Trends in Neosubstrate Degradation by Cereblon-Based Molecular Glues and the Development of Novel Multiparameter Optimization Scores.

Molecular glues enable the degradation of previously "undruggable" proteins via the recruitment of cereblon (CRBN) to the target. One major challenge in designing CRBN E3 ligase modulating compounds (CELMoDs) is the selectivity profile toward neosubstrates, proteins recruited by CRBN E3 ligase agents for degradation. Common neosubstrates include Aiolos, Ikaros, GSPT1, CK1α, and SALL4. Unlike achieving potency and selectivity for traditional small molecule inhibitors, reducing the degradation of these neosubstrates is complicated by the ternary nature of the complex formed between the protein, CRBN, and CELMoD. The standard guiding principles of medicinal chemistry, such as enforcing hydrogen bond formation, are less predictive of degradation efficiency and selectivity. Disclosed is an analysis of our glutarimide CELMoD library to identify interpretable chemical features correlated to selectivity profiles and general cytotoxicity. Included is a simple multiparameter optimization function using only three parameters to predict whether molecules will have undesired neosubstrate activity.

Unique characteristics of the tumor immune microenvironment in young patients with metastatic colorectal cancer.

Introduction: Metastatic colorectal cancer (mCRC) remains a common and highly morbid disease, with a recent increase in incidence in patients younger than 50 years. There is an acute need to better understand differences in tumor biology, molecular characteristics, and other age-related differences in the tumor microenvironment (TME). Methods: 111 patients undergoing curative-intent resection of colorectal liver metastases were stratified by age into those <50 years or >65 years old, and tumors were subjected to multiplex fluorescent immunohistochemistry (mfIHC) to characterize immune infiltration and cellular engagement. Results: There was no difference in infiltration or proportion of immune cells based upon age, but the younger cohort had a higher proportion of programmed death-ligand 1 (PD-L1)+ expressing antigen presenting cells (APCs) and demonstrated decreased intercellular distance and increased cellular engagement between tumor cells (TCs) and cytotoxic T lymphocytes (CTLs), and between TCs and APCs. These trends were independent of microsatellite instability in tumors. Discussion: Age-related differences in PD-L1 expression and cellular engagement in the tumor microenvironment of patients with mCRC, findings which were unrelated to microsatellite status, suggest a more active immune microenvironment in younger patients that may offer an opportunity for therapeutic intervention with immune based therapy.

Multiplex Fluorescent Immunohistochemistry for Preservation of Tumor Microenvironment Architecture and Spatial Relationship of Cells in Tumor Tissues.

The tumor microenvironment (TME), composed of immune cells, antigens, and local soluble factors, is integral to cancer development and progression. Traditional techniques such as immunohistochemistry, immunofluorescence, or flow cytometry limit the analysis of spatial data and cellular interactions within the TME, as they are restricted to colocalization of a small number of antigens or the loss of tissue architecture. Multiplex fluorescent immunohistochemistry (mfIHC) allows for detection of multiple antigens within a single tissue sample, providing a more comprehensive description of tissue composition and spatial interactions within the TME. This technique utilizes antigen retrieval, application of primary and secondary antibodies, followed by a tyramide-based chemical reaction to covalently bind a fluorophore to an epitope of interest and, eventually, stripping of the antibodies. This allows for multiple rounds of antibody application without concern for species cross-reactivity, as well as signal amplification which abrogates the autofluorescence that frequently plagues analysis of fixed tissues. As such, mfIHC can be used to quantify multiple cellular populations and their interactions, in situ, unlocking key biologic data that was previously unavailable. This chapter provides an overview of the experimental design, staining, and imaging strategies using a manual technique in formalin-fixed paraffin-embedded tissue sections.

Machine learning and statistical classification of birdsong link vocal acoustic features with phylogeny.

Birdsong is a longstanding model system for studying evolution and biodiversity. Here, we collected and analyzed high quality song recordings from seven species in the family Estrildidae. We measured the acoustic features of syllables and then used dimensionality reduction and machine learning classifiers to identify features that accurately assigned syllables to species. Species differences were captured by the first 3 principal components, corresponding to basic frequency, power distribution, and spectrotemporal features. We then identified the measured features underlying classification accuracy. We found that fundamental frequency, mean frequency, spectral flatness, and syllable duration were the most informative features for species identification. Next, we tested whether specific acoustic features of species' songs predicted phylogenetic distance. We found significant phylogenetic signal in syllable frequency features, but not in power distribution or spectrotemporal features. Results suggest that frequency features are more constrained by species' genetics than are other features, and are the best signal features for identifying species from song recordings. The absence of phylogenetic signal in power distribution and spectrotemporal features suggests that these song features are labile, reflecting learning processes and individual recognition.

Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Lesions.

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. SIGNIFICANCE: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.

Analysis of donor pancreata defines the transcriptomic signature and microenvironment of early pre-neoplastic pancreatic lesions.

The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathological analysis of the samples revealed PanIN lesions in most individuals irrespective of age. Using a combination of multiplex immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics, we provide the first ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts, and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. Statement of significance: The causes underlying the onset of pancreatic cancer remain largely unknown, hampering early detection and prevention strategies. Here, we show that PanIN are abundant in healthy individuals and present at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell intrinsic factors that restrain, or, conversely, promote, malignant progression.

The Impact of Delays in Interfaculty Transfer on Outcomes in Elderly Trauma Patients.

BACKGROUND: Delay to definitive treatment is a significant and persistent challenge to trauma systems across the United States, especially in rural communities with limited resources. We hypothesized that elderly trauma patients with delay in transfer would have increased morbidity and mortality. This study evaluates the relationship between inter-facility transfer time and outcomes in elderly trauma patients, and the validity of the 4-hour dwell time as a performance improvement benchmark. METHODS: The National Trauma Registry and Emergency Medical Services Database were queried from January 2010 to January 2018. Inclusion criteria included age ≥65, blunt mechanism, and transfer from another facility. Correlation analysis was used to evaluate the association between clinical and demographic variables and transfer time. Multicollinearity was evaluated using the variance inflation factor. RESULTS: 1535 patients were identified. This cohort was further subdivided into 4 cohorts based on dwell time: 0-1.5 hours (n = 384), ≥1.5-1.9 hours (n = 379), 1.9-<2.5 hours (n = 383), and ≥2.5 hours (n = 388). Analysis revealed that shorter dwell time was associated with male gender (P = .0039), higher ISS (injury severity score) (P < .0001), lower RTS (revised trauma score) (P < .0001), higher pre-hospital arrest (P = .0066), lower initial GCS (Glasgow Coma Scale) (P = .0012), higher mortality, longer ICU, and ventilator length of stay (P < .0001). Longer dwell times were associated with discharge from the hospital to home or skilled nursing facility as well as lower mortality (P < .0001). DISCUSSION: Longer dwell time was inversely related to outcome. More severely injured patients were rapidly transferred. This represents a mature rural trauma system. In addition, dwell time should be scrutinized as a meaningful indicator within a performance improvement program.

Where to Draw the Line for Intracranial Hypertension; Opening Pressures and Mortality.

BACKGROUND: Intracranial pressure (ICP) monitoring and treatment is a mainstay of severe TBI management but the relationship between intracranial opening pressure (OP) and outcomes has not been well established. The purpose of our study was to assess the relationship between OP and outcomes in severe TBI patients, with a focus on in-hospital mortality. METHODS: Adult blunt TBI patients with ICP monitoring between 2007 and 2017 were evaluated using sequential multivariable binary logistic modeling. Generalized additive model (GAM) was used to evaluate the relationship between OP and death. Odds ratio (OR) and 95% confidence interval (CI) were calculated for measures of strength of association and precision. RESULTS: A total of 182 patients were identified, with 61 (33.5%) having OP >20 mmHG (overall mean ± OP = 19.4 ± 17.8 mmHG). Forty-eight percent, 9% and 8% of patients were discharged to rehabilitation, skilled nursing institution, and home, respectively. Thirty-five percent died in the hospital. A linear relationship was found between OP and log-odds of mortality. OP (OR = 1.07; 95% CI = 1.04-1.11), age (OR = 1.05;95%CI = 1.02-1.07), and injury severity score (ISS) (OR = 1.06; 95% CI = 1.02-1.10) were independently associated with increased odds of death while adjusting for sex, race, and year. DISCUSSION: Elevated opening pressure is strongly predictive of death in severe TBI. Age and ISS are independent predictors of mortality regardless of OP. These results suggest that maintaining low levels of ICP should result in decreased mortality in severe TBI patients. The patient's age and ISS should be considered in the decision-making processes related to ICP utilization and management.

Direct Peritoneal Resuscitation in Trauma Patients Results in Similar Rates of Intra-Abdominal Complications.

Background: Trauma patients undergoing damage control surgery (DCS) have a propensity for complicated abdominal closures and intra-abdominal complications. Studies show that management of open abdomens with direct peritoneal resuscitation (DPR) reduces intra-abdominal complications and accelerates abdominal closure. This novel study compares intra-abdominal complication rates and the effect of DPR initiation in patients who received DPR and those who did not. Patients and Methods: A retrospective chart review was performed on 120 patients who underwent DCS. Fifty patients were identified as DCS with DPR, and matched to 70 controls by gender, race, age, body mass index (BMI), past medical history, mechanism of trauma, and injury severity score. Results: The two groups of patients, those without DPR (-DPR) and those with DPR (+DPR), were similar in their characteristics. The +DPR group was more likely to have a mesh closure than the -DPR (14% and 3%; p = 0.022). The +DPR group took longer to have a final closure (3.5 ± 2.6 days vs. 2.5 ± 1.8; p = 0.020). Infection complications and mechanical failure of the closure technique were similar among the two groups. Timing of DPR initiation had no effect on closure type but did statistically increase the number of days to closure (initiation at first operation 2.8 ± 1.8 days vs. initiation at subsequent operations 6.0 ± 3.3 days; p ≤ 0.001). Conclusions: The use of DPR did not result in different outcomes in trauma patients. Therefore, traditional resuscitative measures for DCS may not be inferior to DCS with DPR. When choosing to use DPR, initiating it at the first operation could reduce the number of days to closure.

Chemoselective Electrosynthesis Using Rapid Alternating Polarity.

Challenges in the selective manipulation of functional groups (chemoselectivity) in organic synthesis have historically been overcome either by using reagents/catalysts that tunably interact with a substrate or through modification to shield undesired sites of reactivity (protecting groups). Although electrochemistry offers precise redox control to achieve unique chemoselectivity, this approach often becomes challenging in the presence of multiple redox-active functionalities. Historically, electrosynthesis has been performed almost solely by using direct current (DC). In contrast, applying alternating current (AC) has been known to change reaction outcomes considerably on an analytical scale but has rarely been strategically exploited for use in complex preparative organic synthesis. Here we show how a square waveform employed to deliver electric current-rapid alternating polarity (rAP)-enables control over reaction outcomes in the chemoselective reduction of carbonyl compounds, one of the most widely used reaction manifolds. The reactivity observed cannot be recapitulated using DC electrolysis or chemical reagents. The synthetic value brought by this new method for controlling chemoselectivity is vividly demonstrated in the context of classical reactivity problems such as chiral auxiliary removal and cutting-edge medicinal chemistry topics such as the synthesis of PROTACs.

Chemoselective, Scalable Nickel-Electrocatalytic O-Arylation of Alcohols.

The formation of aryl-alkyl ether bonds through cross coupling of alcohols with aryl halides represents a useful strategic departure from classical SN 2 methods. Numerous tactics relying on Pd-, Cu-, and Ni-based catalytic systems have emerged over the past several years. Herein we disclose a Ni-catalyzed electrochemically driven protocol to achieve this useful transformation with a broad substrate scope in an operationally simple way. This electrochemical method does not require strong base, exogenous expensive transition metal catalysts (e.g., Ir, Ru), and can easily be scaled up in either a batch or flow setting. Interestingly, e-etherification exhibits an enhanced substrate scope over the mechanistically related photochemical variant as it tolerates tertiary amine functional groups in the alcohol nucleophile.

Intermolecular Crossed [2 + 2] Cycloaddition Promoted by Visible-Light Triplet Photosensitization: Expedient Access to Polysubstituted 2-Oxaspiro[3.3]heptanes.

This paper describes an intermolecular cross-selective [2 + 2] photocycloaddition reaction of exocyclic arylidene oxetanes, azetidines, and cyclobutanes with simple electron-deficient alkenes. The reaction takes place under mild conditions using a commercially available Ir(III) photosensitizer upon blue light irradiation. This transformation provides access to a range of polysubstituted 2-oxaspiro[3.3]heptane, 2-azaspiro[3.3]heptane, and spiro[3.3]heptane motifs, which are of prime interest in medicinal chemistry as gem-dimethyl and carbonyl bioisosteres. A variety of further transformations of the initial cycloadducts are demonstrated to highlight the versatility of the products and enable selective access to either of a syn- or an anti-diastereoisomer through kinetic or thermodynamic epimerization, respectively. Mechanistic experiments and DFT calculations suggest that this reaction proceeds through a sensitized energy transfer pathway.

Dynamics of perineuronal nets over amphibian metamorphosis.

Extracellular matrix materials known as perineuronal nets (PNNs) have been shown to have remarkable consequences for the maturation of neural circuits and stabilization of behavior. It has been proposed that, due to the possibly long-lived biochemical nature of their components, PNNs may be an important substrate by which long-term memories are stored in the central nervous system. However, little empirical evidence exists that shows that PNNs are themselves stable once established. Thus, the question of their temporal dynamics remains unresolved. We leverage the dramatic morphological and behavioral transformations that occur during amphibian metamorphosis to show that PNNs can be highly dynamic in nature. We used established lectin histochemistry to show that PNNs undergo drastic reconstruction during the metamorphic transition. Pre-metamorphic tadpoles have abundant lectin-labeled pericellular material, which we interpret to be PNNs, surrounding neurons throughout the central nervous system. During the metamorphic transition, these structures degrade, and begin to reform in the months following metamorphosis. We show that PNN sizes and staining intensity further change over metamorphosis, suggesting compositional rearrangement. We found PNNs in brain regions with putative homology to regions in mammals with known PNN function, and in other shared regions where PNN function is unknown. Our results suggest that PNNs are susceptible to remodeling by endogenous mechanisms during development. Interpreting the roles of PNNs in circuit maturation and stability requires understanding their temporal relationship with the neurons and synapses they surround. Our work provides further impetus to investigate this relationship in tandem with developmental and behavioral studies.

Assessing Risk for Mortality in Trauma: The Value of Admission Hemoglobin A1c.

BACKGROUND: Recent efforts have been made to identify admission characteristics of trauma patients that are associated with increased risk of mortality. Contemporary literature has established an increased risk of mortality with admission hyperglycemia. However, the effects of longstanding hyperglycemia, as surrogated by hemoglobin A1c (HbA1c), has not been studied. METHODS: A prospective trauma database was retrospectively reviewed identifying patients with collected HbA1c at admission. Three cohorts were defined by HbA1c: normal (N), <5.7; prediabetic (PD) 5.7-6.5; and diabetic (D) >6.5. Regression models were used to evaluate the risk of increased hospital and intensive care unit (ICU) length of stay (LOS), ventilator days, and mortality. Relative risk (RR) and 95% CI are provided as measures of significance. RESULTS: A total of 2978 patients were included in the analysis (N: n = 1895, PD: n = 744, and D: n = 339). The D cohort was more likely to be older, female, obese, suffered blunt trauma, and triaged at the highest activation acuity level (P < .0001). Mean injury severity score (ISS) was similar between groups. The D group was more likely to have longer ICU-LOS (RR 1.5; 95% CI 1.10-2.07) and ventilator days (RR 1.52; 95% CI 1.03-2.26) than the N group. Relative to the N group, the risk of mortality was 50% higher in the PD (RR 1.49; 95% CI 1.17-1.90) and in the D cohorts (RR 1.50; 95% CI 1.03-2.18). DISCUSSION: Trauma patients with an elevated admission HbA1c have a significantly higher risk of mortality regardless of their history of diabetes. These data add to the body of literature that documents the untoward effect of hyperglycemia on the trauma patient.

Auditory Selectivity for Spectral Contrast in Cortical Neurons and Behavior.

Vocal communication relies on the ability of listeners to identify, process, and respond to vocal sounds produced by others in complex environments. To accurately recognize these signals, animals' auditory systems must robustly represent acoustic features that distinguish vocal sounds from other environmental sounds. Vocalizations typically have spectral structure; power regularly fluctuates along the frequency axis, creating spectral contrast. Spectral contrast is closely related to harmonicity, which refers to spectral power peaks occurring at integer multiples of a fundamental frequency. Although both spectral contrast and harmonicity typify natural sounds, they may differ in salience for communication behavior and engage distinct neural mechanisms. Therefore, it is important to understand which of these properties of vocal sounds underlie the neural processing and perception of vocalizations.Here, we test the importance of vocalization-typical spectral features in behavioral recognition and neural processing of vocal sounds, using male zebra finches. We show that behavioral responses to natural and synthesized vocalizations rely on the presence of discrete frequency components, but not on harmonic ratios between frequencies. We identify a specific population of neurons in primary auditory cortex that are sensitive to the spectral resolution of vocal sounds. We find that behavioral and neural response selectivity is explained by sensitivity to spectral contrast rather than harmonicity. This selectivity emerges within the cortex; it is absent in the thalamorecipient region and present in the deep output region. Further, deep-region neurons that are contrast-sensitive show distinct temporal responses and selectivity for modulation density compared with unselective neurons.SIGNIFICANCE STATEMENT Auditory coding and perception are critical for vocal communication. Auditory neurons must encode acoustic features that distinguish vocalizations from other sounds in the environment and generate percepts that direct behavior. The acoustic features that drive neural and behavioral selectivity for vocal sounds are unknown, however. Here, we show that vocal response behavior scales with stimulus spectral contrast but not with harmonicity, in songbirds. We identify a distinct population of auditory cortex neurons in which response selectivity parallels behavioral selectivity. This neural response selectivity is explained by sensitivity to spectral contrast rather than to harmonicity. Our findings inform the understanding of how the auditory system encodes socially-relevant signals via detection of an acoustic feature that is ubiquitous in vocalizations.

Impact of Stereo- and Regiochemistry on Energetic Materials.

The synthesis, physical properties, and calculated performances of six stereo- and regioisomeric cyclobutane nitric ester materials are described. While the calculated performances of these isomers, as expected, were similar, their physical properties were found to be extremely different. By alteration of the stereo- and regiochemistry, complete tunability in the form of low- or high-melting solids, stand-alone melt-castable explosives, melt-castable explosive eutectic compounds, and liquid propellant materials was obtained. This demonstrates that theoretical calculations should not be the main factor in driving the design of new materials and that stereo- and regiochemistry matter in the design of compounds of potential relevance to energetic formulators.

A Radical Approach to Anionic Chemistry: Synthesis of Ketones, Alcohols, and Amines.

Historically accessed through two-electron, anionic chemistry, ketones, alcohols, and amines are of foundational importance to the practice of organic synthesis. After placing this work in proper historical context, this Article reports the development, full scope, and a mechanistic picture for a strikingly different way of forging such functional groups. Thus, carboxylic acids, once converted to redox-active esters (RAEs), can be utilized as formally nucleophilic coupling partners with other carboxylic derivatives (to produce ketones), imines (to produce benzylic amines), or aldehydes (to produce alcohols). The reactions are uniformly mild, operationally simple, and, in the case of ketone synthesis, broad in scope (including several applications to the simplification of synthetic problems and to parallel synthesis). Finally, an extensive mechanistic study of the ketone synthesis is performed to trace the elementary steps of the catalytic cycle and provide the end-user with a clear and understandable rationale for the selectivity, role of additives, and underlying driving forces involved.