Multi-level regulation of even-skipped stripes by the ubiquitous factor Zelda.

The zinc-finger protein Zelda (Zld) is a key activator of zygotic transcription in early Drosophila embryos. Here, we study Zld-dependent regulation of the seven-striped pattern of the pair-rule gene even-skipped (eve). Individual stripes are regulated by discrete enhancers that respond to broadly distributed activators; stripe boundaries are formed by localized repressors encoded by the gap genes. The strongest effects of Zld are on stripes 2, 3 and 7, which are regulated by two enhancers in a 3.8 kb genomic fragment that includes the eve basal promoter. We show that Zld facilitates binding of the activator Bicoid and the gap repressors to this fragment, consistent with its proposed role as a pioneer protein. To test whether the effects of Zld are direct, we mutated all canonical Zld sites in the 3.8 kb fragment, which reduced expression but failed to phenocopy the abolishment of stripes caused by removing Zld in trans. We show that Zld also indirectly regulates the eve stripes by establishing specific gap gene expression boundaries, which provides the embryonic spacing required for proper stripe activation.

Predictive modeling reveals that higher-order cooperativity drives transcriptional repression in a synthetic developmental enhancer.

A challenge in quantitative biology is to predict output patterns of gene expression from knowledge of input transcription factor patterns and from the arrangement of binding sites for these transcription factors on regulatory DNA. We tested whether widespread thermodynamic models could be used to infer parameters describing simple regulatory architectures that inform parameter-free predictions of more complex enhancers in the context of transcriptional repression by Runt in the early fruit fly embryo. By modulating the number and placement of Runt binding sites within an enhancer, and quantifying the resulting transcriptional activity using live imaging, we discovered that thermodynamic models call for higher-order cooperativity between multiple molecular players. This higher-order cooperativity captures the combinatorial complexity underlying eukaryotic transcriptional regulation and cannot be determined from simpler regulatory architectures, highlighting the challenges in reaching a predictive understanding of transcriptional regulation in eukaryotes and calling for approaches that quantitatively dissect their molecular nature.

Suboptimal Intermediates Underlie Evolution of the Bicoid Homeodomain.

Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. Here we study the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. We focus on the evolution of the HD of AncBcd, which binds to DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, we show that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains (NT, H1, and RH) are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. Our results suggest a multistep pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.

Transcriptional Enhancers in Drosophila.

Key discoveries in Drosophila have shaped our understanding of cellular "enhancers." With a special focus on the fly, this chapter surveys properties of these adaptable cis-regulatory elements, whose actions are critical for the complex spatial/temporal transcriptional regulation of gene expression in metazoa. The powerful combination of genetics, molecular biology, and genomics available in Drosophila has provided an arena in which the developmental role of enhancers can be explored. Enhancers are characterized by diverse low- or high-throughput assays, which are challenging to interpret, as not all of these methods of identifying enhancers produce concordant results. As a model metazoan, the fly offers important advantages to comprehensive analysis of the central functions that enhancers play in gene expression, and their critical role in mediating the production of phenotypes from genotype and environmental inputs. A major challenge moving forward will be obtaining a quantitative understanding of how these cis-regulatory elements operate in development and disease.

Bicoid-Dependent Activation of the Target Gene hunchback Requires a Two-Motif Sequence Code in a Specific Basal Promoter.

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions.

Diphenhydramine-Refractory Antipsychotic-Induced Dystonia in an Adolescent Male With Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome is a rare genetic disorder that results in distinctive craniofacial deformities, developmental delay, hirsutism, and other physical abnormalities. Case reports suggest some of these patients exhibit sensitivity and paradoxical reactions to certain psychoactive drugs. This report of a 16-year-old male with Cornelia de Lange is the first to describe dystonia from a first-generation antipsychotic that did not respond to conventional treatment with diphenhydramine. The patient initially presented to the Emergency Department for agitation, which progressively worsened after administration of diphenhydramine, olanzapine, and intramuscular haloperidol. The patient returned to the Emergency Department the following day because of altered mental status and lethargy that progressed to periodic lip-smacking movements and contraction of his upper extremities. His symptoms continued despite administration of diphenhydramine and loading doses of 3 antiepileptic drugs. His abnormal labs included an elevated creatine kinase and a prolonged QTc interval on his electrocardiogram. His symptoms were later deemed a probable drug-induced dystonic reaction to haloperidol once seizures were excluded by an unremarkable electroencephalogram. This case supports previous reports suggesting an association between Cornelia de Lange and paradoxical drug reactions, and it is recommended that clinicians strongly weigh the risks of prescribing first-generation antipsychotics for this patient population. These medications should be carefully titrated, with close patient monitoring to prevent adverse drug effects and other iatrogenic complications because antidotes may be rendered ineffective by this condition.

Ancient mechanisms for the evolution of the bicoid homeodomain's function in fly development.

The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd's derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd's derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd's transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd's controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution.

Disulfiram-like Reaction Involving Ceftriaxone in a Pediatric Patient.

Several medications have been shown to cause disulfiram-like reactions in patients concomitantly exposed to ethanol, including specific cephalosporin antibiotics that possess a methylthiotetrazole substituent. Within the cephalosporin class, there are few reports of disulfiram-like reactions with ceftriaxone. This case report is the first to involve a pediatric patient, and it describes a mild but likely disulfiram-like reaction manifesting as facial flushing in an 8-year-old male upon receiving a ceftriaxone infusion preceded by a dose of prednisolone elixir (5% ethanol by volume) for presumed community-acquired pneumonia thought to be complicated by an asthma exacerbation. The patient's flushing resolved with intravenous diphenhydramine, did not reappear, and was diagnosed as an allergy to ceftriaxone. Upon further evaluation, a hypersensitivity reaction was considered unlikely, and the allergy history was revised. The patient's antibiotic treatment was switched to azithromycin without steroids, and he had no further issues. This case suggests there is benefit in increased monitoring of pediatric patients receiving certain cephalosporins along with alcohol-containing medications, and it demonstrates how disulfiram reactions can easily be misinterpreted as hypersensitivity reactions. Aside from just alcohol-cephalosporin interactions, this case underscores the need for general vigilance when using alcohol-containing drug preparations in pediatric patients in an effort to prevent adverse effects and potential drug interactions.

A feed-forward relay integrates the regulatory activities of Bicoid and Orthodenticle via sequential binding to suboptimal sites.

The K50 (lysine at amino acid position 50) homeodomain (HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otd's ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes, including otd Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies.

How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. Here, we find that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ('dark' shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback and redundancy to generate cell type-specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.

Usefulness of Three-Dimensional Modeling in Surgical Planning, Resident Training, and Patient Education.

BACKGROUND: Imaging has a critical impact on surgical decision making and three-dimensional (3D) digital models of patient pathology can now be made commercially. We developed a 3D digital model of a cancer of the head of the pancreas by integrating actual CT data with 3D modeling process. After this process, the virtual pancreatic model was also produced using a high-quality 3D printer. PATIENTS AND METHODS: A 56-year-old female with pancreatic head adenocarcinoma presented with biliary obstruction and jaundice. The CT scan showed a borderline resectable tumor with a clear involvement of the gastroduodenal artery but doubtful relationships with the hepatic artery. Our team in collaboration with the Immersive Touch team used multiple series from the CT and segmented the relevant anatomy to understand the physical location of the tumor. An STL file was then developed and printed. RESULTS: Reconstructing and compositing the different series together enhanced the imaging, which allowed clearer observations of the relationship between the mass and the blood vessels, and evidence that the tumor was unresectable. Data files were converted for printing a 100% size rendering model, used for didactic purposes and to discuss with the patient. CONCLUSIONS: This study showed that (1) reconstructing enhanced traditional imaging by merging and modeling different series together for a 3D view with diverse angles and transparency, allowing the observation of previously unapparent anatomical details; (2) with this new technology surgeons and residents can preobserve their planned surgical intervention, explore the patient-specific anatomy, and sharpen their procedure choices; (3) high-quality 3D printed models are increasingly useful not only in the clinical realm but also for personalized patient education.

Teaching professionalism in graduate medical education: What is the role of simulation?

BACKGROUND: We systematically reviewed the literature concerning simulation-based teaching and assessment of the Accreditation Council for Graduate Medical Education professionalism competencies to elucidate best practices and facilitate further research. METHODS: A systematic review of English literature for "professionalism" and "simulation(s)" yielded 697 abstracts. Two independent raters chose abstracts that (1) focused on graduate medical education, (2) described the simulation method, and (3) used simulation to train or assess professionalism. Fifty abstracts met the criteria, and seven were excluded for lack of relevant information. The raters, 6 professionals with medical education, simulation, and clinical experience, discussed 5 of these articles as a group; they calibrated coding and applied further refinements, resulting in a final, iteratively developed evaluation form. The raters then divided into 2 teams to read and assess the remaining articles. Overall, 15 articles were eliminated, and 28 articles underwent final analysis. RESULTS: Papers addressed a heterogeneous range of professionalism content via multiple methods. Common specialties represented were surgery (46.4%), pediatrics (17.9%), and emergency medicine (14.3%). Sixteen articles (57%) referenced a professionalism framework; 14 (50%) incorporated an assessment tool; and 17 (60.7%) reported debriefing participants, though in limited detail. Twenty-three (82.1%) articles evaluated programs, mostly using subjective trainee reports. CONCLUSION: Despite early innovation, reporting of simulation-based professionalism training and assessment is nonstandardized in methods and terminology and lacks the details required for replication. We offer minimum standards for reporting of future professionalism-focused simulation training and assessment as well as a basic framework for better mapping proper simulation methods to the targeted domain of professionalism.

The mRNA-bound proteome of the early fly embryo.

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation.

Morphogen rules: design principles of gradient-mediated embryo patterning.

The Drosophila blastoderm and the vertebrate neural tube are archetypal examples of morphogen-patterned tissues that create precise spatial patterns of different cell types. In both tissues, pattern formation is dependent on molecular gradients that emanate from opposite poles. Despite distinct evolutionary origins and differences in time scales, cell biology and molecular players, both tissues exhibit striking similarities in the regulatory systems that establish gene expression patterns that foreshadow the arrangement of cell types. First, signaling gradients establish initial conditions that polarize the tissue, but there is no strict correspondence between specific morphogen thresholds and boundary positions. Second, gradients initiate transcriptional networks that integrate broadly distributed activators and localized repressors to generate patterns of gene expression. Third, the correct positioning of boundaries depends on the temporal and spatial dynamics of the transcriptional networks. These similarities reveal design principles that are likely to be broadly applicable to morphogen-patterned tissues.

The Impact of a Frailty Education Module on Surgical Resident Estimates of Lobectomy Risk.

BACKGROUND: Frailty is a risk factor for adverse events after surgery. Residents' ability to recognize frailty is underdeveloped. We assessed the influence of a frailty education module on surgical residents' estimates of lobectomy risk. METHODS: Traditional track cardiothoracic surgery residents were randomly allocated to take an online short course on frailty (experimental group) or to receive no training (control group). Residents read a clinical vignette, made an initial risk estimate of major complications for lobectomy, and rated clinical factors on their importance to their estimates. They viewed a video of a standardized patient portraying the patient in the vignette, randomly selected to exhibit either vigorous or frail behavior, and provided a final risk estimate. After rating five vignettes, they completed a test on their frailty knowledge. RESULTS: Forty-one residents participated (20 in the experimental group). Initial risk estimates were similar between the groups. The experimental group rated clinical factors as "very important" in their initial risk estimates more often than did the control group (47.6% versus 38.5%; p < 0.001). Viewing videos resulted in a significant change from initial to final risk estimates (frail 50% ± 75% increase, p = 0.008; vigorous 14% ± 32% decrease, p = 0.043). The magnitude of change in risk estimates was greater for the experimental group (10.0 ± 8.1 versus 5.1 ± 7.7; p < 0.001). The experimental group answered more frailty test questions correctly (93.7% versus 75.2%; p < 0.001). CONCLUSIONS: A frailty education module improved resident knowledge of frailty and influenced surgical risk estimates. Training in frailty may help educate residents in frailty recognition and surgical risk assessment.

Thoracic surgeons' perception of frail behavior in videos of standardized patients.

BACKGROUND: Frailty is a predictor of poor outcomes following many types of operations. We measured thoracic surgeons' accuracy in assessing patient frailty using videos of standardized patients demonstrating signs of physical frailty. We compared their performance to that of geriatrics specialists. METHODS: We developed an anchored scale for rating degree of frailty. Reference categories were assigned to 31 videos of standardized patients trained to exhibit five levels of activity ranging from "vigorous" to "frail." Following an explanation of frailty, thoracic surgeons and geriatrics specialists rated the videos. We evaluated inter-rater agreement and tested differences between ratings and reference categories. The influences of clinical specialty, clinical experience, and self-rated expertise were examined. RESULTS: Inter-rater rank correlation among all participants was high (Kendall's W 0.85) whereas exact agreement (Fleiss' kappa) was only moderate (0.47). Better inter-rater agreement was demonstrated for videos exhibiting extremes of behavior. Exact agreement was better for thoracic surgeons (n = 32) than geriatrics specialists (n = 9; p = 0.045), whereas rank correlation was similar for both groups. More clinical years of experience and self-reported expertise were not associated with better inter-rater agreement. CONCLUSIONS: Videos of standardized patients exhibiting varying degrees of frailty are rated with internal consistency by thoracic surgeons as accurately as geriatrics specialists when referenced to an anchored scale. Ratings were less consistent for moderate degrees of frailty, suggesting that physicians require training to recognize early frailty. Such videos may be useful in assessing and teaching frailty recognition.