IPEx: A gamification tool for learner application of pharmacologic principles of opioid use, misuse, and addiction.

Opioids are often prescribed to treat chronic pain ailments, despite lack of evidence for many conditions. Prescriptions frequently become the gateway to opioid misuse and abuse. In response to the opioid crisis, medical school educators in the state of Pennsylvania developed core competencies pertaining to opioids and addiction for which all medical students should demonstrate proficiency before graduation. To enable students to achieve these competencies, we developed a web-based app (IPEx) that delivers a gamified experience for learners in which they are (re)exposed to opioid competencies and practice applying pharmacologic principles in the context of a series of longitudinal patient scenarios. Learning and application are measured by student responses to application questions before and after each of five modules. Prior to launching the IPEx tool broadly, we wished to test the application questions; thus, we invited fourth year medical students to complete a 45 question quiz based on IPEx module content. Students had no specific preparation prior to taking the quiz but had been exposed to all content elsewhere in the curriculum. A total of 45 of 141 medical students (32%) opted to complete the quiz (mean score was 47% ± 13%; range 18%-73%). Cronbach alpha for the instrument was .74. These results suggest that the instrument has internal validity, and medical students have room for growth when it comes to application of opioid related competencies, a situation that the IPEx tool may be uniquely suited to remedy.

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

A quantification of pathway components supports a novel model of Hedgehog signal transduction.

The secreted protein Hedgehog (Hh) plays a critical instructional role during metazoan development. In Drosophila, Hh signaling is interpreted by a set of conserved, downstream effectors that differentially localize and interact to regulate the stability and activity of the transcription factor Cubitus interruptus. Two essential models that integrate genetic, cell biological, and biochemical information have been proposed to explain how these signaling components relate to one another within the cellular context. As the molar ratios of the signaling effectors required in each of these models are quite different, quantitating the cellular ratio of pathway components could distinguish these two models. Here, we address this important question using a set of purified protein standards to perform a quantitative analysis of Drosophila cell lysates for each downstream pathway component. We determine each component's steady-state concentration within a given cell, demonstrate the molar ratio of Hh signaling effectors differs more than two orders of magnitude and that this ratio is conserved in vivo. We find that the G-protein-coupled transmembrane protein Smoothened, an activating component, is present in limiting amounts, while a negative pathway regulator, Suppressor of Fused, is present in vast molar excess. Interestingly, despite large differences in the steady-state ratio, all downstream signaling components exist in an equimolar membrane-associated complex. We use these quantitative results to re-evaluate the current models of Hh signaling and now propose a novel model of signaling that accounts for the stoichiometric differences observed between various Hh pathway components.

Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2.

The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous human pathologies. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a microtubule-associated Hedgehog signaling complex (HSC). At a minimum, the HSC consists of the Kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). In response to HSC activation, the ratio between repressor and activator forms of Ci is altered, determining the expression levels of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. Here, we describe a functional interaction between Smo and Cos2, which is necessary for Hh signaling. We propose that this interaction is direct and allows for activation of Ci in response to Hh. This work fills in the last major gap in our understanding of the Hh signal transduction pathway by suggesting that no intermediate signal is required to connect Smo to the HSC.

The carboxyl-terminal domain of the protein kinase fused can function as a dominant inhibitor of hedgehog signaling.

The secreted protein hedgehog (Hh) plays a critical role in the developmental patterning of multiple tissues. In Drosophila melanogaster, a cytosolic multiprotein signaling complex appears necessary for Hh signaling. Genes that encode components of this Hh signaling complex (HSC) were originally identified and characterized based on their genetic interactions with hh, as well as with each other. It is only in recent years that the mechanistic functions of these components have begun to be unraveled. Here, we have investigated the relationship between two components of the HSC, the serine/threonine protein kinase Fused (Fu) and the kinesin-related protein Costal2 (Cos2). We have reconstituted a Fu/Cos2 complex in vitro and shown that Fu is able to directly associate with Cos2, forming a complex whose molecular size is similar to a previously described complex found in Drosophila cell extracts. We have also determined that the carboxyl-terminal domain of Fu is necessary and sufficient for the direct binding of Fu to Cos2. To validate the physiological relevance of this interaction, we overexpressed the carboxyl-terminal domain of Fu in wild-type flies. These flies exhibit a phenotype similar to that seen in fu mutants and consistent with an hh loss-of-function phenotype. We conclude that the carboxyl-terminal domain of Fu can function in a dominant negative manner, by preventing endogenous Fu from binding to Cos2. Thus, we provide the first evidence that Hh signaling can be compromised by targeting the HSC for disruption.

Biochemical fractionation of Drosophila cells.

This chapter describes how to perform basic biochemical fractionations of Drosophila cells, and how to begin to characterize the proteins in the resulting fractions. The protocols include maintenance and transfection of Drosophila cell lines (Section 3.1.), hypotonic lysis (Section 3.2.), and separation of cellular lysates into cytosolic and membrane enriched fractions (Section 3.3.). Cytosolic proteins and those extracted from the membrane enriched fraction can be characterized by size exclusion liquid chromatography (Section 3.4.),while the membrane enriched fraction can be subjected to equilibrium density centrifugation to separate different types of cellular membranes from dense, nonmembranous cellular components (Section 3.5.). The resulting fractions can be used to examine the subcellular localization of a given protein, or the activity of a given protein in various subcellular localizations. When the protein of interest is involved in a signaling pathway, its subcellular localization can provide insight into its mechanism of action in the pathway.

Regulation of Hedgehog signaling: a complex story.

The Hedgehog (Hh) signal transduction pathway plays critical instructional roles during development. Activating mutations in human Hh signaling components predispose to a variety of tumor types, and have been observed in sporadic tumors occurring in a wide range of organs. Multiple insights into the regulation of Hh signaling have been achieved through studies using Drosophila melanogaster as a model organism. In Drosophila, regulation of the transcription factor Cubitus interruptus (Ci) is the ultimate target of the Hh pathway. Ci is regulated through communication of the membrane proteins Patched (Ptc) and Smoothened (Smo) to the intracellular Hedgehog Signaling Complex (HSC) in response to a graded concentration of Hh ligand. The HSC consists of the Kinesin Related Protein, Costal2 (Cos2), the serine-threonine protein kinase. Fused (Fu) and Ci. In the absence of Hh stimulation, the HSC is involved in processing of Ci to a truncated repressor protein. In response to Hh binding to Ptc, processing of Ci is blocked to allow for accumulation of full-length Ci activator protein(s). Differential concentrations of Hh ligand stimulate production of Ci transcriptional activators of varying strength, which facilitate activation of distinct subsets of target genes. The mechanism(s) by which Ptc and Smo communicate with the HSC in response to differential ligand concentrations to regulate Ci function are not yet fully elucidated. Here, we review what is known about regulation of individual Hh signaling components, concentrating on the mechanisms by which the Hh signal is propagated through Smo to the HSC.

A philosophy of anti-infectives as a guide in the search for new drugs for tuberculosis.

How we develop antibiotics is shaped by how we view infectious disease. Given the urgent need for new chemotherapeutics for tuberculosis and other infectious diseases, it is timely to reconsider a view of infectious disease that is strongly supported by contemporary evidence but that has rarely been applied in antibiotic development. This view recognizes the importance of nonreplicating bacteria in persistent infections, acknowledges the heterogeneity and stringency of chemical environments encountered by the pathogen in the host, and emphasizes metabolic adaptation of the host and the pathogen during their competition. For example, efforts in our lab are guided by the perspective that Mycobacterium tuberculosis (Mtb) has co-evolved with the human immune response, with the result that Mtb turns host-imposed metabolic adversity to its own advantage. We seek chemotherapeutics that turn Mtb's adversity to the host's advantage.

The Kinesin-related protein Costal2 associates with membranes in a Hedgehog-sensitive, Smoothened-independent manner.

In Drosophila, Hedgehog (Hh) signal transduction has been shown to require a multiprotein complex (Hedgehog signaling complex (HSC)), which includes the Kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). We present evidence that a biologically relevant fraction of the HSC is found in association with cellular membranes. We demonstrate that Cos2 is capable of tethering an exogenous protein to vesicular membranes and that Cos2 association with membranes is Hh-sensitive. In addition, we demonstrate that Cos2 associates with membranes in cells that lack the transmembrane protein Smoothened (Smo) through a domain of Cos2 distinct from its recently characterized Smo binding domain. We suggest that an Hh-regulated membrane binding activity of Cos2 is part of the mechanism by which Cos2 contributes to Hh signaling. We propose a model in which there are two distinct HSCs with discrete subcellular localizations and activities: one is endosome-associated and facilitates production of a repressor form of Ci (HSC-R), and one is Smo-associated and promotes Ci activation (HSC-A). In response to Hh and through interaction with Cos2, Smo mediates both inhibition of the endosome-associated HSC-R and activation of HSC-A at the plasma membrane.