system by modulating the Keap1-Nrf2 interaction either through inhibition at the binding interface or via the covalent modification of Keap1 could provide a powerful therapeutic strategy for a range of diseases. However, despite intensive investigation of the system and significant progress in the development of inhibitory small molecules, there is still much to learn about the pathways associated with the Keap1-Nrf2 system and the structural details underpinning its mechanism of action. In this review, we discuss how a deeper understanding could prove revolutionary in the development of new inhibitors and activators as well as guiding how to best harness Keap1 for targeted protein degradation.

Exploring new strategies for grafting binding peptides onto protein loops using a consensus-designed tetratricopeptide repeat scaffold.

Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra-stable consensus-designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch-like ECH-associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1-binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.

Highly selective inhibition of histone demethylases by de novo macrocyclic peptides.

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.

Sleep to lower elevated blood pressure: study protocol for a randomized controlled trial.

BACKGROUND: Sleep is an essential component of good physical and mental health. Previous studies have reported that poor quality sleep is associated with an increased risk of hypertension and cardiovascular disease. Hypertension is the most common and important risk factor for cardiovascular disease, and even modest reductions in blood pressure can result in significant reductions in the risk of stroke and myocardial infarction. In this trial, we will determine the efficacy of an online sleep intervention in improving blood pressure, in participants with hypertension and poor sleep quality. TRIAL DESIGN: Randomized-controlled, two-group, parallel, blinded, single-center, Phase II trial of 134 participants. Population and recruitment: Primary prevention population of participants with hypertension (systolic blood pressure, 130 to 160 mm Hg; diastolic blood pressure, <110 mm Hg) and poor sleep quality in a community setting. INTERVENTION: Multicomponent online sleep intervention consisting of sleep information, sleep hygiene education, and cognitive behavioral therapy. Comparator: Standardized cardiovascular risk factor and lifestyle-education session (usual care). PRIMARY OUTCOME: Change in mean 24-hour ambulatory systolic blood pressure between baseline and 8-week follow-up. Hypertension has been selected as the primary outcome measure because of its robust association with both poor sleep quality and cardiovascular disease. STATISTICAL ANALYSES: Intention-to-treat analysis by using a linear mixed model. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01809821, registered March 8, 2013.

Turning the tide: how blue carbon and payments for ecosystem services (PES) might help save mangrove forests.

In this review paper, we aim to describe the potential for, and the key challenges to, applying PES projects to mangroves. By adopting a "carbocentric approach," we show that mangrove forests are strong candidates for PES projects. They are particularly well suited to the generation of carbon credits because of their unrivaled potential as carbon sinks, their resistance and resilience to natural hazards, and their extensive provision of Ecosystem Services other than carbon sequestration, primarily nursery areas for fish, water purification and coastal protection, to the benefit of local communities as well as to the global population. The voluntary carbon market provides opportunities for the development of appropriate protocols and good practice case studies for mangroves at a small scale, and these may influence larger compliance schemes in the future. Mangrove habitats are mostly located in developing countries on communally or state-owned land. This means that issues of national and local governance, land ownership and management, and environmental justice are the main challenges that require careful planning at the early stages of mangrove PES projects to ensure successful outcomes and equitable benefit sharing within local communities.

Human UTY(KDM6C) is a male-specific Nϵ-methyl lysyl demethylase.

The Jumonji C lysine demethylases (KDMs) are 2-oxoglutarate- and Fe(II)-dependent oxygenases. KDM6A (UTX) and KDM6B (JMJD3) are KDM6 subfamily members that catalyze demethylation of N(ϵ)-methylated histone 3 lysine 27 (H3K27), a mark important for transcriptional repression. Despite reports stating that UTY(KDM6C) is inactive as a KDM, we demonstrate by biochemical studies, employing MS and NMR, that UTY(KDM6C) is an active KDM. Crystallographic analyses reveal that the UTY(KDM6C) active site is highly conserved with those of KDM6B and KDM6A. UTY(KDM6C) catalyzes demethylation of H3K27 peptides in vitro, analogously to KDM6B and KDM6A, but with reduced activity, due to point substitutions involved in substrate binding. The results expand the set of human KDMs and will be of use in developing selective KDM inhibitors.

Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates.

The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N(ϵ)-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N(ϵ)-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N(ϵ)-methyllysine of an H3K27me3 peptide binds similarly to N(ϵ)-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N(ϵ)-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.

Guidelines, guidelines, guidelines: what are we to do with all of these North American guidelines?

Over the past decade, clinical guidelines for nutrition therapy in the critically ill have been developed by different North American societies. To avoid target audience confusion and uncertainty, there is a need to undergo a review of the content of these guidelines. In this review, the authors compared the grading systems, the levels of evidence used, and the content of North American nutrition clinical guidelines. The 3 clinical guidelines that met their search criteria and hence were included in the comparison are the Canadian Clinical Practice Guidelines, the American Dietetics Association's evidence-based guideline for critical illness, and the Society of Critical Care Medicine and American Society of Parenteral and Enteral Nutrition's joint guideline. Through their comparison, the authors have shown that although there are several topics where there is a similar direction of recommendation across the 3 societies/organizations, there are stark contrasts among many of the recommendations. These major differences can be attributed to the admission of different populations, lower levels of evidence or expert opinion into the guideline production process, lack of clarity in the link between the evidence and the recommendation, and lack of uniformity in the reporting of levels of evidence and grades of recommendation. The authors have identified the need for the North American nutrition organizations to harmonize the development of future nutrition guidelines in a timely way, so that they remain current and up-to-date. Furthermore, guideline users need to be aware of the dissimilarities in these guidelines before applying the recommendations to their daily practice.

Randomized trials in critical care nutrition: look how far we've come! (and where do we go from here?).

BACKGROUND: The purpose of this methodological review is to quantify and qualify critical care nutrition randomized controlled trials (RCTs) that inform our practice, to evaluate their strengths and limitations, and to recommend strategies for improving the design of future trials in this area. METHODS: The literature was systematically reviewed to find all RCTs published between 1980 and December 2008 that evaluated nutrition interventions in critical care. Data were abstracted on the nature and quality of included RCTs. RESULTS: A total of 207 RCTs met the inclusion criteria. Of these, 170 (82.1%) were single-center, and 37 (17.9%) were multicenter. The largest number of trials evaluated intensive insulin therapy (n = 25), arginine-supplemented diets (n = 22), and supplemental parenteral glutamine (n = 17). The first RCTs were published in 1983 (n = 2), and the mean sample size was 39.0. In 2008, there were 26 RCTs, each enrolling an average of 237.1 patients. Excluding 2 cluster RCTs, 62 of 205 (30.2%) trials had concealed randomization, 125 of 205 (61.0%) reported on intention-to-treat analyses, and 69 of 205 (33.7%) had a double-blinded intervention; 18 of 205 (8.8%) studies reported on all 3 design characteristics. Currently, 60 critical care nutrition RCTs (18 multicenter trials) are registered on clinical trials registries. CONCLUSIONS: The future of clinical critical care nutrition research is promising, with more trials of increasing sample size being conducted. Robust trial methodology, transparent reporting, and the development of research networks will help to further advance this important field.

Direct diet quantification indicates low intakes of (n-3) fatty acids in children 4 to 8 years old.

Estimates of essential fatty acid intakes, including (n-3) PUFA, are available in pediatric populations based on limited indirect approaches. Furthermore, recommended intakes for short- and long-chain (LC) (n-3) PUFA have emerged for this population. This study provides direct quantification of fatty acid intakes in children aged 4-8 y. Identical portions of all food and natural health products consumed over 3 d were collected. Duplicate samples were analyzed for energy, macronutrients, and fatty acids, including alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) by high performance capillary GLC. The results for 41 children [25 females, 16 males; 5.8 +/- 0.2 y (mean age +/- SEM)] showed daily energy intakes of 5879 +/- 211 kJ (mean +/- SEM) and (n-3) PUFA intakes in mg/d as follows: ALA, 1161 +/- 108; EPA, 38.4 +/- 9.3; DPA, 26.3 +/- 3.9; and DHA, 54.1 +/- 11.4. Based on the Dietary Reference Intakes from the Institute of Medicine, 61% of the children met the adequate intake for ALA and 22% met the suggested adequate intake for DHA+EPA (10% of the adequate intake for ALA). These intakes were also compared with the recent Australia/New Zealand recommendations for children, where only 51% met the recommended intake for EPA+DPA+DHA. These results demonstrate a moderate shortfall in ALA intake in Canadian children and a nutrient gap for the LC (n-3) PUFA, including DHA, when comparing intakes for this population to suggested and recommended intakes.