Machine Learning Models Identify Inhibitors of New Delhi Metallo-ß-lactamase.

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

Selective esterase-ester pair for targeting small molecules with cellular specificity.

Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations.

Evaluating the Impact of an Evidence-Based Practice Education Program in a Nurse Residency Program on Evidence-Based Practice Beliefs, Implementation, and Competency.

This quality improvement project examined implementation of the John Hopkins Nursing evidence-based practice (EBP) model, a standardized EBP education curriculum, in a hospital-based nurse residency program. We found that EBP education increased nurse residents' EBP beliefs, implementation frequency, and competencies. Our findings suggest that adopting existing EBP curricula is a convenient and effective approach to EBP education. Staff development professionals should continue to support and advocate for the adoption of EBP education within their organizations.

Resources and interventions to support psychological health and wellbeing in the pharmacy workforce: Analysis and use of a health worker 'burnout' toolkit.

Background: Pharmacists have experienced declines in psychological health and wellbeing post-pandemic. The phenomena of moral distress, disengagement and burnout are associated with workforce attrition, unfitness to practice and inferior quality of patient care. A working group of the Canadian Pharmacists Association (CPhA) was formed to identify resources and interventions (R&I) for occupational psychological health and wellbeing. Objective: To characterize R&I from an evidence-based national health worker 'burnout' Toolkit with potential to support the pharmacy workforce. Methods: All R&I included within a draft 'burnout' Toolkit from the Canadian Health Workforce Network (CHWN) were screened to determine relevancy and usefulness for the pharmacy workforce. R&I with higher grades were data-charted to capture information on topic and content delivery. Final R&I were determined through consensus meetings where 'highly rated' R&I were discussed and selected. Results: Of 140 original CHWN Toolkit R&I, 53 (37.8%) were of potential relevance or usefulness to improve well-being for most in the pharmacy workforce. Of those 53 R&I, 28 (20% of original) were final selections. The majority of R&I at each stage were focused on 'preventing burnout' and 'promoting mental health' (>60%) rather than 'addressing burnout', 'supporting recovery' or managing specific issues in the workplace (i.e. stigma, discrimination, bullying, hostility, workload). No R&I were specifically developed or studied within the pharmacy workforce. Conclusions: Health professions may benefit from the CHWN Toolkit and the knowledge translation activity described here. R&I relevant and useful to the pharmacy workforce generally require adaptation for dissemination and/or implementation. The set of final R&I form the basis for orchestrated plans to support the pharmacy workforce with respect to psychological health and wellbeing. There is a relative lack of R&I devoted to addressing and recovering from burnout and workload management issues.

HIV preexposure prophylaxis treatment patterns in a national health plan population.

BACKGROUND: Medication nonadherence diminishes the benefits of preexposure prophylaxis (PrEP) for the 1.2 million Americans at risk for HIV exposure. OBJECTIVE: To describe HIV PrEP treatment patterns among Medicare Advantage Prescription Drug (MAPD) plan and commercially insured beneficiaries. METHODS: This retrospective cohort study identified patients aged 16 to 89 years with at least 1 dispensing of emtricitabine-tenofovir disoproxil fumarate from July 2012, through December 2020, or emtricitabine-tenofovir alafenamide from October 2019 through December 2020, and who were continuously enrolled at least 12 months prior to and following the earliest PrEP claim. Outcomes were HIV PrEP adherence measured by proportion of days covered (PDC) using 2 binary thresholds of 0.60 (4 doses/week) and 0.80 (5-6 doses/week) and duration of index treatment episode, total time on treatment, and total number of prescription fills. RESULTS: The study cohort of 707 (292 MAPD plan, 415 commercial) was predominantly made up of male patients (90.0%) and resided in the South (78.9%) with a mean age of 46.2 years (MAPD plan: 54.5, commercial: 40.4). Both populations engaged in high-risk sexual behavior (All: 18.7%, MAPD plan: 16.8%, commercial: 20.0%) and experienced sexually transmitted infections (All: 3.3%, MAPD plan: 2.1%, commercial: 4.1%). The mean index treatment episode length was 297.0 days (MAPD plan: 283.6, commercial: 306.5). Total time on treatment was 477.3 days (MAPD plan: 450.7, commercial 496.0). At 3 months, 84.9% (MAPD plan: 83.6%, commercial: 85.8%) and at 12 months, 58.7% (MAPD plan: 57.2, commercial: 59.8) of patients achieved a PDC of at least 0.80. At 3 months, 100.0% (MAPD plan: 100.0%, commercial: 100.0%), and at 12 months, 74.3% (MAPD plan: 70.2%, commercial: 76.9%) of patients achieved a PDC of at least 0.60. The cohort had a mean of 16.4 fills of 30 days (MAPD plan: 16.4, commercial: 16.3) supply. CONCLUSIONS: There is an opportunity for clinical programs to focus on improving longer-term PrEP adherence among individuals at risk for HIV exposure.

Association Between Autism Spectrum Disorders and Cardiometabolic Diseases: A Systematic Review and Meta-analysis.

Importance: Although the increased risk of obesity among individuals with autism has been well established, evidence on the association between autism, cardiometabolic disorders, and obesity remains inconclusive. Objective: To examine the association between autism spectrum disorders and cardiometabolic diseases in a systematic review and meta-analysis. Data Sources: PubMed, Scopus, Web of Science, ProQuest, Embase, and Ovid databases were searched from inception through July 31, 2022, without restrictions on date of publication or language. Study Selection: Observational or baseline data of interventional studies reporting the prevalence of cardiometabolic risk factors (ie, diabetes, hypertension, dyslipidemia, atherosclerotic macrovascular disease) among children and/or adults with autism and matched with participants without autism were included. Data Extraction and Synthesis: Screening, data extraction, and quality assessment were performed independently by at least 2 researchers. DerSimonian-Laird random-effects meta-analyses were performed using the meta package in R. Main Outcomes and Measures: Relative risks (RRs) of diabetes, hypertension, dyslipidemia, and atherosclerotic macrovascular disease among individuals with autism were the primary outcomes. Secondary outcomes included the RR of type 1 and type 2 diabetes, heart disease, stroke, and peripheral vascular disease. Results: A total of 34 studies were evaluated and included 276 173 participants with autism and 7 733 306 participants without autism (mean [range] age, 31.2 [3.8-72.8] years; pooled proportion [range] of female individuals, 47% [0-66%]). Autism was associated with greater risks of developing diabetes overall (RR, 1.57; 95% CI, 1.23-2.01; 20 studies), type 1 diabetes (RR, 1.64; 95% CI, 1.06-2.54; 6 studies), and type 2 diabetes (RR, 2.47; 95% CI, 1.30-4.70; 3 studies). Autism was also associated with increased risks of dyslipidemia (RR, 1.69; 95% CI, 1.20-2.40; 7 studies) and heart disease (RR, 1.46; 95% CI, 1.42-1.50; 3 studies). Yet, there was no significantly associated increased risk of hypertension and stroke with autism (RR, 1.22; 95% CI, 0.98-1.52; 12 studies; and RR, 1.19; 95% CI, 0.63-2.24; 4 studies, respectively). Meta-regression analyses revealed that children with autism were at a greater associated risk of developing diabetes and hypertension compared with adults. High between-study heterogeneity was a concern for several meta-analyses. Conclusions and Relevance: Results suggest that the associated increased risk of cardiometabolic diseases should prompt clinicians to vigilantly monitor individuals with autism for potential contributors, signs of cardiometabolic disease, and their complications.

Emerging Therapies for Peanut Allergy.

Peanut allergy (PA) is a food allergy that causes an IgE-mediated type I hypersensitivity reaction. PA has become an increasing public health burden, with 2% of American children reported to have this condition in 2010. Current guidelines recommend allergen avoidance, patient education, and administration of H1 antihistamines, ß2-agonists, or epinephrine based on the severity of reaction. In this review article, emerging therapies for PA are evaluated for their potential role in treating PA. Oral, epicutaneous, and sublingual immunotherapies have completed clinical trials with promising efficacy. In particular, Palforzia (AR101) is an oral immunotherapy that received Food and Drug Administration (FDA)-approval in January 2020 and Viaskin Peanut is an epicutaneous immunotherapy with an anticipated FDA decision date by August 5, 2020. Furthermore, adjuvant combinations with either probiotics or anti-IgE receptor antagonists have shown an improved efficacy and safety profile compared to oral immunotherapy alone. However, immunotherapy-induced adverse reaction rates are high due to the risks associated with intentional allergen exposure. These results suggest that peanut immunotherapy has a promising role in the treatment of PA, although further studies are needed before its incorporation into standard of care.

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics.

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso and Rubin, 2016). Here, we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

Thy1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a for neuronal population imaging in vivo.

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

Cotargeting Ephrin Receptor Tyrosine Kinases A2 and A3 in Cancer Stem Cells Reduces Growth of Recurrent Glioblastoma.

Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023-37. ©2018 AACR.

Sensitive red protein calcium indicators for imaging neural activity.

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo.

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

Metastatic nonfunctioning pancreatic neuroendocrine carcinoma to liver: surgical treatment and outcomes.

BACKGROUND: The surgical treatment of metastatic, nonfunctional pancreatic neuroendocrine carcinoma (nPNEC) is not well defined. Existing series are confounded by inclusion of patients with metastatic functional tumors or gastrointestinal carcinoid. Our hypothesis was that the surgical treatment of metastatic nPNEC provides favorable perioperative and oncologic outcomes. STUDY DESIGN: We performed a retrospective review of all patients undergoing surgical treatment of metastatic nPNEC to the liver from 1987 through 2008 at the Mayo Clinic. Data are presented as medians with ranges. RESULTS: Seventy-two patients were identified, with a median age of 57 years (range 28 to 77 years) and median body mass index (BMI) of 26 kg/m(2) (range 18 to 40 kg/m(2)). Operative intent of resection was curative in 39 (54%) or palliative (≥ 90% tumor debulking) in 32 (44%). Median number of tumors treated and median tumor size were 8 (range 1 to 30) and 4.5 cm (range 0.3 to 20 cm), respectively. Tumor grade was 1 or 2 in 97%, and angioinvasion was identified in 55 (76%) patients. Postoperative morbidity and mortality were 50% and 0%, respectively. Among the 72 patients, overall survivals at 1, 5 and 10 years were 97.1%, 59.9%, and 45.0%, respectively. Among the 39 patients with a complete (R0) resection, the 1- and 5-year disease-free survivals were 53.7% and 10.7%, respectively. For patients undergoing debulking of ≥ 90% tumor burden, the 1- and 5-year survivals free of progression were 58.1% and 3.5%, respectively. CONCLUSIONS: Surgical treatment of metastatic nPNEC to the liver with curative intent or for palliative ≥ 90% debulking provides favorable oncologic outcomes. Despite a high incidence of tumor recurrence, 5-year survival rates are encouraging and appear to justify an aggressive surgical approach in these patients.