Voltage-sensitive gating of the Pannexin-1 channel

Since its discovery just over a decade ago, Pannexin-1 (Px1) has been recognized in a number of important physiological and pathophysiological processes such as taste, inflammation, and tumor suppression. This large-pore, polymodal ion channel was initially identified as ‗voltage-dependent,‘ though there have been no precise studies concerning the gating properties of Px1 to date. Because Px1 is expressed in excitable cells, identifying voltage-gating properties of Px1 was our primary goal. Using the two-electrode voltage clamp technique, we showed for the first time that Px1 is a weakly voltage-gated channel. Depolarizing voltages up to +200 mV revealed half-maximal activation at +51 mV and a weak voltage-dependence through generation of a complete Boltzmann activation curve. We also showed that Px1 activates in \u3c 3.5 ms, consistent with the time frame of action potentials (1-4 ms). Opening rates of Px1 also seemed to be very weakly voltage-dependent. Further, we showed that Px1 displays consistent current decay at depolarizing voltages greater than +100 mV. Additionally, using two cell systems to exogenously express Px1, we observed a marked decrease of functional Px1 expression within ~24 hours of injection. Taken together, our findings suggest that Px1 is a fast opening, voltage-sensitive channel that may have a number of mechanisms in place to prevent uncontrolled conductivity

Evidence for Diversity in Transcriptional Profiles of Single Hematopoietic Stem Cells

Hematopoietic stem cells replenish all the cells of the blood throughout the lifetime of an animal. Although thousands of stem cells reside in the bone marrow, only a few contribute to blood production at any given time. Nothing is known about the differences between individual stem cells that dictate their particular state of activation readiness. To examine such differences between individual stem cells, we determined the global gene expression profile of 12 single stem cells using microarrays. We showed that at least half of the genetic expression variability between 12 single cells profiled was due to biological variation in 44% of the genes analyzed. We also identified specific genes with high biological variance that are candidates for influencing the state of readiness of individual hematopoietic stem cells, and confirmed the variability of a subset of these genes using single-cell real-time PCR. Because apparent variation of some genes is likely due to technical factors, we estimated the degree of biological versus technical variation for each gene using identical RNA samples containing an RNA amount equivalent to that of single cells. This enabled us to identify a large cohort of genes with low technical variability whose expression can be reliably measured on the arrays at the single-cell level. These data have established that gene expression of individual stem cells varies widely, despite extremely high phenotypic homogeneity. Some of this variation is in key regulators of stem cell activity, which could account for the differential responses of particular stem cells to exogenous stimuli. The capacity to accurately interrogate individual cells for global gene expression will facilitate a systems approach to biological processes at a single-cell level

Implementation of interprofessional education (IPE) in 16 U.S. medical schools: Common practices, barriers and facilitators.

BackgroundEnhanced patient outcomes and accreditation criteria have led schools to integrate interprofessional education (IPE). While several studies describe IPE curricula at individual institutions, few examine practices across multiple institutions.PurposeTo examine the IPE integration at different institutions and determine gaps where there is potential for improvement.MethodIn this mixed methods study, we obtained survey results from 16 U.S. medical schools, 14 of which reported IPE activities.ResultsThe most common collaboration was between medical and nursing schools (93%). The prevalent format was shared curriculum, often including integrated modules (57%). Small group activities represented the majority (64%) of event settings, and simulation-based learning, games and role-play (71%) were the most utilized learning methods. Thirteen schools (81.3%) reported teaching IPE competencies, but significant variation existed. Gaps and barriers in the study include limitations of using a convenience sample, limited qualitative analysis, and survey by self-report.ConclusionsMost IPE activities focused on the physician role. Implementation challenges included scheduling, logistics and financial support. A need for effective faculty development as well as measures to examine the link between IPE learning outcomes and patient outcomes were identified

Expression Levels of Genes Previously Associated with HSC Genetic Profile

<div><p>Each single SP cell amplification experiment is represented by a different column (A–L). Genes given a P call by MAS5 are represented in red. Otherwise, they are shown in blue. The white numbers in the boxes represent the logged (base 2) gene expression levels calculated by RMA. The SCE column summarizes the number of P calls across five single-cell-equivalent (see text for explanation) amplification reactions.</p><p>(A) Genes consistently present in all single cell amplifications performed using GSC RT-PCR followed by oligonucleotide array analysis.</p><p>(B) Genes detected less frequently than expected. This can potentially be due to technical limitations or true biological variability among cells.</p></div

Selected Genes Consistently Expressed in SCE (Low Variance of Expression Levels), but Displaying High or Low Variability in Single SP Cells

<p>Genes with low variance in the SCE are likely to be detected accurately by our amplification procedure, since a fraction equivalent to one cell of the same pool of mRNA gives a consistent result across experiments: any variation detected in single cells should therefore reflect true differences in levels of gene expression. Some of these genes have low variation in single cells (B), while others (A) display different levels in distinct stem cells. The latter are likely to be responsible for heterogeneity in behavior of individual HSC (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020159#pgen-0020159-g003" target="_blank">Figure 3</a> for an explanation of the colors and numbers used).</p

Expression Levels Determined by RMA Using GSC RT-PCR Discriminate between Different Cell Populations and Correlate Tightly with Those Determined by Q-PCR

<div><p>(A) The figure represents a dendrogram for genes consistently above background level in more than 50% of the samples. All the genes were used, i.e., no prior selection for genes differentially expressed in the different populations was done. Each 10-cell sample of the lower region of SP cells (LA, LB, and LM), the upper region of SP cells (UN, UO, and UZ), or CD8 T lymphocytes (XB, XC, and XD) with two replicates averaged is represented. Unsupervised hierarchical clustering based on the Euclidean distance between expression levels for each gene separates clearly the three groups of samples.</p><p>(B) Twenty-two genes representing the full range of fold changes were selected for analysis. Relative expression levels (fold changes) obtained by RMA performed on microarrays prepared with our amplification procedure (horizontal axis) were plotted against those calculated using real-time Q-PCR (vertical axis). The regression equation and correlation coefficient are shown in the graph. The 95% confidence intervals for the regression are represented by the dashed lines.</p></div

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs

Summary: We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis. : Gunawardane and colleagues use CRISPR/Cas9 to deliver an excisable cassette to transcriptionally silent loci in hiPSCs, then accomplish excision of the cassette in a second step utilizing Cas9/CRISPR and the MMEJ and NHEJ DNA-repair pathways. Excision results in mEGFP tagging of the targeted loci. Upon differentiation, each of five tagged cell lines appropriately expresses a unique fluorescent fusion protein localized to the sarcomere in live cardiomyocytes. Keywords: CRISPR/Cas9, genome editing, cardiomyocyte differentiation, stem cells, iPSCs, MMEJ, live imaging, endogenous fluorescent tagging, mEGFP, HD