Interactive training with a novel simulation model for upper gastrointestinal endoscopic hemostasis improves trainee technique and confidence.

Background and study aims Endoscopic hemostasis is a life-saving procedure for gastrointestinal bleeding; however, training for it is often performed on real patients and during urgent situations that put patients at risk. Reports of simulation-based training models for endoscopic hemostasis are scarce. Herein, we developed a novel simulator called "Medical Rising STAR-Ulcer type" to practice endoscopic hemostasis with hemoclips and coagulation graspers. This study aimed to evaluate the reproducibility of the clinical difficulty of this model and the effectiveness of simulation-based training for clipping hemostasis. Patients and methods This was a prospective educational study. Fifty gastroenterology residents from Japan and Canada were recruited to participate in a simulation-based training program. The primary outcome was the success rate for clipping hemostasis. We measured differences in trainee subjective assessment scores and evaluated the co-occurrence network based on comments after training. Results The hemostasis success rate of the trainees significantly increased after instruction (64% vs. 86%, P < 0.05). The success rate for ulcers in the upper body of the stomach (59%), a high-difficulty site, was significantly lower than that for ulcers in the antrum, even after feedback and instruction. Trainee self-perceived proficiency and confidence significantly improved after simulation-based training ( P < 0.05). Co-occurrence network analysis showed that trainees valued a structured learning approach, acknowledged simulator limitations, and recognized the need for continuous skill refinement. Conclusions Our study demonstrates the potential of our simulation-based training model as a valuable tool for improving technical skills and confidence in trainees learning to perform endoscopic hemostasis.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System.

A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FALCOscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)-stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.

Spatially compartmentalized phase regulation of a Ca2+-cAMP-PKA oscillatory circuit.

Signaling networks are spatiotemporally organized to sense diverse inputs, process information, and carry out specific cellular tasks. In ß cells, Ca2+, cyclic adenosine monophosphate (cAMP), and Protein Kinase A (PKA) exist in an oscillatory circuit characterized by a high degree of feedback. Here, we describe a mode of regulation within this circuit involving a spatial dependence of the relative phase between cAMP, PKA, and Ca2+. We show that in mouse MIN6 ß cells, nanodomain clustering of Ca2+-sensitive adenylyl cyclases (ACs) drives oscillations of local cAMP levels to be precisely in-phase with Ca2+ oscillations, whereas Ca2+-sensitive phosphodiesterases maintain out-of-phase oscillations outside of the nanodomain. Disruption of this precise phase relationship perturbs Ca2+ oscillations, suggesting the relative phase within an oscillatory circuit can encode specific functional information. This work unveils a novel mechanism of cAMP compartmentation utilized for localized tuning of an oscillatory circuit and has broad implications for the spatiotemporal regulation of signaling networks.

AKAP95 Organizes a Nuclear Microdomain to Control Local cAMP for Regulating Nuclear PKA.

Contrary to the classic model of protein kinase A (PKA) residing outside of the nucleus, we identify a nuclear signaling complex that consists of AKAP95, PKA, and PDE4D5 and show that it forms a functional cyclic AMP (cAMP) signaling microdomain. Locally generated cAMP can accumulate within the vicinity of this complex; however, when cAMP is generated at the plasma membrane, PDE4 serves as a local sink and PDE3 as a barrier to prevent accumulation of cAMP within the microdomain as a means of controlling activation of tethered nuclear PKA.

Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks.

Cellular signaling networks are the foundation which determines the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.

Characterization of Simultaneous Pressure Waves as Biomarkers for Colonic Motility Assessed by High-Resolution Colonic Manometry.

Simultaneous pressure waves (SPWs) in manometry recordings of the human colon have been associated with gas expulsion. Our hypothesis was that the SPW might be a critical component of most colonic motor functions, and hence might act as a biomarker for healthy colon motility. To that end, we performed high-resolution colonic manometry (HRCM), for the first time using an 84-sensor (1 cm spaced) water-perfused catheter, in 17 healthy volunteers. Intraluminal pressure patterns were recorded during baseline, proximal and rectal balloon distention, after a meal and following proximal and rectal luminal bisacodyl administration. Quantification was performed using software, based on Image J, developed during this study. Gas expulsion was always associated with SPWs, furthermore, SPWs were associated with water or balloon expulsion. SPWs were prominently emerging at the termination of proximal high amplitude propagating pressure waves (HAPWs); we termed this motor pattern HAPW-SPWs; hence, SPWs were often not a pan-colonic event. SPWs and HAPW-SPWs were observed at baseline with SPW amplitudes of 12.0 ± 8.5 mmHg and 20.2 ± 7.2 mmHg respectively. The SPW occurrence and amplitude significantly increased in response to meal, balloon distention and luminal bisacodyl, associated with 50.3% anal sphincter relaxation at baseline, which significantly increased to 59.0% after a meal, and 69.1% after bisacodyl. Often, full relaxation was achieved. The SPWs associated with gas expulsion had a significantly higher amplitude compared to SPWs without gas expulsion. SPWs could be seen to consist of clusters of high frequency pressure waves, likely associated with a cluster of fast propagating, circular muscle contractions. SPWs were occasionally observed in a highly rhythmic pattern at 1.8 ± 1.2 cycles/min. Unlike HAPWs, the SPWs did not obliterate haustral boundaries thereby explaining how gas can be expelled while solid content can remain restrained by the haustral boundaries. In conclusion, the SPW may become a biomarker for normal gas transit, the gastrocolonic reflex and extrinsic neural reflexes. The SPW assessment reveals coordination of activities in the colon, rectum and anal sphincters. SPWs may become of diagnostic value in patients with colonic dysmotility.

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

How personal, behavioral, and environmental factors predict working in STEMM vs non-STEMM middle-skill careers.

BACKGROUND: Much of science, technology, engineering, mathematics, and medical (STEMM) education policy and research centers around developing the upper levels of the STEMM workforce sector. However, there are many positions in this workforce, "middle-skill careers," that are largely responsible for executing the innovations and are largely ignored in STEMM education research. RESULTS: Using data from the National Educational Longitudinal Study of 1988, we found differences in what predicts STEMM-related vs. non-STEMM careers across skill-level. For instance, underrepresented minorities and those exhibiting school transgressions are more likely to be working in middle-skill STEMM fields than in middle-skill non-STEMM fields as adults; the same is not true of the high-skill workforce. CONCLUSIONS: One-size-fits-all policies for broadening participation in the STEMM workforce across skill-level are unlikely to be successful. Further, programs that are designed to generate wonder and fascination with STEMM content may be successful in attracting more girls. However, to promote greater participation of individuals from traditionally underrepresented ethnic minority groups in STEMM, programs that support choices toward higher educational attainment, specifically four-year college degree attainment, are more likely to be successful.

Chronic abdominal pain secondary to mesenteric panniculitis treated successfully with endoscopic ultrasonography-guided celiac plexus block: A case report.

Mesenteric panniculitis is a chronic illness that is characterized by fibrosing inflammation of the mesenteries that can lead to intractable abdominal pain. Pain control is a crucial component of the management plan. Most patients will improve with oral corticosteroids treatment, however, some patients will require a trial of other immunosuppressive agents, and a minority of patients will continue to have refractory disease. Endoscopic ultrasound guided celiac plexus block is used frequently to control abdominal pain in patients with pancreatic pathology. To our knowledge there are no case reports describing its use in mesenteric panniculitis patients with refractory abdominal pain.

Integrating fluorescent biosensor data using computational models.

This book chapter provides a tutorial on how to construct computational models of signaling networks for the integration and interpretation of FRET-based biosensor data. A model of cAMP production and PKA activation is presented to provide an example of the model building process. The computational model is defined using hypothesized signaling network structure and measured kinetic parameters and then simulated in Virtual Cell software. Experimental acquisition and processing of FRET biosensor data is discussed in the context of model validation. This data is then used to fit parameters of the computational model such that the model can more accurately predict experimental data. Finally, this model is used to show how computational experiments can interrogate signaling networks and provide testable hypotheses. This simple, yet detailed, tutorial on how to use computational models provides biologists that use biosensors a powerful tool to further probe and evaluate the underpinnings of a biological response.

Scaffold state switching amplifies, accelerates, and insulates protein kinase C signaling.

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.

A presentation of latent tropical sprue in a Canadian hospital.

Tropical sprue (TS) is a chronic diarrheal disease of unknown etiology characterized by malabsorption and small bowel mucosal abnormalities. TS affects residents of, and visitors to, endemic tropical regions. Rarely the disease may remain latent for several years, and to date, few cases of latent TS have been reported in Europe or North America. However, in our increasingly multicultural communities and in a 'global village' where travel is common, clinicians must maintain a high index of suspicion for TS in patients presenting with diarrhea and malabsorption who have traveled to endemic regions. TS may mimic common diarrheal diseases that are seen in developed nations, including celiac disease, Crohn's disease, bacterial overgrowth, and other infectious etiologies. Accordingly, once these more common etiologies have been ruled out, TS must be considered in patients presenting with diarrhea after travel to endemic regions. We present a unique Canadian case of latent TS, with a brief review of the diagnostic approach and treatment.

Triphasic mixture model of cell-mediated enzymatic degradation of hydrogels.

One critical component of engineering living tissue equivalents is the design scaffolds (often made of hydrogels) whose degradation kinetics can match that of matrix production by cells. However, cell-mediated enzymatic degradation of a hydrogel is a highly complex and nonlinear process that is challenging to comprehend based solely on experimental observations. To address this issue, this study presents a triphasic mixture model of the enzyme-hydrogel system, which consists of a solid polymer network, water and enzyme. On the basis mixture theory, the rubber elasticity theory and the Michaelis-Menton kinetics for degradation, the model naturally incorporates a strong coupling between gel mechanical properties, the kinetics of degradation and the transport of enzyme through the gel. The model is then used to investigate the particular problem of a single spherical enzyme-producing cell, embedded in a spherical hydrogel domain, for which the governing equations can be cast within the cento-symmetric assumptions. The governing equations are subsequently solved using an implicit nonlinear finite element procedure to obtain the evolution of enzyme concentration and gel degradation through time and space. The model shows that two regimes of degradation behaviour exist, whereby degradation is dominated either by diffusion or dominated by reaction kinetics. Depending on the enzyme properties and the initial hydrogel design, the temporal and spatial changes in gel cross-linking are dramatically impacted, a feature that is likely to strongly affect new tissue development.

Bigger, better, faster: principles and models of AKAP anchoring protein signaling.

A kinase anchoring proteins (AKAPs) bind multiple signaling proteins and have subcellular targeting domains that allow them to greatly impact cellular signaling. AKAPs localize, specify, amplify, and accelerate signal transduction within the cell by bringing signaling proteins together in space and time. AKAPs also organize higher-order network motifs such as feed forward and feedback loops that may create complex network responses, including adaptation, oscillation, and ultrasensitivity. Computational models have begun to provide an insight into how AKAPs regulate signaling dynamics and cardiovascular pathophysiology. Models of mitogen-activated protein kinase and epidermal growth factor receptor scaffolds have revealed additional design principles and new methods for representing signaling scaffolds mathematically. Coupling computational modeling with quantitative experimental approaches will be increasingly necessary for dissecting the diverse information processing functions performed by AKAP signaling complexes.

Degradation improves tissue formation in (un)loaded chondrocyte-laden hydrogels.

BACKGROUND: Photopolymerizable poly(ethylene glycol) (PEG) hydrogels offer a platform to deliver cells in vivo and support three-dimensional cell culture but should be designed to degrade in sync with neotissue development and endure the physiologic environment. QUESTIONS/PURPOSES: We asked whether (1) incorporation of degradation into PEG hydrogels facilitates tissue development comprised of essential cartilage macromolecules; (2) with early loading before pericellular matrix formation, the duration of load affects matrix production; and (3) dynamic loading in general influences macroscopic tissue development. METHODS: Primary bovine chondrocytes were encapsulated in hydrogels (n = 3 for each condition). The independent variables were hydrogel degradation (nondegrading PEG and degrading oligo(lactic acid)-b-PEG-b-oligo(lactic acid) [PEG-LA]), culture condition (free swelling, unconfined dynamic compressive loading applied intermittently for 1 or 4 weeks), and time (up to 28 days). The dependent variables were neotissue deposition through biochemical contents, immunohistochemistry, and compressive modulus. RESULTS: Degradation led to 2.3- and 2.9-fold greater glycosaminoglycan and collagen contents, respectively; macroscopic cartilage-like tissue formation comprised of aggrecan, collagen II and VI, link protein, and decorin; but decreased moduli. Loading, applied early or throughout culture, did not affect neotissue content in either hydrogel but affected neotissue spatial distribution in degrading hydrogels where 4 weeks of loading appeared to enhance hydrogel degradation resulting in tissue defects. CONCLUSIONS: PEG-LA hydrogels led to macroscopic tissue development comprised of key cartilage macromolecules under loading, but hydrogel degradation requires further tuning. CLINICAL RELEVANCE: PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.

Up-regulated dopamine D1 receptor binding can be detected in vivo following repeated SCH 23390, but not SKF 81297 or 6-hydroxydopamine, treatments.

Three different pharmacological treatments, previously shown to cause dopamine D1 receptor supersensitivity in rats, were studied for changes in the binding of R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) labeled with carbon-11. Rats treated subchronically with the full dopamine D1 receptor agonist R/S-(+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 81297) showed no significant difference in dopamine D1 receptor binding. Similarly, unilateral 6-hydroxydopamine lesioning, followed by apomorphine screening for contralateral rotation, failed to cause significant differences in the rat brain distribution of [11C]SCH 23390 in the lesioned versus the nonlesioned striatal sides. In contrast, repeated exposure with the dopamine D1 receptor antagonist SCH 23390 significantly enhanced the uptake of [11C]SCH 23390 in the dopamine D1 receptor-rich striatum and olfactory tubercles. These results demonstrate that [11C]SCH 23390 can significantly detect enhanced binding in rat brain regions expected to have up-regulated dopamine D1 receptors. The failure of [11C]SCH 23390 to reveal any differences after subchronic agonist or 6-hydroxydopamine treatments suggests that the behavioural supersensitization induced by these treatments is possibly due to changes to the high-affinity state or to components downstream of dopamine D1 receptors in the signal transduction pathway. The present study has implications for studies imaging dopamine D1 receptors in neuropsychiatric disorders with abnormal dopamine stimulation using positron emission tomography.