Training of Non-expert Users Using Remotely Delivered, Point-of-Care Tele-Ultrasound: A Proof-of-Concept Study in 2 Canadian Communities.

ABSTRACT: Many physicians, particularly those practicing in remote regions, lack training opportunities to develop point-of-care ultrasound (POCUS) skills. This pretest-posttest study quantified the skill improvement of learners after participating in a virtual training program that used real-time, remotely delivered point-of-care tele-ultrasound (tele-POCUS) for teaching and learner feedback provision. Ten physicians practicing in an urban tertiary (Kingston, Ontario, Canada, n = 6) or remote care center (Moose Factory, Ontario, Canada, n = 4) completed a 3-week educational program that consisted of e-learning module review, independent image acquisition practice, and expert-guided tele-POCUS consultations. Pretraining and posttraining assessments were performed to evaluate skill enhancement in image acquisition, image quality, and image interpretation for cardiac and lung/pleura POCUS using a 5-point Likert scale. A total of 76 tele-POCUS consultations were performed during the study period. Significant improvements in image quality were noted following remotely delivered mentorship and guidance (all P < 0.01). In cardiac POCUS, pretraining and posttraining comparisons noted significant improvements in image acquisition (means, 2.69-4.33; P < 0.02), quality (means, 2.40-4.03; P < 0.01), and interpretation (means, 2.50-4.40; P < 0.02). In lung/pleura POCUS, significant improvements in image acquisition (means, 3.00-4.43; P < 0.01), quality (means, 3.23-4.37; P < 0.01), and interpretation (means, 3.00-4.40; P < 0.01) were demonstrated. Introductory ultrasound can be taught to novice users using a virtual, live-streamed training format with tele-POCUS while demonstrating significant enhancement in imaging skills.

Inexpensive, High-Fidelity Model to Simulate Ultrasound-Guided Pericardiocentesis for Cardiology Resident Training.

Adult cardiology residency training programs require residents to become proficient at many procedural skills, including pericardiocentesis. However, in many programs, opportunities to perform this procedure are limited. Expensive mannequins have been developed to assist with teaching this skill, however, the associated cost make them impractical for many programs. We hypothesized that a low-cost, high-fidelity pericardiocentesis model could be constructed using items easily accessible to any consumer. We describe a pericardiocentesis model made from pork skin, pork ribs, gelatin, a plastic bag, and an avocado. Total cost was less than CAD$40.00 and preparation time was approximately 60 minutes. The model was evaluated with a survey by 14 senior cardiology and critical care residents as well as 3 experienced senior cardiologists. Imaging results from the ultrasound revealed that the target fluid was easily visualized and all trainees were successful in aspirating fluid. The model was durable and withstood more than a dozen punctures, demonstrating its ability to train multiple residents. Respondents to the survey reported the model as highly realistic. All cardiology residents agreed or strongly agreed that the model should be incorporated into their formal curriculum. This study shows that a low-cost, high-fidelity model can be constructed and easily implemented into the formal curriculum of adult cardiology residency programs. It allows residents the opportunity to practice pericardiocentesis in a low-risk setting on a high-yield device.

Head to head: The role of academic competition in undergraduate anatomical education.

Competition is a key element in many educational games and is often adopted by educators in an effort to motivate and excite their students. Yet, the use of academic competition in educational institutions remains the subject of much debate. Opponents argue that academic competition causes an increase in student anxiety and divides their attention. However, if the contexts of academic competition are defined, could the inclusion of a game-like competition in a university course be a viable and beneficial method of engaging students? Students (n = 67) were recruited from an undergraduate human anatomy course at Western University. Using a crossover design, students were exposed to a competitive tournament either at the time of their first term test or second term test. The anatomical knowledge of participating students was assessed prior to the start of the study using a baseline anatomy test. Following treatment with an online competitive anatomy tournament, student's term test grades and final course grades were analyzed. Both the second term test scores (F(2,64) = 3.743, P = 0.029) and overall course grades (F(2,64) = 3.356, P = 0.041) were found to be significantly different (P < 0.05) for individuals in the competitive group when compared to their non-competing peers. As suggested by the literature where organized competition in the classroom correlates to improved academic performance, this study uncovered significant results pertaining to increased academic performance resulting from participating in tournament-based competition. In light of these positive results, further exploration of the effects of academic competition on student performance across age brackets and disciplines is warranted.

The duplicated retinol-binding protein 7 (rbp7) genes are differentially transcribed in embryos and adult zebrafish (Danio rerio).

Genomic and cDNA sequences coding for two cellular retinol-binding proteins (rbp) in zebrafish were retrieved from DNA sequence databases. Phylogenetic analysis revealed that these proteins were most similar to mammalian RBP7/Rbp7 proteins. Hence, the genes coding for these proteins were named rbp7a and rbp7b. Using a radiation hybrid panel, rbp7a and rbp7b were mapped to the zebrafish chromosomes 23 and 6, respectively. Conserved gene synteny indicated that these genes most likely arose as a result of a fish-specific whole-genome duplication event that had occurred 230-400 million years ago. Whole-mount in situ hybridization to zebrafish embryos detected rbp7a transcripts from the sphere stage (4h post-fertilization (hpf)) in the forerunner cells and the yolk syncytial layer, as well as in Kuppfer's vesicle and the periderm at 12 hpf. The transcripts of rbp7b were seen primarily in the somite stages (10-24 hpf) of zebrafish embryos, but also in the floor plate and hypochord, and did not overlap with the distribution of rbp7a transcripts in embryos. The hybridization signal for rbp7a and rbp7b transcripts was not detected in embryos after 12 hpf and 24 hpf, respectively. While transcripts for both rbp7a and rbp7b were found in all adult tissues assayed by RT-qPCR, the steady-state level of rbp7a transcripts were significantly higher than that of rbp7b transcripts in gill and ovary, whereas rbp7b transcripts were significantly higher than rbp7a transcripts in muscle and brain. The distribution of rbp7a and rbp7b transcripts in embryos and adult zebrafish indicate that the cis-elements that control the transcriptional regulation of the rbp7a and rbp7b genes have diverged considerably since their duplication.

PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma.

Gap junction intercellular communication and cell-cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell-cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin 43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell-cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell-cell adhesion.

Enhanced neurite outgrowth in PC12 cells mediated by connexin hemichannels and ATP.

Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.

Connexin over-expression differentially suppresses glioma growth and contributes to the bystander effect following HSV-thymidine kinase gene therapy.

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.

Nerve growth factor increases connexin43 phosphorylation and gap junctional intercellular communication.

The function of gap junctions is regulated by the phosphorylation state of their connexin subunits. Numerous growth factors are known to regulate connexin phosphorylation; however, the effect of nerve growth factor on gap junction function is not understood. The phosphorylation of connexin subunits is a key event during many aspects of the lifecycle of a connexin, including open/close states, assembly/trafficking, and degradation, and thus affects the functionality of the channel. PC12 cells infected with connexin43 (Cx43) retrovirus were used as a neuronal model to characterize the signal transduction pathways activated by nerve growth factor (NGF) that potentially affect the functional state of Cx43. Immunoblot analysis demonstrated that Cx43 and the mitogen-activated protein kinase (MAPK), ERK-1/2, were phosphorylated in response to TrkA activation via NGF and that phosphorylation could be prevented by treatment with the MEK-1/2 inhibitor U0126. The effects of NGF on gap junction intercellular communication were examined by monitoring fluorescence recovery after photobleaching PC12-Cx43 cells preloaded with calcein. Fluorescence recovery in the photobleached area increased after NGF treatment and decreased when pretreated with the MEK-1/2 inhibitor U0126. These data are the first to show a direct signaling link between neurotrophins and the phosphorylation of connexin proteins through the MAPK pathway resulting in increased gap junctional intercellular communication. Neurotrophic regulation of connexin activity provides a novel mechanism of regulating intercellular communication between neurons during nervous system development and repair.

Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication.

The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.