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1.  Gap Junction-Mediated Death of Retinal Neurons Is Connexin and Insult Specific: A Potential Target for Neuroprotection 
The Journal of Neuroscience  2014;34(32):10582-10591.
Secondary cell death via gap junctions (GJs) plays a role in the propagation of neuronal loss under a number of degenerative disorders. Here, we examined the role of GJs in neuronal death in the retina, which has arguably the most diverse expression of GJs in the CNS. Initially, we induced apoptotic death by injecting single retinal ganglion cells and glia with cytochrome C and found that this resulted in the loss of neighboring cells to which they were coupled via GJs. We next found that pharmacological blockade of GJs eradicated nearly all amacrine cell loss and reduced retinal ganglion cell loss by ∼70% after induction of either excitotoxic or ischemic insult conditions. These data indicate that the GJ-mediated secondary cell death was responsible for the death of most cells. Whereas genetic deletion of the GJ subunit Cx36 increased cell survivability by ∼50% under excitotoxic condition, cell loss in Cx45 knock-out mouse retinas was similar to that seen in wild-type mice. In contrast, ablation of Cx45 reduced neuronal loss by ∼50% under ischemic insult, but ablation of Cx36 offered no protection. Immunolabeling of the connexins showed differential changes in protein expression consistent with their differing roles in propagating death signals under the two insults. These data indicate that secondary cell death is mediated by different cohorts of GJs dependent on the connexins they express and the type of initial insult. Our results suggest that targeting specific connexins offers a novel therapeutic strategy to reduce progressive cell loss under different neurodegenerative conditions.
doi:10.1523/JNEUROSCI.1912-14.2014
PMCID: PMC4200109  PMID: 25100592
bystander effect; cell death; connexin; gap junctions; neuroprotection; retina
2.  Instability of Delay Classification and Determination of Early Intervention Eligibility in the First Two Years of Life 
Research in developmental disabilities  2013;35(1):10.1016/j.ridd.2013.10.017.
The purpose of this study was to determine the effectiveness of the Bayley Scales of Infant Development, Third Edition (Bayley-III) to track development and classify delays in low- and high-risk infants across the first two years of life. We assessed cognitive, language, and motor development in 24 low-risk full-term and 30 high-risk preterm infants via seven assessments performed between 3 and 24 months corrected age. The Bayley-III resulted in highly unstable delay classifications, low sensitivities, and poor positive predictive values across time. The results highlight that early intervention professionals, researchers, and policy makers should: (1) emphasize clinical opinion and prevalence of risk factors rather than standardized assessment findings when classifying delays and determining eligibility for services, and (2) develop more effective developmental assessments for infants and young children.
doi:10.1016/j.ridd.2013.10.017
PMCID: PMC3863394  PMID: 24176257
assessment; early intervention; eligibility; developmental delay; preterm; Bayley Scales of Infant Development; Third Edition
3.  Consumer Participation in Quality Improvements for Chronic Disease Care: Development and Evaluation of an Interactive Patient-Centered Survey to Identify Preferred Service Initiatives 
Background
With increasing attention given to the quality of chronic disease care, a measurement approach that empowers consumers to participate in improving quality of care and enables health services to systematically introduce patient-centered initiatives is needed. A Web-based survey with complex adaptive questioning and interactive survey items would allow consumers to easily identify and prioritize detailed service initiatives.
Objective
The aim was to develop and test a Web-based survey capable of identifying and prioritizing patient-centered initiatives in chronic disease outpatient services. Testing included (1) test-retest reliability, (2) patient-perceived acceptability of the survey content and delivery mode, and (3) average completion time, completion rates, and Flesch-Kincaid reading score.
Methods
In Phase I, the Web-based Consumer Preferences Survey was developed based on a structured literature review and iterative feedback from expert groups of service providers and consumers. The touchscreen survey contained 23 general initiatives, 110 specific initiatives available through adaptive questioning, and a relative prioritization exercise. In Phase II, a pilot study was conducted within 4 outpatient clinics to evaluate the reliability properties, patient-perceived acceptability, and feasibility of the survey. Eligible participants were approached to complete the survey while waiting for an appointment or receiving intravenous therapy. The age and gender of nonconsenters was estimated to ascertain consent bias. Participants with a subsequent appointment within 14 days were asked to complete the survey for a second time.
Results
A total of 741 of 1042 individuals consented to participate (71.11% consent), 529 of 741 completed all survey content (78.9% completion), and 39 of 68 completed the test-retest component. Substantial or moderate reliability (Cohen’s kappa>0.4) was reported for 16 of 20 general initiatives with observed percentage agreement ranging from 82.1%-100.0%. The majority of participants indicated the Web-based survey was easy to complete (97.9%, 531/543) and comprehensive (93.1%, 505/543). Participants also reported the interactive relative prioritization exercise was easy to complete (97.0%, 189/195) and helped them to decide which initiatives were of most importance (84.6%, 165/195). Average completion time was 8.54 minutes (SD 3.91) and the Flesch-Kincaid reading level was 6.8. Overall, 84.6% (447/529) of participants indicated a willingness to complete a similar survey again.
Conclusions
The Web-based Consumer Preferences Survey is sufficiently reliable and highly acceptable to patients. Based on completion times and reading level, this tool could be integrated in routine clinical practice and allows consumers to easily participate in quality evaluation. Results provide a comprehensive list of patient-prioritized initiatives for patients with major chronic conditions and delivers practice-ready evidence to guide improvements in patient-centered care.
doi:10.2196/jmir.3545
PMCID: PMC4285719  PMID: 25532217
ambulatory care; health care surveys; patient-centered care; consumer participation; medical oncology; chronic disease; cardiology; neurology
4.  Aminoterminal Amphipathic α-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production 
PLoS Pathogens  2014;10(11):e1004501.
Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.
Author Summary
With an estimated 180 million chronically infected individuals, hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. HCV is a positive-strand RNA virus that builds its replication complex on rearranged intracellular membranes, designated as membranous web. HCV nonstructural protein 4B (NS4B) is a key organizer of HCV membranous web and replication complex formation. Here, we provide a detailed structure-function analysis of an N-terminal amphipathic α-helix of NS4B, named AH1, and demonstrate that it plays key roles in shaping the membranous web as well as in virus production. We also show that the N-terminal part of NS4B adopts a dual membrane topology in a replicative context, possibly reflecting the different roles of this protein in the viral life cycle.
doi:10.1371/journal.ppat.1004501
PMCID: PMC4231108  PMID: 25392992
5.  A Business Case for Tele-Intensive Care Units 
The Permanente Journal  2014;18(4):76-84.
A tele-intensive care unit (tele-ICU) uses telemedicine, in an intensive care unit (ICU) setting, to care for critically ill patients by off-site clinical resources. This literature review examined a large number of studies of implementation in hospitals. The evidence supporting cost savings was mixed. Implementation of a tele-ICU system was associated with cost savings, shorter lengths of stay, and decreased mortality. However, two studies suggested increased hospital cost after implementation. Intensivists working these systems are able to more effectively treat ICU patients, providing better clinical outcomes for patients at lower costs compared with hospitals without a tele-ICU.
Objectives:
A tele-intensive care unit (tele-ICU) uses telemedicine in an intensive care unit (ICU) setting, applying technology to provide care to critically ill patients by off-site clinical resources. The purpose of this review was to examine the implementation, adoption, and utilization of tele-ICU systems by hospitals to determine their efficiency and efficacy as identified by cost savings and patient outcomes.
Methods:
This literature review examined a large number of studies of implementation of tele-ICU systems in hospitals.
Results:
The evidence supporting cost savings was mixed. Implementation of a tele-ICU system was associated with cost savings, shorter lengths of stay, and decreased mortality. However, two studies suggested increased hospital cost after implementation of tele-ICUs is initially expensive but eventually results in cost savings and better clinical outcomes.
Conclusions:
Intensivists working these systems are able to more effectively treat ICU patients, providing better clinical outcomes for patients at lower costs compared with hospitals without a tele-ICU.
doi:10.7812/TPP/14-004
PMCID: PMC4206175
6.  A Novel, Highly Sensitive Method for Assessing Gap Junctional Coupling 
Journal of neuroscience methods  2013;220(1):10.1016/j.jneumeth.2013.08.007.
To assess gap junctional intercellular communication we have developed a tracer-based methodology which is both highly sensitive and potentially adaptable for in vivo measurements. We found that injection of serotonin revealed significantly more intercellular communication than that injection of the most permeant synthetic tracer currently in use, neurobiotin. Furthermore, mechanical tracer loading steps can be replaced by transfection with human serotonin transporter and the inclusion of serotonin in the medium. Tracer and transporter are detected using immunocytochemical techniques and the presence of cells that are tracer-positive but transporter-negative indicates junctional communication. Tracer loading in vivo using transgenesis, electroporation or viral transduction to direct expression of transporter should be more easily accomplished than with mechanical loading methods.
doi:10.1016/j.jneumeth.2013.08.007
PMCID: PMC3808728  PMID: 23958747
gap junction; connexin; dye transfer; serotonin
7.  Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment 
Journal of Virology  2013;87(19):10612-10627.
Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.
doi:10.1128/JVI.01370-13
PMCID: PMC3807400  PMID: 23885072
8.  Complementary and Alternative Medicine use in women during pregnancy: do their healthcare providers know? 
Background
The National Institutes of Health reported in 2007 that approximately 38% of United States adults have used at least one type of Complementary and Alternative Medicine (CAM). There are no studies available that assess general CAM use in US pregnant women.
The objectives of our study were to determine the prevalence and type of CAM use during pregnancy at one medical center; understand who is using CAM and why they are using it; and assess the state of patients’ CAM use disclosure to their obstetrical providers.
Methods
A cross-sectional survey study of post-partum women was done to assess self-reported CAM use during pregnancy. Results of this survey were compared to results from a previous survey performed by this research team in 2006. Data were analyzed using binary logistic regression.
Results
In 2013, 153 women completed the survey, yielding a response rate of 74.3%. Seventy-two percent and 68.5% of participants reported CAM use during their pregnancies in 2006 and 2013 respectively. The percentage of participants who reported discussing CAM use with their obstetrical providers was less than 1% in 2006 and 50% in 2013. Increased use of different CAM therapies was associated with increased maternal age, primagravida, being US-born, and having a college education (p ≤ 0.05). However, these factors were poor predictors of CAM use.
Conclusions
Given the frequency of CAM use and the difficulty in predicting who is using it, obstetrical providers should consider being informed about CAM and incorporating discussions about its use into routine patient assessments.
doi:10.1186/1472-6882-14-85
PMCID: PMC3945795  PMID: 24592860
Complementary and Alternative Medicine; Pregnancy; Maternal-fetal health; Patient-physician communication; Self-care; Prevalence; Cross-sectional study
9.  Survey on Amacrine Cells Coupling to Retrograde-Identified Ganglion Cells in the Mouse Retina 
Purpose.
Retinal amacrine cells (ACs) may make inhibitory chemical synapses and potentially excitatory gap junctions on ganglion cells (GCs). The total number and subtypes of ACs coupled to the entire GC population were investigated in wild-type and three lines of transgenic mice.
Methods.
GCs and GC-coupled ACs were identified by the previously established LY-NB (Lucifer yellow–Neurobiotin) retrograde double-labeling technique, in conjunction with specific antibodies and confocal microscopy.
Results.
GC-coupled ACs (NB-positive and LY-negative) comprised nearly 11% of displaced ACs and 4% of conventional ACs in wild-type mice, and were 9% and 4% of displaced ACs in Cx45−/− and Cx36/45−/− mice, respectively. Their somas were small in Cx36/45−/− mice, but variable in other strains. They were mostly γ-aminobutyric acid (GABA)-immunoreactive (IR) and located in the GC layer. They comprised only a small portion in the AC subpopulations, including GABA-IR, glycine-IR, calretinin-IR, 5-HT-accumulating, and ON-type choline acetyltransferase (ChAT) ACs in wild-type and ChAT transgenic mice (ChAT- tdTomato). In the distal 80% of the inner plexiform layer (IPL), dense GC dendrites coexisted with rich glycine-IR and GABA-IR. In the inner 20% of the IPL, sparse GC dendrites presented with a major GABA band and sparse glycine-IR.
Conclusions.
Various subtypes of ACs may couple to GCs. ACs of the same immunoreactivity may either couple or not couple to GCs. Cx36 and Cx45 dominate GC-AC coupling except for small ACs. The overall potency of GC-AC coupling is moderate, especially in the proximal 20% of the IPL, where inhibitory chemical signals are dominated by GABA ACs.
Retrograde double-labeling was used to survey the populations of retinal amacrine cells (ACs) coupled to the ganglion cell (GC) populations in retinas from wild-type and three lines of transgenic mice. Less than 11% of ACs are found to couple to GCs, indicating generally moderate GC-AC coupling.
doi:10.1167/iovs.13-11774
PMCID: PMC3732024  PMID: 23821205
retrograde labeling; connexin; GABA; NOS; 5-HT; ChAT
10.  Möbius-strip-like columnar functional connections are revealed in somato-sensory receptive field centroids 
Receptive fields of neurons in the forelimb region of areas 3b and 1 of primary somatosensory cortex, in cats and monkeys, were mapped using extracellular recordings obtained sequentially from nearly radial penetrations. Locations of the field centroids indicated the presence of a functional system in which cortical homotypic representations of the limb surfaces are entwined in three-dimensional Möbius-strip-like patterns of synaptic connections. Boundaries of somatosensory receptive field in nested groups irregularly overlie the centroid order, and are interpreted as arising from the superposition of learned connections upon the embryonic order. Since the theory of embryonic synaptic self-organization used to model these results was devised and earlier used to explain findings in primary visual cortex, the present findings suggest the theory may be of general application throughout cortex and may reveal a modular functional synaptic system, which, only in some parts of the cortex, and in some species, is manifest as anatomical ordering into columns.
doi:10.3389/fnana.2014.00119
PMCID: PMC4215792  PMID: 25400552
cortical column; cortical development; synaptic organization; cortical response properties; neuromicrocircuitry; S1 segregates
11.  Urinary Tract Symptoms (LUTS) Secondary to Benign Prostatic Hyperplasia (BPH) and LUTS/BPH with Erectile Dysfunction in Asian Men: A Systematic Review Focusing on Tadalafil 
The World Journal of Men's Health  2013;31(3):193-207.
This review assesses lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) with or without erectile dysfunction (ED) and related therapies focusing on tadalafil. A literature search was obtained and reviewed for the epidemiology, treatment therapies, pathophysiology, and efficacy and safety of phosphodiesterase type 5 inhibitor (PDE5i) tadalafil in patients with LUTS/BPH. Approximately 42% of men aged 51 to 60 years have BPH. Approximately 90% of men aged 45 to 80 years have LUTS. Occurrence of LUTS increases with age for almost all racial/ethnic groups (range, 32% to 56%) with prevalence of LUTS highest among Hispanic men, then Blacks, Caucasians, and Asians. There is an independent relationship with LUTS/BPH and ED, with approximately 70% of men with LUTS/BPH having ED with severity of one disease often correlating with the other. The European Urological Association guidelines include the use of the PDE5i tadalafil. Tadalafil is the only therapy recommended for treatment of co-existing BPH and ED, while other therapies have unwanted ED side effects. The mode of action of tadalafil may involve different areas of the lower urinary tract such as smooth muscle cell relaxation in the bladder neck, prostate, and urethra, but there may also be resulting modulation of the afferent nerve activity. Tadalafil (5 mg) in Asian men with LUTS/BPH, similar to global studies, is efficacious and safe. Tadalafil (5 mg) improves co-existing LUTS/BPH and ED, independently. Men with LUTS/BPH likely also have ED. Asian men with LUTS/BPH have similar incidence rates, co-existing ED, comorbid diseases, and risks as non-Asian men. Tadalafil can improve co-existing LUTS/BPH and ED.
doi:10.5534/wjmh.2013.31.3.193
PMCID: PMC3888888  PMID: 24459652
Erectile dysfunction; Pharmacology; Phosphodiesterase 5 inhibitors; Prostatic hyperplasia; Tadalafil
12.  The Epigenetic Effects of a High Prenatal Folate Intake in Male Mouse Fetuses Exposed In Utero to Arsenic 
Toxicology and applied pharmacology  2012;264(3):439-450.
Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never been thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring.
doi:10.1016/j.taap.2012.08.022
PMCID: PMC3478409  PMID: 22959928
Arsenic; folate; epigenetics; DNA methylation; transplacental exposure; CD1 mice
13.  Visual responses in the lateral geniculate evoked by Cx36-independent rod pathways 
Vision research  2010;51(2):280-287.
Emerging evidence indicates rods can communicate with retinal ganglion cells (RGCs) via pathways that do not involve gap-junctions. Here we investigated the significance of such pathways for central visual responses, using mice lacking a key gap junction protein (Cx36−/−) and carrying a mutation that disrupts cone phototransduction (Gnat2cpfl3). Electrophysiological recordings spanning the lateral geniculate revealed rod-mediated ON and OFF visual responses in virtually every cell from all major anatomical sub-compartments of this nucleus. Hence, we demonstrate that one or more classes of RGC receive input from Cx36-independent rod pathways and drive extensive ON and OFF responses across the visual thalamus.
doi:10.1016/j.visres.2010.08.010
PMCID: PMC3741614  PMID: 20709095
mouse; scotopic; melanopsin; electroretinogram; connexin
14.  Gap Junctions Are Essential for Generating the Correlated Spike Activity of Neighboring Retinal Ganglion Cells 
PLoS ONE  2013;8(7):e69426.
Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets.
doi:10.1371/journal.pone.0069426
PMCID: PMC3720567  PMID: 23936012
15.  Field Trips as a Novel Means of Experiential Learning in Ambulatory Pediatrics 
Background
Parents and caregivers look to pediatric health care providers for guidance on feeding, safety issues, and child-care products for children, but trainees have infrequent first-hand exposure to child products marketed to parents.
Objective
To conduct a pilot study to assess an experiential field trip as a novel method of enhancing medical knowledge in ambulatory pediatric feeding and safety.
Methods
Resident physicians and medical students visited a local children's store, where they took part in an interactive store tour, product discussions, and product demonstrations led by a physician educator. Participants also completed a 20-question pretest and a 20-question posttest related to common ambulatory pediatric feeding and safety issues, based on recent American Academy of Pediatrics (AAP) policy statements and practice guidelines.
Results
Sixty-seven medical students and resident physicians participated in the study. Overall, participants' short-term knowledge significantly increased from 9.9 ± 2.6 to 15.4 ± 2.2 questions correct (P  =  .001), with statistically significant gains (P < .001) on both the feeding and safety sections of the test. There were no differences in improvement based on participant's student or resident status, residency program type, program year, sex, or parental status. Ninety-five percent of the participants believed that their knowledge was enhanced by this approach, and participants uniformly agreed that this field trip was valuable to their pediatric training and that such field trip sessions should continue.
Conclusions
The inclusion of experiential learning through an interactive field trip in the curriculum of medical training was acceptable and feasible and showed short-term improvements in knowledge of AAP safety and feeding concepts.
doi:10.4300/JGME-D-11-00173.1
PMCID: PMC3399621  PMID: 23730450
16.  Architecture and biogenesis of plus-strand RNA virus replication factories 
World Journal of Virology  2013;2(2):32-48.
Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virus-induced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.
doi:10.5501/wjv.v2.i2.32
PMCID: PMC3785047  PMID: 24175228
Viral replication factory; Viral replication complex; Plus-strand RNA virus; Membrane remodeling; Virus-host interaction; Alphavirus; Enterovirus; Coronavirus; Flavivirus; Hepatitis C virus
17.  Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid 
An X-ray crystallographic structure is described for unliganded Vaccinia virus poly(A) polymerase monomer (VP55), showing the first domain-level structural isoforms among either VP55, it’s processivity factor VP39, or the VP55-VP39 heterodimer. The occurrence of domain-level motion specifically in monomeric VP55 is consistent with the finding that the monomeric protein undergoes saltatory translocation whereas the heterodimer does not.
Vaccinia virus poly(A) polymerase (VP55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssNA). Previously, its structure has only been solved in the context of the VP39 processivity factor. Here, a crystal structure of unliganded monomeric VP55 has been solved to 2.86 Å resolution, showing the first backbone structural isoforms among either VP55 or its processivity factor (VP39). Backbone differences between the two molecules of VP55 in the asymmetric unit indicated that unliganded monomeric VP55 can undergo a ‘rocking’ motion of the N-terminal domain with respect to the other two domains, which may be ‘rigidified’ upon VP39 docking. This observation is consistent with previously demonstrated experimental molecular dynamics of the monomer during translocation with respect to nucleic acid and with different mechanisms of translocation in the presence and absence of processivity factor VP39. Side-chain conformational changes in the absence of ligand were observed at a key primer contact site and at the catalytic center of VP55. The current structure completes the trio of possible structural forms for VP55 and VP39, namely the VP39 monomer, the VP39–VP55 heterodimer and the VP55 monomer.
doi:10.1107/S0907444913000346
PMCID: PMC3606039  PMID: 23519670
VP55; Vaccinia virus poly(A) polymerase; polymerases
18.  On the dynamics of cortical development: synchrony and synaptic self-organization 
We describe a model for cortical development that resolves long-standing difficulties of earlier models. It is proposed that, during embryonic development, synchronous firing of neurons and their competition for limited metabolic resources leads to selection of an array of neurons with ultra-small-world characteristics. Consequently, in the visual cortex, macrocolumns linked by superficial patchy connections emerge in anatomically realistic patterns, with an ante-natal arrangement which projects signals from the surrounding cortex onto each macrocolumn in a form analogous to the projection of a Euclidean plane onto a Möbius strip. This configuration reproduces typical cortical response maps, and simulations of signal flow explain cortical responses to moving lines as functions of stimulus velocity, length, and orientation. With the introduction of direct visual inputs, under the operation of Hebbian learning, development of mature selective response “tuning” to stimuli of given orientation, spatial frequency, and temporal frequency would then take place, overwriting the earlier ante-natal configuration. The model is provisionally extended to hierarchical interactions of the visual cortex with higher centers, and a general principle for cortical processing of spatio-temporal images is sketched.
doi:10.3389/fncom.2013.00004
PMCID: PMC3573321  PMID: 23596410
synchronous oscillation; cortical development; synaptic organization; cortical response properties; cortical information flow
19.  Asystole after Orthotopic Lung Transplantation: Examining the Interaction of Cardiac Denervation and Dexmedetomidine 
Case Reports in Anesthesiology  2012;2012:203240.
Dexmedetomidine is an α2-receptor agonist commonly used for sedation and analgesia in ICU patients. Dexmedetomidine is known to provide sympatholysis and also to have direct atrioventricular and sinoatrial node inhibitory effects. In rare instances, orthotopic lung transplantation has been associated with disruption of autonomic innervation of the heart. The combination of this autonomic disruption and dexmedetomidine may be associated with severe bradycardia and/or asystole. Since orthotopic lung transplant patients with parasympathetic denervation will not respond with increased heart rate to anticholinergic therapy, bradyarrhythmias must be recognized and promptly treated with direct acting beta agonists to avoid asystolic cardiac events.
doi:10.1155/2012/203240
PMCID: PMC3472541  PMID: 23091739
20.  The differential effects of maternal age, race/ethnicity and insurance on neonatal intensive care unit admission rates 
Background
Maternal race/ethnicity, age, and socioeconomic status (SES) are important factors determining birth outcome. Previous studies have demonstrated that, teenagers, and mothers with advanced maternal age (AMA), and Black/Non-Hispanic race/ethnicity can independently increase the risk for a poor pregnancy outcome. Similarly, public insurance has been associated with suboptimal health outcomes. The interaction and impact on the risk of a pregnancy resulting in a NICU admission has not been studied. Our aim was, to analyze the simultaneous interactions of teen/advanced maternal age (AMA), race/ethnicity and socioeconomic status on the odds of NICU admission.
Methods
The Consortium of Safe Labor Database (subset of n = 167,160 live births) was used to determine NICU admission and maternal factors: age, race/ethnicity, insurance, previous c-section, and gestational age.
Results
AMA mothers were more likely than teenaged mothers to have a pregnancy result in a NICU admission. Black/Non-Hispanic mothers with private insurance had increased odds for NICU admission. This is in contrast to the lower odds of NICU admission seen with Hispanic and White/Non-Hispanic pregnancies with private insurance.
Conclusions
Private insurance is protective against a pregnancy resulting in a NICU admission for Hispanic and White/Non-Hispanic mothers, but not for Black/Non-Hispanic mothers. The health disparity seen between Black and White/Non-Hispanics for the risk of NICU admission is most evident among pregnancies covered by private insurance. These study findings demonstrate that adverse pregnancy outcomes are mitigated differently across race, maternal age, and insurance status.
doi:10.1186/1471-2393-12-97
PMCID: PMC3495040  PMID: 22985092
21.  Metyrapone in treatment-resistant depression 
Depression affects a significant proportion of the population, with 1-year and lifetime prevalence of 3–5% and 10–30% respectively. Full remission is achieved in only a third of patients following treatment with first-line antidepressant. There is a need for novel treatments for treatment-resistant depression (TRD). Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis has been described in patients with depression. There is persistent rise in the levels of cortisol (end product of the HPA axis) and impairment of the negative feedback inhibition mechanism of the HPA axis. Dysregulation of the HPA axis has been found to be linked to nonresponse to antidepressants and relapse following successful treatment. The efficacy of pharmacological agents that intervene with the mechanisms involved in dysregulation of cortisol synthesis and release are being explored in depression, particularly in TRD. Studies have been carried out with these drugs as augmenting agents for antidepressants or as monotherapy. The strongest evidence has come from studies using metyrapone, a cortisol synthesis inhibitor, and this has been described in detail in this review. The most robust evidence for its antidepressant efficacy in depression comes from a double-blind, randomized, placebo-controlled study of augmentation of serotonergic antidepressants with metyrapone. A 3-week augmentation of serotonergic antidepressants with 1 g metyrapone daily was shown to be superior to placebo in reducing the Montgomery–Asberg Depression Rating Scale by 50%, 5 weeks following initiation of treatment. The mechanism of the antidepressant action of metyrapone is not clear but the evidence for various potential mechanisms is discussed.
doi:10.1177/2045125312436597
PMCID: PMC3736936  PMID: 23983967
antidepressant; antiglucocorticoid; depression; hypothalamic–pituitary–adrenal axis; metyrapone; treatment resistant
22.  Deletion of oligodendrocyte Cx32 and astrocyte Cx43 causes white matter vacuolation, astrocyte loss and early mortality 
Glia  2011;59(7):1064-1074.
CNS glia exhibit a variety of gap junctional interactions: between neighboring astrocytes, between neighboring oligodendrocytes, between astrocytes and oligodendrocytes, and as ‘reflexive’ structures between layers of myelin in oligodendrocytes. Together, these junctions are thought to form a network facilitating absorption and removal of extracellular K+ released during neuronal activity. In mice, loss of the two major oligodendrocyte connexins causes severe demyelination and early mortality, while loss of the two major astrocyte connexins causes mild dysmyelination and sensorimotor impairment, suggesting that reflexive and/or oligo-oligo coupling may be more important for the maintenance of myelin than other forms. To further explore the functional relationships between glial connexins, we generated double knockout mice lacking one oligodendrocyte and one astrocyte connexin. Cx32-Cx43 dKO animals develop white matter vacuolation without obvious ultrastructural abnormalities in myelin. Progressive loss of astrocytes but not oligodendrocytes or microglia accompanies sensorimotor impairment, seizure activity and early mortality at around 16 weeks of age. Our data reveal an unexpected role for connexins in the survival of white matter astrocytes, requiring the expression of particular isoforms in both oligodendrocytes and astrocytes.
doi:10.1002/glia.21179
PMCID: PMC3094483  PMID: 21538560
Gap junction; connexin; myelin; astrocyte; oligodendrocytes; Cx47; Cx32; Cx43
23.  Paradoxical persistence through mixed-system dynamics: towards a unified perspective of reversal behaviours in evolutionary ecology 
Counterintuitive dynamics of various biological phenomena occur when composite system dynamics differ qualitatively from that of their component systems. Such composite systems typically arise when modelling situations with time-varying biotic or abiotic conditions, and examples range from metapopulation dynamics to population genetic models. These biological, and related physical, phenomena can often be modelled as simple financial games, wherein capital is gained and lost through gambling. Such games have been developed and used as heuristic devices to elucidate the processes at work in generating seemingly paradoxical outcomes across a spectrum of disciplines, albeit in a field-specific, ad hoc fashion. Here, we propose that studying these simple games can provide a much deeper understanding of the fundamental principles governing paradoxical behaviours in models from a diversity of topics in evolution and ecology in which fluctuating environmental effects, whether deterministic or stochastic, are an essential aspect of the phenomenon of interest. Of particular note, we find that, for a broad class of models, the ecological concept of equilibrium reactivity provides an intuitive necessary condition that must be satisfied in order for environmental variability to promote population persistence. We contend that further investigations along these lines promise to unify aspects of the study of a range of topics, bringing questions from genetics, species persistence and coexistence and the evolution of bet-hedging strategies, under a common theoretical purview.
doi:10.1098/rspb.2010.2074
PMCID: PMC3061147  PMID: 21270032
stochastic dynamics; persistence; coexistence; paradoxical games; reversal behaviour; reactivity
24.  Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels 
Molecular Biology of the Cell  2011;22(23):4503-4512.
The mechanisms regulating assembly of gap junctions are not well defined. It is shown that assembly of gap junctions containing Cx50 requires both a PDZ domain–binding motif at the connexin C-terminus and the scaffolding protein ZO-1.
The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.
doi:10.1091/mbc.E11-05-0438
PMCID: PMC3226470  PMID: 21965293
25.  Functional Heterotypic Interactions Between Astrocyte and Oligodendrocyte Connexins 
Glia  2011;59(1):26-34.
Human genetic diseases and mouse knockouts illustrate that the maintenance of central nervous system myelin requires connexin expression by both astrocytes and oligodendrocytes. Because these cell types express nonoverlapping sets of connexins, the intercellular channels formed between them must be asymmetric with regard to connexin content, defined as heterotypic. Here, we show that oligodendrocyte Cx47 can form heterotypic channels with astrocyte Cx43 or Cx30 but not Cx26, whereas oligodendrocyte Cx32 can functionally interact with astrocyte Cx30 or Cx26 but not Cx43. Thus, as many as four types of intercellular channels could be formed between astrocytes and oligodendrocytes.
doi:10.1002/glia.21073
PMCID: PMC3022368  PMID: 21046554
gap junction; connexin; myelin; glia

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