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Background Clinical trials initiated during emerging infectious disease outbreaks must quickly enroll participants to identify treatments to reduce morbidity and mortality. This may be at odds with enrolling a representative study population especially when the population affected is undefined. Methods We evaluated the utility of CDC COVID-19-Associated Hospitalization Surveillance Network (COVID-NET), COVID-19 Case Surveillance System (CCSS), and 2020 U.S. Census data to determine demographic representation in the four stages of the Adaptive COVID-19 Treatment Trial (ACTT). We compared the cumulative proportion of participants by sex, race, ethnicity, and age enrolled at U.S. ACTT sites, with the respective 95% confidence intervals, to the reference data in forest plots. Results U.S. ACTT sites enrolled 3,509 adults hospitalized with COVID-19. When compared with COVID-NET, ACTT enrolled a similar or higher proportion of Hispanic or Latino and White participants depending on the stage, and a similar proportion of African American participants in all stages. In contrast, ACTT enrolled a higher proportion of these groups when compared with U.S. Census and CCSS. The proportion of participants ages 65 years was either similar or lower than COVID-NET and higher than CCSS and U.S. Census. The proportion of females enrolled in ACTT was lower than the proportion of females in the reference datasets. Conclusions While surveillance data of hospitalized cases may not be available early in an outbreak, it is a better comparator than U.S. Census data and surveillance of all cases, which may not reflect the population affected and at higher risk of severe disease.

The value of research mentorship in academic medicine is well-recognized, yet there is little practical advice for how to develop and sustain effective mentoring partnerships. Gaining research skill and mentorship is particularly critical to success in academic surgery, yet surgeon scientists are challenged in their mentorship efforts by time constraints and lack of education on how to mentor. To address this gap, this study explored the strategies that award-winning faculty mentors utilize in collaborating with their medical student mentees in research.For this qualitative study, the authors invited physician recipients of an institution-wide mentorship award to participate in individual, semi-structured interviews during July and August 2018. Following interview transcription, the authors independently coded the text and collaboratively identified common mentoring strategies and practices via a process of thematic analysis.Nine physician mentors, representing a mix of genders, medical specialties and types of research (basic science, clinical, translational, and health services), participated in interviews. The authors identified 12 strategies and practices from the interview transcripts that fell into 5 categories: Initiating the partnership; Determining the research focus; Providing project oversight; Developing mentee research competence; and Supporting mentee self-efficacy.Award-winning mentors employ a number of shared strategies when mentoring medical trainees in research. These strategies can serve as a guide for academic surgeons who wish to improve their research mentoring skills.

Pharmacodynamic studies of everolimus and sapanisertib treated PDX-PNETs using Western blot analysis of mTOR pathway targets.

Events upstream AID-mediated switch lesion are not affected in Nbs1deltaB/deltaB Atm -/- VAV B cells

Flow cytometric analysis of haematopoietic tissues

PDF file - 180K, Supplemental Figure 1: Complete deletion of Dlg1 in three independent Cre-expression systems permits development of mature, peripheral B cell subsets.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel “pre-leukemic” stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers. Cancer Immunol Res; 1(6); 426–37. ©2013 AACR.

PDF file - 181K, Supplemental Figure 5: Histopathology analysis of lymph node and spleen of p53 KO, DKO, and B6/CTRL mice.

PDF file - 88K, Supplemental Table 1: Primers used for qRT-PCR.

PDF file - 61K, Supplemental Figure 4: Diminished expression of endogenous PTEN protein in BCR-Abl transformed Dlg1 deficient B cells.

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as MEN1 and PTEN), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET.

Sμ-Sγ1 junctions

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress.Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

PDF file - 81K

PDF file - 115K, Supplemental Figure 6: Chromosomal translocations visualized by Spectral Karyotyping (SKYTM) analysis of splenic tumors.

PDF file - 132K, Supplemental Figure 3: Dissemination of oncogenically- transformed pre-B cells in the in vivo model of retrovirally- induced BCR-ABL-positive B-ALL.

BackgroundContinued high incidence of HIV and other STIs, paired with rising antibiotic resistance to a number of existing treatments, warrants the development of new pharmaceutical approaches for STI prevention. Multipurpose prevention technologies (MPTs) offer an innovative approach for expanding HIV/STI prevention. The majority of MPT product candidates currently in development include HIV prevention, while only half include compounds active against non-HIV STIs.MethodsThis narrative review focuses on compounds in preclinical development (in vitro and in vivo) through phase 3 clinical trials with activity against one or more of the following infections: HIV, HSV-1, HSV-2, Chlamydia trachomatis,Neisseria gonorrhoeae,Treponema pallidum, andTrichomonas vaginalis.Bacterial vaginosis is included due to its association with increased risk of STIs. The focus is on compounds with novel mechanisms of action and prophylactic and/or therapeutic potential. Articles published in PubMed between 2011 and 2021, NIH RePorter and conference abstracts and proceedings between 2020 and 2021 were searched. Excluded from the review are compounds that are already being used in MPT product candidates.Main resultsThere is a growing pipeline of compounds targeting viral STIs, many of which have successfully transitioned from preclinical to clinical stages of development. However, the product development pipeline remains limited for compounds that target bacterial STIs.ConclusionsThe paucity of new pharmaceutical approaches for STI prevention, particularly non-HIV STIs, remains a public health gap. Future funding priorities should include STI prevention research. Despite limited attention to STI prevention in the development of MPTs, many research institutions worldwide are working on discoveries of new compounds, exploring new indications for existing drugs or on innovative drug delivery mechanisms. Our findings can be used to connect researchers across the globe to advance the development of compounds that have potential as active pharmaceutical ingredients in future MPTs.

Objective:To evaluate broad-spectrum intravenous antibiotic use before and after the implementation of a revised febrile neutropenia management algorithm in a population of adults with hematologic malignancies.Design:Quasi-experimental study.Setting and population:Patients admitted between 2014 and 2018 to the Adult Malignant Hematology service of an acute-care hospital in the United States.Methods:Aggregate data for adult malignant hematology service were obtained for population-level antibiotic use: days of therapy (DOT), C. difficile infections, bacterial bloodstream infections, intensive care unit (ICU) length of stay, and in-hospital mortality. All rates are reported per 1,000 patient days before the implementation of an febrile neutropenia management algorithm (July 2014–May 2016) and after the intervention (June 2016–December 2018). These data were compared using interrupted time series analysis.Results:In total, 2,014 patients comprised 6,788 encounters and 89,612 patient days during the study period. Broad-spectrum intravenous (IV) antibiotic use decreased by 5.7% with immediate reductions in meropenem and vancomycin use by 22 (P = .02) and 15 (P = .001) DOT per 1,000 patient days, respectively. Bacterial bloodstream infection rates significantly increased following algorithm implementation. No differences were observed in the use of other antibiotics or safety outcomes including C. difficile infection, ICU length of stay, and in-hospital mortality.Conclusions:Reductions in vancomycin and meropenem were observed following the implementation of a more stringent febrile neutropenia management algorithm, without evidence of adverse outcomes. Successful implementation occurred through a collaborative effort and continues to be a core reinforcement strategy at our institution. Future studies evaluating patient-level data may identify further stewardship opportunities in this population.

A sufficient β-cell mass is crucial for preventing diabetes, and perinatal β-cell proliferation is important in determining the adult β-cell mass. However, it is not yet known how perinatal β-cell proliferation is regulated. Here, we report that serotonin regulates β-cell proliferation through serotonin receptor 2B (HTR2B) in an autocrine/paracrine manner during the perinatal period. In β-cell–specific Tph1 knockout (Tph1 βKO) mice, perinatal β-cell proliferation was reduced along with the loss of serotonin production in β-cells. Adult Tph1 βKO mice exhibited glucose intolerance with decreased β-cell mass. Disruption of Htr2b in β-cells also resulted in decreased perinatal β-cell proliferation and glucose intolerance in adulthood. Growth hormone (GH) was found to induce serotonin production in β-cells through activation of STAT5 during the perinatal period. Thus, our results indicate that GH-GH receptor-STAT5-serotonin-HTR2B signaling plays a critical role in determining the β-cell mass by regulating perinatal β-cell proliferation, and defects in this pathway affect metabolic phenotypes in adults.

Objectives The selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model. Methods Pharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7. Results Meropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone. Conclusions Simulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

Objective:To determine the placebo component of treatment responses in patients with intellectual disability (ID).Methods:A statistical meta-analysis comparing bias-corrected effect sizes (Hedges g) of drug responses in open-label vs placebo-controlled clinical trials was performed, as these trial types represent different certainty of receiving genuine treatment (100% vs 50%). Studies in fragile X, Down, Prader-Willi, and Williams syndrome published before June 2015 were considered.Results:Seventeen open-label trials (n = 261, 65% male; mean age 23.6 years; mean trial duration 38 weeks) and 22 placebo-controlled trials (n = 721, 62% male; mean age 17.1 years; mean trial duration 35 weeks) were included. The overall effect size from pre to post treatment in open-label studies was g = 0.602 (p = 0.001). The effect of trial type was statistically significant (p = 0.001), and revealed higher effect sizes in studies with 100% likelihood of getting active drug, compared to both the drug and placebo arm of placebo-controlled trials. We thus provide evidence for genuine placebo effects, not explainable by natural history or regression toward the mean, among patients with ID.Conclusions:Our data suggest that clinical trials in patients with severe cognitive deficits are influenced by the certainty of receiving genuine medication, and open-label design should thus not be used to evaluate the effect of pharmacologic treatments in ID, as the results will be biased by an enhanced placebo component.

Antimicrobial resistance (AMR) causes millions of illnesses every year, threatening the success of lifesaving antibiotic therapy and, thus, public health. To examine the rise and spread of antimicrobial resistance around the world, our study performs a multivariate statistical analysis of antimicrobial resistance gene data from eight different countries: the US, the UK, China, Brazil, Mexico, Canada, Australia, and South Africa. Multi-dimensional data points were projected onto a two-dimensional plane using principal component analysis and organized into a dendrogram utilizing hierarchical clustering to identify significant AMR genes and pathogens. Outlier genes/pathogens were typically involved in high occurrences of antimicrobial resistance, and they were able to indicate the trend of antimicrobial resistance in the future. Statistical analysis of the data identified: (1) tet(A), aph(3&rsquo, &rsquo, )-Ib, aph(6)-Id, blaEC, blaTEM-1, qacEdelta1, sul1, sul2, and aadA1 as the nine most common AMR genes among the studied countries, (2) Salmonella enterica and E. coli and Shigella as the most common AMR foodborne pathogens, and (3) chicken as the most prevalent meat carrier of antimicrobial resistance. Our study shows that the overall number of reported antimicrobial resistance cases in foodborne pathogens is generally rising. One potential contributing factor for this is the increasing antimicrobial usage in the growing livestock industry.

BackgroundMechanisms of arrhythmogenicity in hypertrophic cardiomyopathy (HCM) are not well understood.ObjectiveTo characterize an electrophysiological substrate of HCM in comparison to ischemic cardiomyopathy (ICM), or healthy individuals.MethodsWe conducted a prospective case-control study. The study enrolled HCM patients at high risk for ventricular tachyarrhythmia (VT) (n=10; age 61±9 y; left ventricular ejection fraction (LVEF) 60±9%), and three comparison groups: healthy individuals (n=10; age 28±6 y; LVEF>70%), ICM patients with LV hypertrophy (LVH) and known VT (n=10; age 64±9 y; LVEF 31±15%), and ICM patients with LVH and no known VT (n=10; age 70±7y; LVEF 46±16%). All participants underwent 12-lead ECG, cardiac CT or MRI, and 128-electrode body surface mapping (BioSemi ActiveTwo, Netherlands). Non-invasive voltage and activation maps were reconstructed using the open-source SCIRun (University of Utah) inverse problem-solving environment.ResultsIn the epicardial basal anterior segment, HCM patients had the greatest ventricular activation dispersion [16.4±5.5 vs. 13.1±2.7 (ICM with VT) vs. 13.8±4.3 (ICM no VT) vs. 8.1±2.4 ms (Healthy); P=0.0007], the largest unipolar voltage [1094±211 vs. 934±189 (ICM with VT) vs. 898±358 (ICM no VT) vs. 842±90 µV (Healthy); P=0.023], and the greatest voltage dispersion [median(interquartile range) 215(161-281) vs. 189(143-208) (ICM with VT) vs. 158(109-236) (ICM no VT) vs. 110(106-168)µV (Healthy); P=0.041]. Differences were also observed in other endo-and epicardial basal and apical segments.ConclusionHCM is characterized by a greater activation dispersion in basal segments, a larger voltage, and a larger voltage dispersion through LV.

Objective We examined the impact of video editing and rater expertise in surgical resident evaluation on operative performance ratings of surgical trainees. Design Randomized independent review of intraoperative video. Setting Operative video was captured at a single, tertiary hospital in Boston, MA. Participants Six common general surgery procedures were video recorded of 6 attending-trainee dyads. Full-length and condensed versions (n = 12 videos) were then reviewed by 13 independent surgeon raters (5 evaluation experts, 8 nonexperts) using a crossed design. Trainee performance was rated using the Operative Performance Rating Scale, System for Improving and Measuring Procedural Learning (SIMPL) Performance scale, the Zwisch scale, and ten Cate scale. These ratings were then standardized before being compared using Bayesian mixed models with raters and videos treated as random effects. Results Editing had no effect on the Operative Performance Rating Scale Overall Performance (-0.10, p = 0.30), SIMPL Performance (0.13, p = 0.71), Zwisch (-0.12, p = 0.27), and ten Cate scale (-0.13, p = 0.29). Additionally, rater expertise (evaluation expert vs. nonexpert) had no effect on the same scales (-0.16 (p = 0.32), 0.18 (p = 0.74), 0.25 (p = 0.81), and 0.25 (p = 0.17). Conclusions There is little difference in operative performance assessment scores when raters use condensed videos or when raters who are not experts in surgical resident evaluation are used. Future validation studies of operative performance assessment scales may be facilitated by using nonexpert surgeon raters viewing videos condensed using a standardized protocol.

Geminin (Gmnn) is a coiled-coil nuclear protein originally identified as a DNA replication inhibitor. In addition, nuclear Gmnn regulates gene expression by interacting (through a domain distinct from the Cdt1-interacting domain) with Brg1, the catalytic subunit of the SWI/SNF chromatin remodeling complex. In this context, Gmnn regulates expression of key transcription factors involved in differentiation. The dual role of Gmnn in the cell cycle and differentiation has been viewed as a critical mechanism for linking replication and differentiation. We wanted to test its role in human pancreas development. We found a robust Gmnn expression in the human fetal pancreas but its expression slightly decreased with age. Remarkably the fraction of cells expressing both Gmnn and Ki67 declined much faster with age. We isolated 13WD human fetal pancreas and studied the proportion of replicating Sox9+cells. We compared control human fetal explants with fetal explants transduced with a shRNA lentivirus directed against Gmnn (Gmnnsh) and discovered that Gmnnshexplants display a decrease in proliferation compared to controls after 2 days in culture. RNA sequencing comparing Gmnnshexplants to controls revealed a marked decrease in transcripts associated with endocrine differentiation. To evaluate the effects on endocrine differentiation, human fetal pancreases were isolated at 13WD and treated for 5 days with lentiviruses or casein kinase inhibitors. We detected a significant reduction in the number of insulin+cells after treatment with the casein kinase inhibitor TBB and a significant reduction in the number of glucagon+cells after treatment with TBB or after siCsnk2a1. Overexpression of Gmnn significantly increased the proportion of Neurog3+cells compared to the control explants. Finally, we found that inhibition of Gmnn or casein kinase 2 was capable of significantly reducing expression of Nkx6.1 compared to controls. We conclude that Gmnn may play a critical role pancreas cell replication and endocrine specification in human development. Disclosure L. Baeyens: None. D.W. Scheel: None. K. Yang: None. M. German: Advisory Panel; Self; Encellin. Stock/Shareholder; Self; Encellin, Viacyte, Inc. Funding JDRF

Quantitation of drug target engagement in single cells has proven difficult, often leaving unanswered questions in the drug development process. Here we show that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitate the dynamics of target engagement of covalent BTK inhibitors as well as reversible PARP inhibitors in populations of single cells as two examples using a single companion imaging probe for each target. We then determine average in vivo tumor concentrations and show marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

Aim: Ki67 is a prognostic and/or predictive biomarker in patients with malignancies. Flow cytometry is a powerful technology for single-cell multiparameter analysis. Results: We developed and validated a multicolor quantitative flow cytometry assay for detection of intracellular Ki67 expression in various immune cell subsets from human blood. The assay was optimized and showed excellent precisions. Assessment of the sample stability indicated that percentage changes from the fresh sample for the reportable results of interest were within 20%, up to 72 h after blood collection in the Cyto-Chex® BCT tube. Conclusion: The validated assay is sufficiently robust to analyze clinical samples. Easy access to peripheral blood enables continuous monitoring of Ki67 expression in blood as a biomarker, for example, for immunotherapy studies.

A mechanics education testbed comprised of instructional modules written in Mathematica was developed in order to increase the effectiveness of course delivery through an integrated computation, visualization, and animation approach with the aim of accelerating the learning process. Overall, 25 modules were developed for Statics and another 20 modules for Mechanics of Materials during the AY2017-18. The testbed complements class instruction and motivates self-learning by illustrating basic mechanics concepts relative to homework problems and textbook examples. Its features include visualization, parametrization, and trial-and-error simulation capabilities that enable students to view simulated phenomena from different perspectives, thereby leaving a lasting impression which facilitates quicker absorption of the mechanics concepts than the traditional approach. The parametrization feature enables variation of boundary and initial conditions, as well as other parameters that affect the simulated phenomenon to demonstrate each parameter’s influence on the system response. These capabilities enable students to employ the trial-and-error approach in acquiring an intuitive understanding of the visualized phenomenon, validated through direct calculation. Student comments are summarized and survey results are analyzed and related to individual student performance. Student assessment confirms the testbed’s effectiveness and provides helpful suggestions for further enhancement/development. Representative modules are presented to demonstrate their functions and capabilities.

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as MEN1 and PTEN), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET.

Continuous subcutaneous insulin infusion (CSII) therapy is routinely used in diabetes management for patients expecting to use their insulin pumps during hospitalizations. For hospital personnel, this is unfamiliar and complex technology, which necessitates implementing policies and guidelines for standardizing CSII use in the inpatient setting. This study aims to examine adherence to an institutional CSII policy and safety glucometrics for hospitalized patients using CSII. We performed an IRB-exempt retrospective review of CSII user records from January to June 2017. Policy adherence was evaluated for appropriate provider and nurse order set use and documentation in the electronic medical record. 81 patients with 111 admissions on CSII were evaluated. Mean age was 42±16.9 years, 62% were female and 82.7% had type 1 diabetes. Median LOS was 5 days for 788 total patient days, HgbA1c 8.3 ± 1.78, and baseline admission blood glucose (BG) was 175 mg/dL. CSII was continued in 62% of admissions with 12.6% discontinuing use due to medical and surgical contraindications. Overall adherence to the policy was high with documented endocrinology consultations in 100%, provider specified basal rates in 96%, specified bolus doses in 92%, specified correction doses in 83%, specified target BG in 90%, and nurse documented verified doses on the MAR in 93%. Nurse documentation of CSII in the Lines/Drains/Airways flowsheets was 16%. Hypoglycemia (BG299mg/dL) were present in 10%, 0.8%, and 10.5% of patient days, respectively. Rates of DKA in CSII users was 0.9%. Policy adherence was effectively achieved for CSII use in hospitals. Rates of hypoglycemia and uncontrolled hyperglycemia were high with very low incidence of severe hypoglycemia or DKA episodes. These results suggest that CSII is possible to safely continue in hospitalized patients, but future studies are necessary to determine how to modify pump rates and optimize glycemic control. Disclosure K. Yang: None. M.B. Ateya: Stock/Shareholder; Self; AbbVie Inc.. R. Gianchandani: None.

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation., Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods. Here, we report that trichloroacetic acid increases the MS signal of tobramycin. To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed. Aliquots of 25 μL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid. The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode. ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification. The calibration curve concentration range was 50–25000 ng/mL. Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold). The method is simple, fast, and reliable. Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.

Surface plasmon resonance (SPR ) enables real-time, label-free detection of ligand binding to target receptors immobilized on a sensing surface. SPR has emerged as a promising technique for extracellular vesicle (EV) characterization with its label-free detection scheme, and exquisite sensitivity. Among the various system configurations, nanohole-based SPR sensors are of particular interest because of their simple optical setup, tunability, and scalability. Here, we describe the characterization of circulating EVs or exosomes from human clinical samples using a nanohole-based SPR sensor, named nPLEX (nano-plasmonic exosome) sensing.

Insulin production by the pancreatic β-cell is required for normal glucose homeostasis. While key transcription factors that bind to the insulin promoter are known, relatively little is known about the upstream regulators of insulin transcription. Using a whole-genome RNA interference screen, we uncovered 26 novel regulators of insulin transcription that regulate diverse processes including oxidative phosphorylation, vesicle traffic, and the unfolded protein response (UPR). We focused on Spry2—a gene implicated in human type 2 diabetes by genome-wide association studies but without a clear connection to glucose homeostasis. We showed that Spry2 is a novel UPR target and its upregulation is dependent on PERK. Knockdown of Spry2 resulted in reduced expression of Serca2, reduced endoplasmic reticulum calcium levels, and induction of the UPR. Spry2 deletion in the adult mouse β-cell caused hyperglycemia and hypoinsulinemia. Our study greatly expands the compendium of insulin promoter regulators and demonstrates a novel β-cell link between Spry2 and human diabetes.

To support the development of a dynamic in vitro human Pharmacokinetic/Pharmacodynamic simulation model for biofilm-mediated infections and study stability of meropenem, an LC-MS/MS method for the determination of meropenem in Luria Bertani (LB) media was developed and validated in an API2000 LC-MS/MS system. A partial validation was also performed in M9 media. Sample aliquots of 100 μL (or 25 μL for M9 media) were mixed with the internal standard (IS) ceftazidime and filtered. The filtrate was directly injected onto a C8 column eluted with ammonium formate (10mM, pH4) and acetonitrile (0.1% formic acid) in a gradient mode. ESI+ and MRM with ion pair m/z 384 → 68 for meropenem and m/z 547 → 468 for the IS were used for quantification. The calibration curve concentration range was 50 to 25,000 ng/mL. The recovery was over 98%. In LB media, significant signal suppression was observed throughout the time period of detection when compared with mobile phase solvents, but the matrix effect was compensated well with the IS. In M9 media, much less signal suppression was observed. The method is simple, fast, and reliable. Using the method, stability of meropenem in LB and M9 media were tested. No significant degradation was observed for at least 8 hours in both LB media (37 °C) and M9 media (30 °C), but more than 15% degradation was observed overnight (∼20hr). The method was transferred to an API5000 LC-MS/MS system using meropenem-d6 as the IS.

Background Delays in time to appropriate management and antimicrobial therapy in patients with Staphylococcus aureus bacteremia (SAB) lead to dramatic increases in mortality, cost, and length of hospital stay. This study assesses the impact of antimicrobial stewardship pharmacist (ASP)-led Verigene education sessions paired with a physician targeted EPIC best practice alert (BPA) on time to appropriate therapy and rate of infectious diseases (ID) consult for patients SAB. Methods This single-center pre–post study included adult patients with SAB from October 2016 through January 2018. A BPA was implemented in August 2017, and fired for any patient with SAB and no ID consult. The BPA provided four recommendations: (1) repeat blood cultures till clearance, (2) obtain ID consult, (3) start vancomycin for SAB with mecA gene (i.e., MRSA) and nafcillin or cefazolin for SAB without mecA gene (i.e., MSSA), and (4) obtain echocardiogram. The ASP also provided education on antimicrobial therapy choices and optimization to clinical pharmacists and ID physicians. The hospital utilized Verigene Gram-positive blood culture nucleic acid test during both study periods and ASP review of SAB cases without an ID consult in the preintervention phase. The primary outcome was time to appropriate therapy defined as the time a positive blood culture was drawn to the time of first appropriate antibiotic administration. Results A total of 223 patients with SAB were included; 134 were in the 10-month historic group (October 2016–July 2017) and 89 were in the 5-month postintervention (PI) group (August 2017–January 2018). The BPA fired for 86% (n = 77) of patients in the PI group. Average time to appropriate therapy for all SAB patients and patients with MSSA significantly improved following the intervention (35.1 vs. 20.4 hours, P = 0.004; 53.2 vs. 30.3 hours, P = 0.001). During the intervention phase, therapy was more frequently changed between the time of Verigene results and antibiotic susceptibilities (77.6% vs. 86.5%, P = 0.254). The rate of ID consult also significantly improved following the intervention (89.6% vs. 97.8%, P < 0.02). Conclusion Implementing an SAB BPA and education on interpretation of Verigene results for SAB significantly improved time to appropriate therapy for all patients with SAB, patients with MSSA bacteremia, and rate of ID consult. Disclosures All authors: No reported disclosures.

IHVR-19029 (6) is a lead endoplasmic reticulum α-glucosidases I and II inhibitor, which efficiently protected mice from lethal Ebola and Marburg virus infections via injection route, but suffered from low bioavailability and off-target interactions with gut glucosidases when administered orally. In an effort to improve efficacious exposure levels and avoid side effects, we designed and synthesized ester prodrugs. Not only were the prodrugs stable in simulated gastric and intestinal fluids and were inactive against glucosidases but they also exhibited antiviral activities against dengue virus infection in a cell based assay. Further in vitro evaluation showed that the bioconversion of the prodrugs is species dependent: in mice, the prodrugs were converted to 6 in the plasma and liver; while in human, the conversion occurred mainly in liver. An in vivo pharmacokinetic study in mice demonstrated that the tetrabutyrate prodrug 8 achieved the most improved overall exposure of 6 upon both oral and intravenous a...

Surface plasmon resonance (SPR ) enables real-time, label-free detection of ligand binding to target receptors immobilized on a sensing surface. SPR has emerged as a promising technique for extracellular vesicle (EV) characterization with its label-free detection scheme, and exquisite sensitivity. Among the various system configurations, nanohole-based SPR sensors are of particular interest because of their simple optical setup, tunability, and scalability. Here, we describe the characterization of circulating EVs or exosomes from human clinical samples using a nanohole-based SPR sensor, named nPLEX (nano-plasmonic exosome) sensing.

Background: Drug-drug interactions (DDIs) with warfarin and antimicrobial agents are a common cause of international normalized ratio (INR) instability, which can affect the risk for bleeding and thrombotic events. Objective: The purpose of this study was to assess the impact of a comprehensive guideline for the management of warfarin-antimicrobial DDIs across transitions of care. The guideline emphasizes improving identification of significant antimicrobial-warfarin DDIs during hospitalization, empirical warfarin dose modification based on DDI and baseline INR, patient education, documentation of the DDI, communication with outpatient providers regarding the DDI and anticipated antimicrobial stop date, and warfarin dose adjustment on discontinuation of antimicrobial. Methods: This retrospective, single-center, quasiexperimental, pre-post study compared end points 3 months before and after guideline implementation. The primary outcome was time within therapeutic range (TTR). Results: The study included 78 preguideline and 31 postguideline patients; baseline characteristics were similar between groups. Implementation of the guideline resulted in greater in-hospital TTR (72% vs 50%, P = 0.04) and improved TTR across transition of care (70% vs 46%, P = 0.01). Documentation of DDI in the pharmacy anticoagulation discharge summary significantly improved in the postguideline group (40% vs 14%, P = 0.02) and numerically improved within the daily pharmacy progress notes (94% vs 82%, P = 0.13). The implementation of the guideline was associated with a nonsignificant, numerical reduction in bleeding events compared with the preguideline group (0 vs 4 events, P = 0.11). Conclusion: This single-center approach to optimize the comprehensive management of significant antimicrobial-warfarin DDIs resulted in improved communication with outpatient providers and improved INR TTR.