Using Drosophila Oogenesis in the Classroom to Increase Student Participation in Biomedical Research.

Students that participate in undergraduate research benefit in multiple ways, including improved learning outcomes, increased enthusiasm for science, technology, engineering, and mathematics (STEM) fields, and increased likelihood of continuation into a STEM career. These benefits are even more pronounced for students that are traditionally under-represented in STEM, although these students often face barriers to participation in traditional apprenticeship-style research experiences. Course-based undergraduate research experiences (CUREs) are a promising and increasingly popular approach to increase undergraduate participation in research in a way that is inclusive of all students. Here, we describe how Drosophila oogenesis can be used as the basis for CUREs in a wide variety of courses. We provide an overview of our own oogenesis-based CURE, as well as suggestions for how this CURE could be adapted to accommodate a variety of schedules, course sizes, and institution types. Our goal is to simplify the process for CURE implementation in the hopes that a greater number of instructors choose to implement a CURE in their own courses.

Patient-centered Reporting in Radiology: A Single-site Survey Study of Lung Cancer Screening Results.

PURPOSE: This study aimed to assess whether patients preferred traditional or patient-friendly radiology reports and, secondarily, whether one reporting style led to a subjective improvement in patients' understanding of their imaging results and next steps in their clinical care. MATERIALS AND METHODS: This randomized study included patients who had previously enrolled in an institutional comprehensive lung cancer screening program. Three hundred patients were randomly selected from the program database to receive both traditional and patient-centered radiology reports. Randomization also occurred at both the risk level of the fictitious test results (low, intermediate, or high) and the order in which the reports were read by each participant. Participants completed a survey providing demographic information and indicating which report style was preferred and which report style led to a better understanding of screening results and future options. In addition, each report style was rated (from 1 to 5) for clarity, understandability, attractiveness, and helpfulness. RESULTS: A total of 46 responses for report preference data and 41 responses for attribute rating data were obtained. Overall, participants demonstrate a preference for patient-friendly reports (65.2%) over traditional reports (21.7%). On a 5-point scale, average ratings for patient-friendly reports were higher than traditional reports by 1.2 (P<0.001) for clarity, 1.5 (P<0.001) for understandability, 1.5 (P<0.001) for attractiveness, and 1.0 (P<0.001) for helpfulness. CONCLUSION: Data suggest that patients prefer patient-friendly reports over traditional reports and find them to be clearer, more comprehensible, more attractive, and more helpful.

Using Drosophila Motor Mutants to Teach Neurodevelopment in an Undergraduate Neurobiology Lab.

Most undergraduate neuroscience courses include a neurodevelopment component. Typically, the focus is on development of the mammalian central nervous system, including the concepts of neurulation, patterning of the neural tube, and differentiation of the various cells required to build a functional nervous system. However, it can be challenging to design an affordable undergraduate laboratory exercise to reinforce these concepts for students outside of lecture. Here we describe a laboratory exercise that takes advantage of the high level of conservation in neurodevelopmental pathways using Drosophila as a model organism to illuminate the connection between cell differentiation and nervous system function. Following a lesson discussing spinal cord development, students use Drosophila larvae to assess the effects of mutations in highly conserved motor neuron differentiation genes on motor behaviors such as crawling. As outcomes of this laboratory, students are able to master important neurodevelopmental concepts, connect neurodevelopment to nervous system function, and gain experience with experimental design and data analysis.

Patient-centered Radiology Reporting for Lung Cancer Screening.

Medicine is slowly transitioning toward a more patient-centered approach, with patients taking a more central role in their own care. A key part of this movement has involved giving patients increased access to their medical record and imaging results via electronic health portals. However, most patients lack the knowledge to fully understand medical documents, which are generally written above their comprehension level. Radiology reports, in particular, utilize complex terminology due to radiologists' historic function as consultants to other physicians, with little direct communication to patients. As a result, typical radiology reports lack standardized formatting, and they are often inscrutable to patients. Numerous studies examining patient preference also point to a trend for more accessible radiology reports geared toward patients. Reports designed with an infographic format, combining simple pictures and standardized text, may be an ideal format that radiologists can pursue to provide patient-centered care. Our team, through feedback from patient advisory groups, developed a patient-friendly low-dose computed tomography lung cancer screening report with an infographic format that is both visually attractive and comprehensible to the average patient. The report is designed with sections including a description of low-dose computed tomography, a section on individualized patient results, the meaning of the results, and a list of the next steps in their care. We believe that this form of the report has the potential to serve as a bridge between radiologists and patients, allowing for a better patient understanding of their health and empowering patients to participate in their health and health care.

Flexible opto-electronics enabled microfluidics systems with cloud connectivity for point-of-care micronutrient analysis.

In developing countries, the deployment of medical diagnostic technologies remains a challenge because of infrastructural limitations (e.g. refrigeration, electricity), and paucity of health professionals, distribution centers and transportation systems. Here we demonstrate the technical development and clinical testing of a novel electronics enabled microfluidic paper-based analytical device (EE-µPAD) for quantitative measurement of micronutrient concentrations in decentralized, resource-limited settings. The system performs immune-detection using paper-based microfluidics, instrumented with flexible electronics and optoelectronic sensors in a mechanically robust, ultrathin format comparable in size to a credit card. Autonomous self-calibration, plasma separation, flow monitoring, timing and data storage enable multiple devices to be run simultaneously. Measurements are wirelessly transferred to a mobile phone application that geo-tags the data and transmits it to a remote server for real time tracking of micronutrient deficiencies. Clinical tests of micronutrient levels from whole blood samples (n=95) show comparable sensitivity and specificity to ELISA-based tests. These results demonstrate instantaneous acquisition and global aggregation of diagnostics data using a fully integrated point of care system that will enable rapid and distributed surveillance of disease prevalence and geographical progression.

Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication.

In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E-Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E-Cdk2 activity are not fully understood. Here, we show that the Drosophila Cyclin E-Cdk2 inhibitor Dacapo (Dap) is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. We propose from this model and our in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E-Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy.

Lateral flow assay with near-infrared dye for multiplex detection.

BACKGROUND: Lateral flow assays (LFAs) are popular point-of-care diagnostic tools because they are rapid and easy to use. Nevertheless, they often lack analytical sensitivity and quantitative output and may be difficult to multiplex, limiting their usefulness in biomarker measurement. As a proof-of-concept study, we detail the design of a quantitative, multiplex LFA with readily available near-infrared (NIR) detection to improve analytical sensitivity. METHODS: NIR dye was conjugated to selected antibodies and incorporated into LFAs. We used singleplex, optimized NIR-LFAs to measure interleukin (IL)-6 from 0 to 200 pg/mL and developed duplex assays to simultaneously measure IL-6 from 0 to 100 pg/mL (0 to 4.5 pmol/L) and C-reactive protein (CRP) from 50 to 2500 ng/mL (0.4 to 20 nmol/L) on a single test strip. Assays were tested on 60 different spiked samples and compared to ELISA results. RESULTS: NIR-LFAs detected IL-6 in a 10% plasma matrix with a limit of detection of 4 pg/mL (182 fmol/L) and a CV <7%. Duplex NIR-LFAs quantitatively measured IL-6 and CRP concentrations simultaneously. Values strongly correlated to ELISA measurements, with R(2) values of 0.9825 and 0.9711 for IL-6 and CRP, respectively. CONCLUSIONS: NIR-LFAs exhibit quantitative measurement at pg/mL concentrations owing to a high signal-to-background ratio and robust detection antibody clearance through the test strip. Moreover, NIR-LFAs are able to detect molecules present at vastly different concentrations in multiplex format and compare favorably to ELISAs. LFAs with direct NIR detection may be a valuable tool for biomarker evaluation in the point-of-care setting.

The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses.

Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene: Larval lethality and male fertility.

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that "denticleless" is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.

Sparkling insights into enhancer structure, function, and evolution.

This review focuses on a single cis-regulatory element: the sparkling eye enhancer of the Drosophila dPax2 gene. sparkling responds to Notch and EGFR signaling, along with other direct regulatory inputs, to drive gene expression that is restricted to cone cells of the developing fly retina. Functional, genetic, biochemical, evolutionary, and bioinformatic analyses have revealed surprising properties of sparkling, which may provide new insights into cis-regulatory logic and mechanisms of transcriptional activation. These properties include: a very high density of regulatory information and a correspondingly low "junk" content; an unexpectedly complex combinatorial code; tight functional constraints on enhancer organization, paradoxically coupled with high turnover of DNA sequence and binding site position; a requirement for weak binding of the transcription factor Su(H) to low-affinity sites in order to maintain a cell-type-specific response to Notch signaling; and multiple specialized regulatory sequences conferring functionally distinct activation activities, all of which are required in concert to achieve proper gene expression in vivo.

Rapid evolutionary rewiring of a structurally constrained eye enhancer.

BACKGROUND: Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as-in some cases-the arrangement and spacing of binding sites for these factors. Here, we examine evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye. RESULTS: Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. Our data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that we observe. Furthermore, stereotypical spatial relationships between certain binding sites ("grammar elements") can be identified in all sparkling orthologs-although the sites themselves are often recently derived. We also find that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in noncone cells. CONCLUSIONS: Rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. Our findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale.

Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer.

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? Here, we examine in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. Our results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others.

Tyrosine kinases as targets for the treatment of rheumatoid arthritis.

As critical regulators of numerous cell signaling pathways, tyrosine kinases are implicated in the pathogenesis of several diseases, including rheumatoid arthritis (RA). In the absence of disease, synoviocytes produce factors that provide nutrition and lubrication for the surrounding cartilage tissue; few cellular infiltrates are seen in the synovium. In RA, however, macrophages, neutrophils, T cells and B cells infiltrate the synovium and produce cytokines, chemokines and degradative enzymes that promote inflammation and joint destruction. In addition, the synovial lining expands owing to the proliferation of synoviocytes and infiltration of inflammatory cells to form a pannus, which invades the surrounding bone and cartilage. Many of these cell responses are regulated by tyrosine kinases that operate in specific signaling pathways, and inhibition of a number of these kinases might be expected to provide benefit in RA.

Suprasegmental characteristics of speech produced during simultaneous communication by inexperienced signers.

This study investigated suprasegmental variables of syllable stress and intonation contours in contextual speech produced during simultaneous communication (SC) by inexperienced signers. Ten hearing inexperienced sign language users were recorded under SC and speech-alone (SA) conditions speaking a set of sentences containing stressed versus unstressed versions of the same syllables and a set of sentences containing interrogative versus declarative versions of the same words. Results indicated longer sentence durations for SC than SA for all speech materials. Vowel duration and fundamental frequency differences between stressed and unstressed syllables as well as intonation contour differences between declarative and interrogative sentences were essentially the same in both SC and SA conditions. The conclusion that prosodic rules were not violated by inexperienced signers in SC is consistent with previous research indicating that temporal alterations produced during SC do not involve degradation of other temporal or spectral characteristics of English speech.

A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies.