Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Australia's continental-scale acoustic tracking database and its automated quality control process.

Our ability to predict species responses to environmental changes relies on accurate records of animal movement patterns. Continental-scale acoustic telemetry networks are increasingly being established worldwide, producing large volumes of information-rich geospatial data. During the last decade, the Integrated Marine Observing System's Animal Tracking Facility (IMOS ATF) established a permanent array of acoustic receivers around Australia. Simultaneously, IMOS developed a centralised national database to foster collaborative research across the user community and quantify individual behaviour across a broad range of taxa. Here we present the database and quality control procedures developed to collate 49.6 million valid detections from 1891 receiving stations. This dataset consists of detections for 3,777 tags deployed on 117 marine species, with distances travelled ranging from a few to thousands of kilometres. Connectivity between regions was only made possible by the joint contribution of IMOS infrastructure and researcher-funded receivers. This dataset constitutes a valuable resource facilitating meta-analysis of animal movement, distributions, and habitat use, and is important for relating species distribution shifts with environmental covariates.

Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity.

Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compounds for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized molecules. In this manuscript we assess the ability of several experimental and computational approaches to predict GSH t1/2 for a range of cysteine targeting warheads, including a novel method based on pKa. Furthermore, matched molecular pairs analysis has been performed against our internal compound collection, revealing structure-activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.

Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors.

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Humeral elevation reduces the dynamic control ratio of the shoulder muscles during internal rotation.

OBJECTIVES: To determine the differences in the dynamic control ratio of the glenohumeral joint rotators, during internal rotation at 20° and 60° of humeral elevation in the scapular plan. Dynamic control ratio (DCR) is defined as the ratio between eccentric action of the lateral rotators and the concentric action of the medial rotators. DESIGN: A cross-sectional laboratory study. METHODS: Thirty asymptomatic participants (men n=14, women n=16, mean age=29.4±8.9years, BMI: 24.1±5.4) were tested. Peak torque generated by the concentric action of the MR and the eccentric action of the LR of the shoulder joint and the DCR were evaluated on the dominant arm using an isokinetic dynamometer at 20° and 60° of humeral elevation at a speed of 20°/s. RESULTS: There was a significant decrease in the DCR at 60° humeral elevation when compared to 20° humeral elevation (p<0.05). This decrease was due to the significant decrease in eccentric peak torques at 60° humeral elevation when compared to 20° (p<0.05). However, there was no significant difference in the concentric peak torques between 20° and 60° (p>0.05). CONCLUSIONS: The significant decrease in the DCR as a consequence of a decrease in the eccentric peak torque of the LR when the humerus is in a more elevated position suggests that the introduction of humeral elevation can be used as a progression for improving the eccentric action of the shoulder LR and subsequently the dynamic control of the shoulder.

Combining photochemistry and catalysis: rapid access to sp3 - rich polyheterocycles from simple pyrroles.

Use of FEP flow reactor technology allows access to gram quantities of photochemically-generated tricyclic aziridines. These undergo a range of novel palladium-catalyzed ring-opening and cycloaddition reactions, likely driven by their inherent strain, allowing incorporation of further functionality by fusing additional heterocyclic rings onto these already complex polycyclic cores. This rapid, 2-step access to complex sp3 - rich heterocycles should be of interest to those in the fields of drug discovery and natural product synthesis.

Shared Medical Appointments: A Portal for Nutrition and Culinary Education in Primary Care-A Pilot Feasibility Project.

INTRODUCTION: Diseases linked to obesity such as cardiovascular disease, diabetes, degenerative joint disease, gastroesophageal reflux, and sleep apnea constitute a large portion of primary care visits. Patients with these conditions often lack knowledge, skills, and support needed to maintain health. Shared medical appointments (SMAs) that include culinary skills and nutrition education offer a novel, cost-effective way to address these diseases in primary care. METHODS: Adult patients in a primary care practice at a large academic hospital in Boston, Massachusetts, who had at least 1 cardiovascular risk factor were invited to participate in SMAs that included cooking demonstrations and teaching about nutrition in addition to medical management of their conditions. Sessions were conducted by a physician and an assistant in a conference room of a traditional primary care practice as part of a pilot feasibility project. RESULTS: Seventy patients, contributing a total of 156 patient visits, attended 17 nutrition-focused SMAs over a 4-year period. Patients were surveyed after each visit and indicated that they enjoyed the SMAs, would consider alternating SMAs with traditional one-on-one visits, and would recommend SMAs to others. Half would pay out of pocket or a higher copay to attend SMAs. Financially, the practice broke even compared with traditional one-onone office visits. CONCLUSION: In this feasibility study, chronic disease SMAs conducted with a culinary/nutrition focus were feasible, cost-effective, and well received by patients. Follow-up studies are needed to evaluate short- and long-term outcomes of this SMA model on obesity-related diseases.

Nutrition Education in an Era of Global Obesity and Diabetes: Thinking Outside the Box.

In an era when rates of obesity, diabetes, and other lifestyle-related diseases challenge medical educators and governments worldwide, it is necessary to consider novel educational strategies, both didactic and experiential, whereby current and future health professionals can be better prepared to proactively advise and teach patients enhanced self-care skills (e.g., diet, movement, stress management, and enhanced behavioral change).In this Perspective, the authors summarize current circumstances involving rising rates of obesity and diabetes worldwide, the lack of nutrition- and lifestyle-related curricular requirements for professional medical certification, societal trends regarding modern food culture and food availability in health care settings, and the misalignment of financial incentives to promote health.The authors assess what elements of self-care should or should not be required within future curricula and certification exams. They consider how best to educate trainees about diet and how to "translate" nutrition, exercise, and behavioral science knowledge into practical advice. They explore several ideas for reforming nutrition education, including "teaching kitchens" as required laboratory classes for nutrition and lifestyle instruction, wearable technologies for tracking behaviors and physiological data relating to lifestyle choices, and the prospect of hospitals and other medical venues serving as exemplars of healthy, delicious food options. Finally, the authors argue that "salutogenesis"-the study of the creation and maintenance of health and well-being-should assume its rightful position alongside the study of "pathogenesis"-disease diagnosis and treatment-in medical education and practice.

Physiological trajectory of patients pre and post ICU discharge.

The intensive care unit (ICU) admits the most severely ill patients, and the goal of the unit can be interpreted as stabilizing patient physiology. Once these patients are discharged from the ICU to a step-down ward, they continue to have their vital signs monitored by nursing staff. Early detection of physiological deterioration has been highlighted as a key step to reduce ICU readmission and improve patient outcomes. Vital signs were collected for a dataset of 98 patients admitted to an ICU and who survived to hospital discharge after their stay on a step-down ward. A model of physiological normality was developed using data from the day of hospital discharge, and patients were retrospectively evaluated throughout their stay using this model. We show that the physiology of patients being cared for in the ICU improves very rapidly in the three days prior to discharge, and furthermore, that this recovery continues during their stay on the ward, albeit at a slower rate.

Novel and versatile synthesis of disubstituted 1,2-dihydro-1,2,4-triazol-3-ones.

A novel method for the synthesis of a wide range of 1,5-disubstituted 1,2-dihydro-1,2,4-triazol-3-ones is described. The key step involves a reaction between a dilithiated BOC-hydrazine and a N-alkoxycarbonylcarboximidothioate. A broad range of aryl and alkyl functional groups are tolerated, providing a versatile route for the synthesis of triazolones.

Parkinsonian tremor identification with multiple local field potential feature classification.

This paper explores the development of multi-feature classification techniques used to identify tremor-related characteristics in the Parkinsonian patient. Local field potentials were recorded from the subthalamic nucleus and the globus pallidus internus of eight Parkinsonian patients through the implanted electrodes of a Deep brain stimulation (DBS) device prior to device internalization. A range of signal processing techniques were evaluated with respect to their tremor detection capability and used as inputs in a multi-feature neural network classifier to identify the activity of Parkinsonian tremor. The results of this study show that a trained multi-feature neural network is able, under certain conditions, to achieve excellent detection accuracy on patients unseen during training. Overall the tremor detection accuracy was mixed, although an accuracy of over 86% was achieved in four out of the eight patients.

Identifying tremor-related characteristics of basal ganglia nuclei during movement in the Parkinsonian patient.

Local field potential (LFP) and Electromyographic (EMG) signals were recorded from 12 Parkinsonian patients with tremor-dominant symptoms as they performed passive and voluntary movements. The LFP signals were categorised into episodes of tremorous and atremorous activity (identified through EMG power spectra), then divided into delta (2-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), and beta (13-30 Hz) frequency bands. Modulation of LFP oscillatory activity in these frequency bands were compared between the subthalamic nucleus (STN) and the globus pallidus internus (GPi) to determine if differential tremor-related characteristics were identifiable for either target. Our results suggest that such local characteristic activity is identifiable in the STN, and thus could be a target for initial development of a closed-loop demand driven stimulator device which capitalises on such activity to trigger stimulation, even during voluntary movement activity.

Prediction of Parkinson's disease tremor onset using a radial basis function neural network based on particle swarm optimization.

Deep Brain Stimulation (DBS) has been successfully used throughout the world for the treatment of Parkinson's disease symptoms. To control abnormal spontaneous electrical activity in target brain areas DBS utilizes a continuous stimulation signal. This continuous power draw means that its implanted battery power source needs to be replaced every 18-24 months. To prolong the life span of the battery, a technique to accurately recognize and predict the onset of the Parkinson's disease tremors in human subjects and thus implement an on-demand stimulator is discussed here. The approach is to use a radial basis function neural network (RBFNN) based on particle swarm optimization (PSO) and principal component analysis (PCA) with Local Field Potential (LFP) data recorded via the stimulation electrodes to predict activity related to tremor onset. To test this approach, LFPs from the subthalamic nucleus (STN) obtained through deep brain electrodes implanted in a Parkinson patient are used to train the network. To validate the network's performance, electromyographic (EMG) signals from the patient's forearm are recorded in parallel with the LFPs to accurately determine occurrences of tremor, and these are compared to the performance of the network. It has been found that detection accuracies of up to 89% are possible. Performance comparisons have also been made between a conventional RBFNN and an RBFNN based on PSO which show a marginal decrease in performance but with notable reduction in computational overhead.