Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.

Top 10 Tips on Safety From the Air Medical Transport Industry.

The air medical transport industry places a high value on developing and maintaining a culture of safety due to the higher risk nature of its operations. The dynamic nature of response and transport, inherent risks involved with flight, lack of supporting resources, weather conditions, and austere nature of the transport environment are all factors that highlight the need for enhanced safety. As such, the air medical transport industry has developed a robust and unique approach to provider and patient safety involving many tactics not otherwise used in other areas of health care. This article describes some of the unique safety features and approaches that are commonplace in the air medical transport industry and proposes a means for these initiatives to other areas of the health care system.

Short-Term Outcomes of Expedited Arthroscopic Tensionable Knotless Biologic Tuberoplasty for Massive Irreparable Rotator Cuff Tears.

Background: Massive irreparable rotator cuff tears in the nonarthritic patient are challenging because of the high failure rate and technical difficulty of intraoperative repair. We examined the outcomes of expedited arthroscopic tensionable knotless biologic tuberoplasty for massive irreparable rotator cuff tears. Methods: Eleven patients with an average follow-up of 8.2 months were included in the analysis. Patient-reported outcome measures were the visual analog scale (VAS) pain score, American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES) score, Single Assessment Numeric Evaluation (SANE) score, and Veterans RAND 12-Item Health Survey (VR-12) physical component score and mental component score. Results: In comparison to the preoperative mean, mean VAS pain scores were significantly reduced at 2 weeks, 6 weeks, 3 months, 6 months, and 1 year. The mean VAS pain scores decreased from 6.9 ± 1.3 preoperatively to 0.2 ± 0.4 at 1 year (P<0.001). Mean ASES scores and SANE scores were both significantly improved at 3 months, 6 months, and 1 year. Mean ASES scores increased from 40.3 ± 17 preoperatively to 93.0 ± 5.5 at 1 year (P=0.001), and mean SANE scores increased from 40.7 ± 23.7 preoperatively to 85.6 ± 8.9 at 1 year (P=0.007). The mean VR-12 physical component score was significantly improved at 6 months and 1 year postoperatively. The mean VR-12 mental component score was clinically improved at 6 months and 1 year postoperatively. Conclusion: Arthroscopic tensionable knotless biologic tuberoplasty is an effective treatment for massive irreparable rotator cuff tears and resulted in statistically significant improvements in VAS pain, ASES, SANE, and the VR-12 physical component scores and clinically significant improvements in the VR-12 mental component score in our patient cohort.

Urinary neopterin and total neopterin measurements allow monitoring of oxidative stress and inflammation levels of knee and hip arthroplasty patients.

Knee and hip arthroplasty are common surgeries within an aging population. Some data has suggested that knee arthroplasty is more traumatic to the body than hip arthroplasty due to the increased complexity and load bearing nature of the joint. Here, we compare the stress of the two surgeries by measuring urinary neopterin and total neopterin as biomarkers of oxidative stress and inflammation. Urinary neopterin and total neopterin (neopterin + 7,8-dihydroneopterin) levels were analysed in 28 knee and 22 hip arthroplasty patients pre- and post-operatively to determine oxidative stress and inflammation levels. Total neopterin was 31.1% higher with knee arthroplasty (p<0.05). Urinary neopterin was 32.8% higher in the knee arthroplasty group versus hips. The increase in neopterin and total neopterin following a post-surgical decrease in levels was significant in both groups. Levels of neopterin and total neopterin were varied between patients, but all increased following surgery and subsided by day 28. The increased levels of urinary neopterin and total neopterin from knee arthroplasty indicate that knee osteoarthritis and arthroplasty is a more significant trauma to the body than hip osteoarthritis and arthroplasty surgery. This is also shown by faster inflammatory resolution following hip arthroplasty.

Neopterin formation through radical scavenging of superoxide by the macrophage synthesised antioxidant 7,8-dihydroneopterin.

Clinical measurement of neopterin has been extensively used as a marker of inflammation but the in vivo mechanism generating neopterin is poorly understood. Neopterin is described as the oxidation product of 7,8-dihydroneopterin, a potent antioxidant generated by monocyte/macrophages in response to interferon-γ. While peroxyl and hydroxyl scavenging generates dihydroxanthopterin, hypochlorite efficiently oxidises 7,8-dihydroneopterin into neopterin, but this reaction alone does not explain the high levels of neopterin seen in clinical data. Here, we examine whether superoxide scavenging by 7,8-dihydroneopterin generates neopterin. U937 cells incubated with oxLDL showed a time dependent increase superoxide and 7,8-dihydroneopterin oxidation to neopterin. Neopterin generation in oxLDL or phorbol ester treated U937 cells or human monocytes was inhibited by apocynin and PEG-SOD. Addition of the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide (ABAH) had no effect of the superoxide generation or neopterin formation. 7,8-Dihydroneopterin reacted with superoxide/hydroxy radical mixtures generated by X-ray radiolysis to give neopterin. Formation of neopterin by superoxide derived from the xanthine/xanthine oxidase system was inhibited by superoxide dismutase. Neopterin formation was inhibited by apocynin in phorbol ester treated human carotid plaque rings in tissue culture. These results indicate that 7,8-dihydroneopterin scavenges superoxide and is subsequently oxidised into neopterin in cellular and cell-free experimental systems.

Pediatric Orthopedic Trauma.

The management of pediatric orthopedic trauma continues to evolve rapidly. Whereas the strong healing potential of pediatric patients often allows for the nonoperative treatment of most conditions, many injuries require urgent operative treatment to ensure that patients may return to all activities without disability. Some injuries may require additional follow-up and interventions, as complications such as growth arrests or deformity may occur. This article summarizes the most common fractures and orthopedic injuries of the pediatric patient. The keys to diagnosis, acute management, nonoperative and operative treatments, and complications are discussed. The detection and management of nonaccidental trauma are also examined.

Pterins as Diagnostic Markers of Mechanical and Impact-Induced Trauma: A Systematic Review.

We performed a systematic review of the literature to evaluate pterins as biomarkers of mechanical and impact-induced trauma. MEDLINE and Scopus were searched in March 2019. We included in vivo human studies that measured a pterin in response to mechanical or impact-induced trauma with no underlying prior disease or complication. We included 40 studies with a total of 3829 subjects. Seventy-seven percent of studies measured a significant increase in a pterin, primarily neopterin or total neopterin (neopterin + 7,8-dihydroneopterin). Fifty-one percent of studies measured an increase within 24 h or trauma, while 46% measured increases beyond 48 h. Pterins also showed promise as predictors of post-trauma complications such as sepsis, multi-organ failure and mortality. Exercise-induced trauma and traumatic brain injury caused an immediate increase in neopterin or total neopterin, while patients of multiple trauma had elevated pterin levels that remained above baseline for several days. Pterin concentration changes in response to surgery were variable with patients undergoing cardiac surgery having immediate and sustained pterin increases, while hysterectomy, liver resection or hysterectomy showed no change. This review provides systematic evidence that pterins, in particular neopterin and total neopterin, increase in response to multiple forms of mechanical or impact-induced trauma.

Ethical issues in patients referred for palliative radiation therapy.

BACKGROUND: As patients with advanced cancer approach end of life, ethical issues may arise. We describe ethical issues encountered by radiation oncologists in this setting. METHODS: A prospective, survey-based study assessed 162 consecutive consults for palliative radiation therapy (PRT) over 4 months at 3 hospitals. Consulting radiation clinicians completed a survey assessing palliative care issues encountered, based on national guidelines. Ethical issues included 5 subthemes (conflict between clinicians, caregiver-clinician conflict, internal conflict, feeling unable to do what was best for the patient, and violation of personal morals), an option for unclassified issues, and open-ended responses. Multivariate analyses (MVA) assessed potential patient-related predictors of ethical issues: gender, performance status (PS), PRT indication, physical symptoms, and presence of psychosocial, goals of care, care coordination, cultural, or spiritual issues. RESULTS: Of 162 surveys, 140 were completed (response rate =86%). Overall, 14 (10%) surveys identified ethical issues relevant to care; 11 of 14 (78%) identified more than 1 ethical issue. Half (7; 50%) involved conflict between clinicians and clinician-caregiver conflict; 6 (43%) involved clinician distress or internal conflict; and 2 (14%) felt impeded from doing what they felt was best for the patient. Open-ended responses revealed differences in opinion between medical specialties (n=6, 43%), and conflict related to coordination of care among clinicians (n=3, 21%). On UVA, ethical issues were associated with PRT referrals for bleeding, dyspnea, or dysphagia due to visceral metastases (30%) versus CNS indications such as brain metastases or cord compression (7%) or for bony metastases (4%) P<0.001. On MVA, ethical issues were associated with PRT for visceral metastases (OR 13.0; 95% CI, 2.3-74.6; P<0.001) and presence of spiritual issues (OR 4.0; 95% CI, 1.1-14.5; P=0.04). CONCLUSIONS: At least 1 in 10 referrals for PRT involve ethical issues. Further studies are warranted to assess the ability of radiation oncologists to manage ethical issues.

The glutathione cycle shapes synaptic glutamate activity.

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.

Knee replacement surgery significantly elevates the urinary inflammatory biomarkers neopterin and 7,8-dihydroneopterin.

CONTEXT: Knee arthroplasty surgery is significant trauma, leading to an activated immune system causing inflammation and oxidative stress. Many current biomarkers are invasive, costly, and often slow to analyse, limiting their use for rapid inflammatory measurements. OBJECTIVES: We have examined the use of urinary neopterin and total neopterin in knee arthroplasty patients to non-invasively measure oxidative stress and inflammation from immune system activation. We aim to validate the use of these biomarkers for quick, cost effective and predictive measurements of post-surgical inflammation assessment. METHODOLOGY: 19 Knee arthroplasty patients were analysed pre-operatively and for a defined post-operative period to determine the urinary levels of neopterin and total neopterin (neopterin +7,8-dihydroneopterin) using high performance liquid chromatography with fluorescence detection. These results were then compared to a control group of 20 participants with normal knee function. RESULTS: 7,8-Dihydroneopterin was stable in urine over 12 h when refrigerated. Knee arthritis was associated with an increase in pre-operative neopterin (oxidative stress) and total neopterin (inflammation). The subsequent arthroplasty surgery generated a significant increase neopterin and total neopterin. Both biomarkers were reduced immediately post-operatively, before becoming elevated on the following days. There was no clear evidence of an association between initial neopterin and total neopterin levels and a patient's level of inflammation during in-hospital recovery. CONCLUSIONS: The stability of 7,8-dihydroneopterin in urine allows for its use as an inflammatory marker. Urinary neopterin and total neopterin provided a fast, non-invasive, and simple measure of oxidative stress and inflammation after knee arthroplasty.

Neopterin, Inflammation, and Oxidative Stress: What Could We Be Missing?

Neopterin has been extensively used as a clinical marker of immune activation during inflammation in a wide range of conditions and stresses. However, the analysis of neopterin alone neglects the cellular reactions that generate it in response to interferon-γ. Neopterin is the oxidation product of 7,8-dihydroneopterin, which is a potent antioxidant generated by interferon-γ-activated macrophages. 7,8-Dihydroneopterin can protect macrophage cells from a range of oxidants through a scavenging reaction that generates either neopterin or dihydroxanthopterin, depending on the oxidant. Therefore, plasma and urinary neopterin levels are dependent on both macrophage activation to generate 7,8-dihydroneopterin and subsequent oxidation to neopterin. This relationship is clearly shown in studies of exercise and impact-induced injury during intense contact sport. Here, we argue that neopterin and total neopterin, which is the combined value of 7,8-dihydroneopterin and neopterin, could provide a more comprehensive analysis of clinical inflammation than neopterin alone.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Supply and demand: How does variation in atmospheric oxygen during development affect insect tracheal and mitochondrial networks?

Atmospheric oxygen is one of the most important atmospheric component for all terrestrial organisms. Variation in atmospheric oxygen has wide ranging effects on animal physiology, development, and evolution. This variation in oxygen has the potential to affect both respiratory systems (the supply side) and mitochondrial networks (the demand side) in animals. Insect respiratory systems supplying oxygen to tissues in the gas phase through blind ended tracheal systems are particularly susceptible to this variation. While the large conducting tracheae have previously been shown to respond developmentally to changes in rearing oxygen, the effect of oxygen on the tracheolar network has been relatively unexplored, especially in adult insects. Similarly, mitochondrial networks that meet energy demand in insects and other animals are dynamic and their enzyme activities have been shown to vary in the presence of oxygen. These two systems together should be under selective pressure to meet the aerobic metabolic requirements of insects. To test this hypothesis, we reared Mito-YFP Drosophila under three different oxygen concentrations hypoxia (12%), normoxia (21%), and hyperoxia (31%) and imaged their tracheolar and mitochondrial networks within their flight muscle using confocal microscopy. In terms of oxygen supply, hypoxia increased mean (mid-length) tracheolar diameters, tracheolar tip diameters, the number of tracheoles per main branch and affected tracheal branching patterns, while the opposite was observed in hyperoxia. In terms of oxygen demand, hypoxia increased mitochondrial investment and mitochondrial to tracheolar volume ratios; while the opposite was observed in hyperoxia. Generally, hypoxia had a stronger effect on both systems than hyperoxia. These results show that insects are capable of developmentally changing investment in both their supply and demand networks to increase overall fitness.

Burden of palliative care issues encountered by radiation oncologists caring for patients with advanced cancer.

PURPOSE: Radiation oncologists frequently provide care for patients with advanced cancer who are in their last months or weeks of life. This study examined the previously not well-characterized types and frequencies of palliative care issues encountered in consultations for palliative radiation therapy (PRT). METHODS AND MATERIALS: This prospective, survey-based study assessed consecutive consults for PRT from May 19, 2014, to September 26, 2014 at 3 Boston-area community and academic, hospital-based centers. Participating physicians and nurse practitioners completed a survey to identify and rank the relevance (5-point Likert scale, not at all to extremely) of palliative care issues. Eight domains adapted from national palliative care guidelines (physical symptoms, psychosocial issues, cultural considerations, spiritual needs, care coordination, advance care planning, goals of care, and ethical and legal issues) were evaluated. A total of 162 consecutive consultations were surveyed with 140 responses received (86% response rate). RESULTS: Most (82%) consults had 2 or more palliative care domains ranked as highly (very or extremely) relevant to care. The domains of physical symptoms (91%), care coordination (70%), goals of care (59%), and psychosocial issues (52%) were the most commonly reported domains as highly relevant to care. Forty-six percent of consults involved a high palliative care burden (4 or more palliative care domains identified as highly relevant to care). Predictors of high palliative care burden in multivariable analysis were Eastern Cooperative Oncology Group performance status >2 (odds ratio, 3.57; P = .047), a plan for no further anticancer therapy after PRT (odds ratio, 3.46; P = .03), and a recommendation against PRT (odds ratio, 4.80; P = .01). CONCLUSIONS: Radiation oncology clinicians encounter multiple palliative care issues when consulting on patients for PRT. Clinicians identified physical symptoms, care coordination, and goals of care as the most relevant palliative care domains. These findings can help guide palliative care development within radiation oncology, including education and structures of care delivery.

Mapped Grid Methods Applied to the Slow Variable Discretization--Enhanced Renormalized Numerov Approach.

We introduce a hyperspherical coordinate mapping procedure to the slow variable discretization-enhanced renormalized Numerov method that allows for more accurate and cost-effective calculations of cold and ultracold atom-dimer scattering. The mapping procedure allows optimization of the numerical grid point spacing by adjusting to the shape of the interaction potential. We show results for elastic scattering in HeH2 and compare the results to previous MOLSCAT calculations by Forrey et al. [ Phys. Rev. A 1999, 59, 2146 ].

Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs.

Lung disease is a major cause of death in the United States, with current therapeutic approaches serving only to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSCs) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system, which significantly improved the efficiency of this correction. The corrected iPSCs were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches.

State-to-state three-atom time-dependent reactive scattering in hyperspherical coordinates.

We present a time-dependent, hyperspherical wave packet method for calculating three-atom state-to-state S-matrix elements. The wave packet is propagated in time using adiabatically adjusting, principal axes hyperspherical coordinates that treat all arrangement channels equivalently, allowing the simultaneous analysis of the products in all three arrangement channels. We take advantage of the symmetry of the potential energy surface and decompose the initial wave packet into its component irreducible representations, propagating each component separately. Each irreducible representation component of the wave packet is analyzed by projecting it onto the hyperspherical basis at a fixed, asymptotic hyperradius, and irreducible representation dependent S-matrix elements are obtained by matching the hyperspherical projections to symmetry-adapted Jacobi coordinate boundary conditions. We obtain arrangement channel-dependent S-matrix elements as linear combinations of the irreducible representation dependent elements. State-to-state H + H(2) and F + H(2) results for zero total angular momentum are presented.

Geometric phase for collinear conical intersections. I. Geometric phase angle and vector potentials.

We present a method for properly treating collinear conical intersections in triatomic systems. The general vector potential (gauge theory) approach for including the geometric phase effects associated with collinear conical intersections in hyperspherical coordinates is presented. The current study develops an introductory method in the treatment of collinear conical intersections by using the phase angle method. The geometric phase angle, η, in terms of purely internal coordinates is derived using the example of a spin-aligned quartet lithium triatomic system. A numerical fit and thus an analytical form for the associated vector potentials are explicitly derived for this triatomic A(3) system. The application of this methodology to AB(2) and ABC systems is also discussed.