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Eastern Australia was subject to its hottest and driest year on record in 2019. This extreme drought resulted in massive canopy die-back in eucalypt forests. The role of hydraulic failure and tree size on canopy die-back in three eucalypt tree species during this drought was examined. We measured pre-dawn and midday leaf water potential (Ψleaf ), per cent loss of stem hydraulic conductivity and quantified hydraulic vulnerability to drought-induced xylem embolism. Tree size and tree health was also surveyed. Trees with most, or all, of their foliage dead exhibited high rates of native embolism (78-100%). This is in contrast to trees with partial canopy die-back (30-70% canopy die-back: 72-78% native embolism), or relatively healthy trees (little evidence of canopy die-back: 25-31% native embolism). Midday Ψleaf was significantly more negative in trees exhibiting partial canopy die-back (-2.7 to -6.3 MPa), compared with relatively healthy trees (-2.1 to -4.5 MPa). In two of the species the majority of individuals showing complete canopy die-back were in the small size classes. Our results indicate that hydraulic failure is strongly associated with canopy die-back during drought in eucalypt forests. Our study provides valuable field data to help constrain models predicting mortality risk.

AimsDetermination of the number of plasma cells in bone marrow biopsies is required for the diagnosis and ongoing evaluation of plasma cell neoplasms. We developed an automated digital enumeration platform to assess plasma cells identified by antigen expression in whole bone marrow sections in multiple myeloma, and compared it with manual assessments.MethodsBone marrow trephine biopsy specimens from 91 patients with multiple myeloma at diagnosis, remission and relapse were stained for CD138 and multiple myeloma oncogene 1 (MUM1). Manual assessment and digital quantification were performed for plasma cells in the entire trephine section. Concordance rates between manual and digital methods were evaluated for each antigen by intraclass correlation analyses (ICC) with associated Spearman’s correlations.ResultsThe digital platform counted 16 484–1 118 868 cells and the per cent CD138 and MUM1-positive plasma cells ranged from 0.05% to 93.5%. Overall concordance between digital and manual methods was 0.63 for CD138 and 0.89 for MUM1. Concordance was highest with diffuse plasma cell infiltrates (MUM1: ICC=0.90) and lowest when in microaggregates (CD138: ICC=0.13). Manual counts exceeded digital quantifications for both antigens (CD138: mean=26.4%; MUM1: mean=9.7%). Diagnostic or relapse threshold counts, as determined by CD138 manual assessments, were not reached with digital counting for 16 cases (18%).ConclusionsAutomated digital enumeration of the entire, immunohistochemically stained bone marrow biopsy section can accurately determine plasma cell burden, irrespective of pattern and extent of disease (as low as 0.05%). This increases precision over manual visual assessments which tend to overestimate plasma burden, especially for CD138, and when plasma cells are in clusters.

Shifts in the timing, intensity and/or frequency of climate extremes, such as severe drought and heatwaves, can generate sustained shifts in ecosystem function with important ecological and economic impacts for rangelands and managed pastures. The Pastures and Climate Extremes experiment (PACE) in Southeast Australia was designed to investigate the impacts of a severe winter/spring drought (60% rainfall reduction) and, for a subset of species, a factorial combination of drought and elevated temperature (ambient +3°C) on pasture productivity. The experiment included nine common pasture and Australian rangeland species from three plant functional groups (C3 grasses, C4 grasses and legumes) planted in monoculture. Winter/spring drought resulted in productivity declines of 45% on average and up to 74% for the most affected species (Digitaria eriantha) during the 6-month treatment period, with eight of the nine species exhibiting significant yield reductions. Despite considerable variation in species’ sensitivity to drought, C4 grasses were more strongly affected by this treatment than C3 grasses or legumes. Warming also had negative effects on cool-season productivity, associated at least partially with exceedance of optimum growth temperatures in spring and indirect effects on soil water content. The combination of winter/spring drought and year-round warming resulted in the greatest yield reductions. We identified responses that were either additive (Festuca), or less-than-additive (Medicago), where warming reduced the magnitude of drought effects. Results from this study highlight the sensitivity of diverse pasture species to increases in winter and spring drought severity similar to those predicted for this region, and that anticipated benefits of cool-season warming are unlikely to be realized. Overall, the substantial negative impacts on productivity suggest that future, warmer, drier climates will result in shortfalls in cool-season forage availability, with profound implications for the livestock industry and natural grazer communities.

In the face of a changing climate, research indicates that more frequent and severe drought conditions are critical problems that will constrain production of high-quality forage and influence the performance of grazing animals in the future. In addition, the duration of drought and potential trade-offs between plant morphology and nutritional composition may influence plant drought adaptation strategies across pasture species, and the consequences for forage quality are not well understood. Here we present the results of a study investigating the effects of drought on biomass productivity, dead material, leaf:stem biomass allocation and nutritional composition (whole-plant and tissue-specific) across nine diverse pasture species. For this, we conducted a field experiment exposing species to a 6-month period of simulated severe drought (60% rainfall reduction during winter and spring) and samples were collected at multiple harvests. We found that drought had different, harvest-specific effects on plant biomass structure and nutritional composition among pasture species. The severity of drought impacts on productivity, but not on nutritional quality, increased with drought duration. In general, drought strongly reduced productivity, increased the percentage of dead material and had mixed effects (increases, decreases and no effect) on leaf:stem ratio and concentrations of crude protein, non-structural carbohydrates, neutral detergent fibre and lignin. Changes in plant-level nutritional quality were driven by simultaneous changes in both leaf and stem tissues for most, but not all, species. Our findings may be especially helpful for selection of adapted species/cultivars that could minimize potential drought risks on forage, thereby optimising pasture performance under future drought scenarios.

Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here, we describe a new imaging flow cytometry method whereby fluorescence in situ hybridization (FISH) is integrated with cell phenotyping. The method, called "immuno-flowFISH," provides an exciting new dimension for the analysis of genomic changes in cytological samples (e.g., blood, bone marrow). Cells are analyzed in suspension without any requirement for prior cell isolation or separation. Multiple antibodies and FISH probes, each with a unique fluorophore, can be added and many thousands of cells analyzed. Specific cell populations are identified by their antigenic profile and then analyzed for the presence of chromosomal defects. Immuno-flowFISH was applied to the assessment of chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis and treatment requirements. This integrated immunophenotyping and multi-probe FISH strategy could detect both structural and numerical chromosomal changes involving chromosomes 12 and 17 in CLL cells. Given that many thousands of cells were analyzed and the leukemic cells were positively identified by their immunophenotype, this multi-probe method adds precision to the cytogenomic analysis of CLL. © 2021 Wiley Periodicals LLC.

Purpose Results of a study of rates of acute kidney injury (AKI) in pediatric patients treated with vancomycin plus piperacillin–tazobactam or vancomycin plus alternative antipseudomonal β-lactams (APBLs) are reported. Methods A retrospective, single-center cohort study was performed. Pediatric patients were included in the study cohort if they received combination therapy for at least 48 hours, had documented baseline and follow-up serum creatinine levels, and had a documented serum vancomycin trough concentration. The primary outcome was the frequency of AKI, defined as a 50% or greater increase in serum creatinine concentration from baseline or an increase of at least 0.5 mg/dL from baseline. The secondary outcome was time to AKI onset. Results A total of 474 patients were included. Among 100 patients who received vancomycin plus piperacillin–tazobactam, the rate of AKI was higher than the rate in the group treated with vancomycin plus alternative APBLs (27% versus 7%, p < 0.0001). The median time to AKI onset was shorter in the piperacillin–tazobactam group versus the alternative APBL group (3.8 versus 7.9 days, p = 0.0065). Patients who were administered piperacillin–tazobactam were almost 6 times as likely to develop AKI (odds ratio [OR], 5.955; 95% confidence interval [CI], 2.774–12.784), and patients who had a maximum vancomycin trough concentration greater than 20 mg/L were 7.5 times as likely to develop AKI (OR, 7.552; 95% CI, 3.625–15.734). Conclusion Pediatric patients treated with concomitant vancomycin and piperacillin–tazobactam had a higher rate of AKI, with faster AKI onset, than those who received vancomycin in combination with other APBLs.

Flow cytometry provides robust, multi-parametric and quantitative information on single cells which also exhibits enormous potential as a tool for small particle characterisation. Small extracellular vesicle (sEV) detection by flow cytometry remains compromised due to the high prevalence of swarm detection, which is defined by the simultaneous illumination of more than one sEV, recorded as a single event. Detection of sEVs by imaging flow cytometry presents a major advantage by having the ability to resolve single particles from swarm detection based on the image features recorded for each event. In this study, we provide a simplified protocol that facilitates the removal of both swarm events and aggregated particles to improve the accuracy of sEV analysis. Our results indicate that imaging flow cytometry should be at the forefront as a robust and sensitive technique for sEV characterisation.

Shifts in the timing, intensity and/or frequency of climate extremes, such as severe drought and heatwaves, can generate sustained shifts in ecosystem function with important ecological and economic impacts for rangelands and managed pastures. The Pastures and Climate Extremes experiment (PACE) in Southeast Australia was designed to investigate the impacts of a severe winter/spring drought (60% rainfall reduction) and, for a subset of species, a factorial combination of drought and elevated temperature (ambient +3 °C) on pasture productivity. The experiment included nine common pasture and Australian rangeland species from three plant functional groups (C3 grasses, C4 grasses and legumes) planted in monoculture. Winter/spring drought resulted in productivity declines of 45% on average and up to 74% for the most affected species (Digitaria eriantha) during the 6-month treatment period, with eight of the nine species exhibiting significant yield reductions. Despite considerable variation in species’ sensitivity to drought, C4 grasses were more strongly affected by this treatment than C3 grasses or legumes. Warming also had negative effects on cool-season productivity, associated at least partially with exceedance of optimum growth temperatures in spring and indirect effects on soil water content. The combination of winter/spring drought and year-round warming resulted in the greatest yield reductions. We identified responses that were either additive such that there was only as significant warming effect under drought (Festuca), or less-than-additive, where there was no drought effect under warming (Medicago), compared to ambient plots. Results from this study highlight the sensitivity of diverse pasture species to increases in winter and spring drought severity similar to those predicted for this region, and that anticipated benefits of cool-season warming are unlikely to be realised. Overall, the substantial negative impacts on productivity suggest that future, warmer, drier climates will result in shortfalls in cool-season forage availability, with profound implications for the livestock industry and natural grazer communities.

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells. Here we report the development and use of imaging flow cytometry to assess chromosomes by FISH in phenotyped CLL cells in suspension. Thousands of CLL cells, identified by their phenotype, are assessed for specific FISH probe signals using an automated, high throughput imaging flow cytometer. The "extended depth of field" capability of the imaging flow cytometer enables FISH probe signals ("spots") to be resolved and localised within the (stained) nucleus of the immunophenotyped cells. We report the development of the automated "immuno-flowFISH" on normal blood using the Amnis ImageStreamX mark II platform and illustrate the clinical application of the method for the assessment of chromosome 12 in CLL. It is a powerful new method which has potential to be applied at diagnosis for disease stratification, and following treatment to assess residual disease. These applications will assist clinicians in optimising therapeutic decision making and thereby improve patient outcome.

Objective. To determine the extent to which students can recreate a recently completed examination from memory. Methods. After two mid-term examinations, students were asked, as a class, to recreate recently completed examinations. Students were given 48 hours to recreate the examination, including details about the questions and answer choices. The results were compared to the original examination to determine the accuracy of students’ recall and reproduction of content. Results. The students were able to collectively recreate 90% of the questions on the two examinations. For the majority of questions (51%), students also recreated the question as well as the correct response and at least one incorrect response. The majority of questions that the students recreated were of medium to high accuracy as they contained detailed phrasing that aligned with the original question on the examination. Conclusion. The collective memory of a group of students may allow them to accurately recreate the majority of a completed examination from memory. Based on the findings of this study and tenets of social psychology, faculty should consider the potential implications for examination security, whether to provide feedback to students on examinations, and whether completed examinations should be released to students following the examination.

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders characterized by well-known driver mutations [1]. The mutational status of Janus kinase 2 (JAK2), calretic...

Objective. To describe the development and implementation of an innovative, comprehensive, multi-day module focused on assessing and providing feedback on student cognitive and interpersonal skill development and practice readiness after the first year (PY1) of a Doctor of Pharmacy (PharmD) curriculum. Methods. A multi-day capstone assessment was developed to evaluate first-year students' knowledge of course content, ability to find and apply information, and interpersonal skills, including teamwork and adaptability. The PY1 Capstone consisted of four parts. Knowledge was assessed using 130 multiple-choice items on first-year course content and 50 fill-in-the-blank items on Top 200 brand and generic drug names. The ability to find and apply information was assessed using a 45-question open-book test. Interpersonal skills were assessed using a specially designed multiple mini-interview (MMI). The final part of the assessment was a debriefing session that provided rapid-cycle feedback on capstone performance and a bridge between students' recently completed first-year coursework and an upcoming 2-month experiential immersion. Results. The average score on the closed-book and open-book assessments were 75% and 68%, respectively. Most students displayed satisfactory interpersonal skills based on the MMI. Students viewed the assessment positively based on post-assessment survey responses (>75%). Most students (98%) reported not studying for the assessment, indicating that the results should reflect students' retention of knowledge and skills. Conclusion. The capstone assesses students on knowledge and skills and provides students with feedback on areas to focus on during their early immersion. Continued work is needed to ensure the process is transparent and cost-effective.

Jack Benny became one of the most influential entertainers of the 20th century - by being the top radio comedian, when the comics ruled radio, and radio was the most powerful and pervasive mass medium in the US. In 23 years of weekly radio broadcasts, by aiming all the insults at himself, Benny created Jack, the self-deprecating “Fall Guy” character. He indelibly shaped American humor as a space to enjoy the equal opportunities of easy camaraderie with his cast mates, and equal ego deflation. Benny was the master of comic timing, knowing just when to use silence to create suspense or to have a character leap into the dialogue to puncture Jack’s pretentions. Jack Benny was also a canny entrepreneur, becoming one of the pioneering “showrunners” combining producer, writer and performer into one job. His modern style of radio humor eschewed stale jokes in favor informal repartee with comic hecklers like his valet Rochester (played by Eddie Anderson) and Mary Livingstone his offstage wife. These quirky characters bouncing off each other in humorous situations created the situation comedy. In this career study, we learn how Jack Benny found ingenious ways to sell his sponsors’ products in comic commercials beloved by listeners, and how he dealt with the challenges of race relations, rigid gender ideals and an insurgent new media industry (TV). Jack Benny created classic comedy for a rapidly changing American culture, providing laughter that buoyed radio listeners from 1932’s depths of the Great Depression, through World War II to the mid-1950s.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.

Entrustable Professional Activities (EPAs) are workplace responsibilities that directly impact patient care. The use of EPAs allows pharmacy faculty and preceptors to provide learners with feedback and assessment in the clinical setting. Because they focus assessment on a learner’s execution of professional activities which requires integration of the respective competencies, EPAs help provide a more holistic picture of a learner’s performance. Using EPAs to backwards design classroom learning for those competencies is highly encouraged, but instructors cannot or should not assess performance and make entrustment decisions using EPAs in the classroom setting for several reasons: a learner’s classroom performance usually does not predict clinical performance very well, assessment of EPAs require direct observation of the learner performing the EPAs, EPA assessment requires multiple observations of the learner with different patients with varying level of acuity, and most importantly, EPA assessment must result in a decision to trust the learner to perform the clinical activity with limited supervision. By ensuring all entrustment decisions are made in a clinical or experiential setting, students will receive an accurate assessment and benchmark of their performance that will lead them one step closer to becoming independent practitioners.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

Aims Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved. Methods The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2 V617F / CALR mutation status and platelet count. Results The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR -mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2 V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR -mutated cases. Conclusions Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target.

The novel coronavirus identified in 2019 (COVID-19) pandemic has impacted pharmacy graduate and postgraduate education. This crisis has resulted in a cosmic shift in the administration of these programs to ensure core values are sustained. Adjustments may be needed at a minimum to ensure that postgraduate trainees complete program requirements while maintaining safety. Moving forward, additional issues may arise that will need to be addressed such as admissions and program onboarding, acclimating students to new training environments, and managing inadequate resources for distance education, distance practice, and remote versus in-person research opportunities.

The coronavirus identified in 2019 (COVID-19) has caused dramatic disruptions in pharmacy experiential education. Administrators and programs have worked to help external preceptors, faculty members, and students cope with the new realities of virtual or remote experiences and new or increased use of telemedicine. Clear and effective lines of communication as well as well-reasoned and resourced alternative plans are necessary to help manage the current issues and prepare for future challenges. Doctor of Pharmacy programs should enhance their focus not just on the physical health and well-being of students, faculty members, and external preceptors, but also on their mental and emotional health. The full scope of the impact of the pandemic on experiential education in pharmacy is still unclear, but this situation should serve as a stimulus for innovation and rethinking the paradigm of how pharmacy programs educate and prepare students for pharmacy practice.

Student engagement during classes includes behavioural, cognitive and emotional components, and is a pre-requisite for successful active learning environments. A novel approach to measuring student engagement was developed, involving triangulation of real-time student-self report, observation by trained observers and heart rate measurement. The self-report instrument was evaluated in four separate cohorts (n = 123) at Monash University and the University of North Carolina. The six item self-report demonstrated good reliability (Cronbach’s alpha values ranged from 0.7–0.81). The self-report showed predictive validity in that small group activities were rated as significantly more engaging than didactic lecturing. Additionally, there was significant inter-instructor variability and within-class variability, indicating good discrimination between classroom activities. This self-report may prove useful to academic teaching staff in evaluating and refining their active learning activities. Independent observation was not found to correlate with student self-report, due in part to students who were pretending to engage being rated as engaged by an observer. Strikingly, students reported that they were pretending to engage for 23% of class time, even for highly regarded instructors. Individual participants were rated as engaged for 42 of the 46 intervals for which they reported that they had “pretended to engage”, indicating that the two observers were unable to detect disengagement during periods in which students pretended to engage. Instructors should be aware that student cues such as eye contact and nodding may indicate pretending to engage. One particular self-report item; “I tried a new approach or way of thinking about the content”, correlated positively with heart rates, and a controlled study reproduced this finding during two activities that required students to try a new approach to understanding a concept. Agreement with this item also correlated with superior performance on two in-class written assessment tasks (n = 101, p

In recent years, there has been increasing use of BRAF-inhibiting drugs for the treatment of various malignancies, including melanoma. However, these agents are associated with the development of other nonmelanoma skin lesions, in particular squamoproliferative lesions such as keratoacanthomas (KAs), squamous cell carcinomas, and BRAF inhibitor-associated verrucous keratoses. The molecular pathogenesis of these lesions is of interest, not only for therapeutic reasons, but also for the insight it might provide into the development of similar lesions in a sporadic setting. We used next-generation sequencing to compare the mutational profiles of lesions after treatment with a BRAF inhibitor, with similar lesions arising sporadically. HRAS mutations were common among the BRAF inhibitor-induced lesions, being identified in 56%, compared with 14% of lesions in the sporadic group (P = 0.002). Thus, despite similar histomorphological appearances, the underlying molecular mechanisms may be different. In addition, within the BRAF inhibitor-associated group, the lesions designated as KAs and BRAF inhibitor-associated verrucous keratoses had a similar mutational profile (mutations in PIK3CA, APC, and HRAS), which was distinct to that seen in squamous cell carcinomas (FGFR3, CDKN2A, and STK11). We have previously noted histological overlap between KAs and BRAF inhibitor-associated verrucous keratoses, and this finding supports the notion that they may represent morphological or temporal variants of a single lesion type.

This case study traces the evolution of Jack Benny's comedy practices as he moved across entertainment systems from vaudeville to radio in the early 1930s. The core of his comic identity endured—his self-deprecating humor—but other facets of his comic identity shifted and changed in reaction to the new environment. Facing the daunting challenge of filling radio's unprecedented, ferocious demand for new content, Jack Benny struggled but ultimately thrived by developing new approaches to comedy. Benny and writer Harry Conn began to craft a personality-based radio variety program that moved from Benny's vaudeville monologues to expanding the narrative world of the show with dialogue and skits. They created characters out of the show's performers, put them in situations, and turned Benny into a “fall guy” character, the butt of his cast's insulting jokes.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r2 = >0.99, P