Early Introduction of Dermatology Clinical Skills in Medical Training.

BACKGROUND: Skin diseases are among the most common diseases encountered by healthcare professionals. Despite this, dedicated dermatology teaching is limited in most Canadian medical school training programs. This is especially true of clinical skills training. OBJECTIVES: To determine the impact of early introduction of dermatology clinical skills in Canadian undergraduate medical training. Secondary objectives included examining the impact of having sessions co-led by a clinician with expertise in the field of dermatology. METHODS: A half-day dermatology clinical skills session was introduced in the University of Toronto preclerkship medical school during the dermatology week. Sessions were co-led by staff or resident dermatologists. Sessions were evaluated using student pre- and postclinical skills test scores, as well as experience questionnaires completed by both tutors and students. RESULTS: The clinical skills session was well received by both students and tutors, with no cumulative score less than 4.34 on a 5-point Likert scale. The majority of students agreed or strongly agreed that the introduction of clinical skills in the early years of the curriculum facilitated learning consolidation (99.5%). Comparison of pre- and post-test scores showed that students' dermatology comprehension improved by an average of 12.1% following completion of the session. In questionnaire responses, students and nondermatology co-tutors emphasized the impact of having an expert in the field of dermatology co-lead the session. CONCLUSIONS: Students valued early clinical skills exposure to dermatology and felt it enhanced their knowledge and abilities. This study demonstrates learning consolidation when dermatologic didactic teachings are paired with practical clinical skills sessions.

Drug-Induced Photosensitivity-An Update: Culprit Drugs, Prevention and Management.

Photosensitive drug eruptions are cutaneous adverse events due to exposure to a medication and either ultraviolet or visible radiation. In this review, the diagnosis, prevention and management of drug-induced photosensitivity is discussed. Diagnosis is based largely on the history of drug intake and the appearance of the eruption primarily affecting sun-exposed areas of the skin. This diagnosis can also be aided by tools such as phototesting, photopatch testing and rechallenge testing. The mainstay of management is prevention, including informing patients of the possibility of increased photosensitivity as well as the use of appropriate sun protective measures. Once a photosensitivity reaction has occurred, it may be necessary to discontinue the culprit medication and treat the reaction with corticosteroids. For certain medications, long-term surveillance may be indicated because of a higher risk of developing melanoma or squamous cell carcinoma at sites of earlier photosensitivity reactions. A large number of medications have been implicated as causes of photosensitivity, many with convincing clinical and scientific supporting evidence. We review the medical literature regarding the evidence for the culpability of each drug, including the results of phototesting, photopatch testing and rechallenge testing. Amiodarone, chlorpromazine, doxycycline, hydrochlorothiazide, nalidixic acid, naproxen, piroxicam, tetracycline, thioridazine, vemurafenib and voriconazole are among the most consistently implicated and warrant the most precaution by both the physician and patient.

Optimistic honesty: understanding surgeon and patient perspectives on hopeful communication in pancreatic cancer care.

BACKGROUND: Prognosis conversations between surgical oncologists and patients with pancreatic cancer are critically important and challenging. Surgeons and their patients often have discrepant understandings of prognosis despite extensive conversations. Little is known about how surgeons approach prognosis conversations with these patients; patients' experiences with these conversations are also not well understood. This qualitative study sought to better understand surgeon and patient perspectives on communication in pancreatic cancer care with a view toward improvement. METHODS: Grounded theory methodology was used. Semi-structured interviews were conducted with surgical oncologists and patients who had undergone surgical resection with curative intent for periampullary cancer. Data were collected and analyzed inductively and iteratively to the point of theoretical saturation. RESULTS: 10 surgeons and 10 patients participated. Three inter-linking concepts were found to drive surgeon-patient conversations: understanding, trust and hope. Surgeons delicately and purposefully tailored information for patients, striving to deliver essential though honest, empathetic and hopeful messages. Patients desired simple, truthful explanations that demonstrated caring and fostered optimism. CONCLUSION: Surgeons and patients with pancreatic cancer value optimistic honesty in tailored prognosis conversations. Perceived discrepancies in surgeon-patient understanding must be contextualized within efforts to establish a sufficient understanding, high level of trust, and optimistic stance of hope.

Management of scalp psoriasis: current perspectives.

Psoriasis is a chronic inflammatory condition. The age of onset, chronicity, physical, and psychosocial consequences of the disease cause psoriasis to have a significant impact on patient quality of life. Scalp psoriasis is no different, and effective treatment results in an improvement in quality of life. Successful management of scalp psoriasis includes topical therapies that are acceptable to the patient for mild-to-moderate disease, and systemic therapies for recalcitrant or moderate-to-severe disease. The most effective topical therapies are corticosteroid products, or combination products with calcipotriol and corticosteroid. Newer vehicle options provide more attractive and pleasing products for patients and may improve adherence. The current perspectives for management of scalp psoriasis are discussed including available data for systemic therapy of severe disease.

FGF23: mediator of poor prognosis in a sizeable subgroup of patients with castration-resistant prostate cancer presenting with severe hypophosphatemia?

Castration-resistant prostate cancer (CRPC) is an advanced and incurable stage of the second most frequently diagnosed malignancy in men globally. Current treatment options improve survival modestly but eventually fail due to intrinsic or acquired therapeutic resistance. A hypothesis is presented wherein circulating levels of fibroblast growth factor 23 (FGF23), an endocrine member of the fibroblast growth factor family with phosphaturic properties, are proposed as a prognostic and predictive marker to identify CRPC patients with poor prognosis that are amenable to FGF23 antibody therapy (FGF23i) or treatment with fibroblast growth factor receptor inhibitors (FGFRi). With respect to the latter, FGF23 may also serve as a pharmacodynamic marker enabling individualized FGFRi dosing. We recently discovered that the development of severe and sustained hypophosphatemia in CRPC patients undergoing zoledronic acid therapy for bone metastases was associated with markedly worse prognosis compared to patients without or with only mild and transient hypophosphatemia. Severe hypophosphatemia is a typical manifestation of tumor-induced hypophosphatemic osteomalacia (TIO), a paraneoplastic condition mediated by FGF23 overexpression in most instances. While the postulated tumor-promoting role of FGF23 in CRPC or other malignancies has not yet been studied, several lines of evidence suggest that FGF23 may mediate both severe hypophosphatemia (via its endocrine properties) and aggressive CRPC behavior (via autocrine and paracrine activities): (i) FGF23 and the necessary signalling machinery (i.e. members of the fibroblast growth factor receptor [FGFR] family and the essential co-receptor α-KLOTHO [KL]) are highly expressed in a sizeable subgroup of CRPC patients; (ii) FGF/FGFR signalling plays important roles in prostate cancer; (iii) FGF23 can induce its own expression via a positive autocrine feedback loop involving FGFR1; and (iv) this positive feedback loop may be triggered by bone-targeted therapies frequently used for the treatment of CRPC-associated bone metastases. While there is a lack of personalized treatment strategies in the management of CRPC to date, FGF23 targeted therapy has the potential to fill this unmet clinical need in the not-so-distant future. In fact, FGFRi are currently in advanced clinical testing for a number of malignancies such as kidney and lung cancer, but there is a lack of conclusive data on FGFRi therapy in patients selected for FGF/FGFR pathway activation.

Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy.

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4B(C74A)in vitro and in vivo, we show that the effects of ATG4B(C74A) are cell type, treatment, and context-dependent. ATG4B(C74A) expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.

A phosphoproteomics approach to identify candidate kinase inhibitor pathway targets in lymphoma-like primary cell lines.

Mass spectrometry-based technologies are increasingly utilized in drug discovery. Phosphoproteomics in particular has allowed for the efficient surveying of phosphotyrosine signaling pathways involved in various diseases states, most prominently in cancer. We describe a phosphotyrosine-based proteomics screening approach to identify signaling pathways and tyrosine kinase inhibitor targets in highly tumorigenic human lymphoma-like primary cells. We identified several receptor tyrosine kinase pathways and validated SRC family kinases (SFKs) as potential drug targets for targeted selection of small molecule inhibitors. BMS-354825 (dasatinib) and SKI-606 (bosutinib), second and third generation clinical SFK/ABL inhibitors, were found to be potent cytotoxic agents against tumorigenic cells with low toxicity to normal pediatric stem cells. Both SFK inhibitors reduced ERK1/2 and AKT phosphorylation and induced apoptosis. This study supports the adaptation of high-end mass spectrometry techniques for the efficient identification of candidate tyrosine kinases as novel therapeutic targets in primary cancer cell lines.

CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope.

The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

Pooled lentiviral shRNA screening for functional genomics in mammalian cells.

Genome sequencing efforts have reformed the nature of biological inquiry, prompting the development of technologies for the functional annotation of mammalian genes. Based on methodologies originally discovered in plants and Caenorhabditis elegans, RNA interference has offered cell biologists an effective and reproducible approach to perturb gene function in mammalian cells and whole organisms. Initial application of RNA interference libraries targeting the human and mouse genomes relied on arrayed screening approaches, whereby each unique RNA interference reagent is arrayed into individual wells of a microtiter plate. These screens are not trivial to perform, requiring a substantial investment in infrastructure. In the past decade, many technological advances have been made that make genome-wide RNA interference screening more accessible to researchers and more feasible to perform in nonspecialized laboratories. Here, we describe a comprehensive protocol for pooled short-hairpin RNA screening, including methodologies for pooled lentivirus production, cell infection, genome-wide negative selection screening and resources for pooled screen deconvolution, and data analysis. As a technique, pooled shRNA screening is still in its infancy, but the methodology has already been successfully applied to probe diverse signaling pathways, as a means of drug target identification, and to identify essential genes in normal and cancer cell lines.

Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole.

Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.

A comprehensive platform for highly multiplexed mammalian functional genetic screens.

BACKGROUND: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. RESULTS: Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens. CONCLUSION: Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.

Selective targeting of neuroblastoma tumour-initiating cells by compounds identified in stem cell-based small molecule screens.

Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.

System-level analysis of neuroblastoma tumor-initiating cells implicates AURKB as a novel drug target for neuroblastoma.

PURPOSE: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. EXPERIMENTAL DESIGN: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. RESULTS: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. CONCLUSION: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets.

A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants.

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.