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1.  Antibiotic Use in Neonatal Intensive Care Units and Adherence with Centers for Disease Control and Prevention 12 Step Campaign to Prevent Antimicrobial Resistance 
The Centers for Disease Control and Prevention (CDC) 12-Step Campaign to Prevent Antimicrobial Resistance was launched to educate clinicians about antimicrobial resistance and provide strategies to improve clinical practice, including antimicrobial utilization.
A multicenter retrospective observational study of antibiotic use was performed in 4 tertiary care NICUs to assess adherence to the guidelines defined by the CDC 12-Step Campaign using predetermined criteria. Fifty infants per NICU were identified who received intravenous antibiotics at greater than 72 hours of age. Antibiotic regimens, clinical and microbiologic data, and indications for initiation and continuation of antibiotics (after 72 hours of use) were recorded. Inappropriate utilization was characterized at initiation, continuation, by agent, and by CDC 12-Step.
Two hundred neonates received 323 antibiotic courses totaling 3344 antibiotic-days. Ninety (28%) courses and 806 (24%) days were judged to be nonadherent to a CDC 12-Step. Inappropriate use was more common with continuation of antibiotics (39%) than with initiation (4%) of therapy. Vancomycin was the most commonly used drug (n = 895 antibiotic-days) of which 284 (32%) days were considered inappropriate. Carbapenems were used less frequently (n = 310 antibiotic-days), and 132 (43%) of these days were inappropriate. Common reasons for nonadherence at the time of continuation included failure to narrow antibiotic coverage after microbiologic results were known and prolonged antibiotic prophylaxis after surgery with chest tube placement.
The CDC 12-Step Campaign can be modified for neonatal populations. Inappropriate antibiotic prescribing was common in the study NICUs. Improvement efforts should target antibiotic use 72 hours after initiation, particularly focusing on narrowing therapy and instituting protocols to limit prophylaxis.
PMCID: PMC4526135  PMID: 19858773
antibiotic usage; neonates; resistance; intensive care units; CDC program
2.  NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication 
mBio  2015;6(4):e00759-15.
Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.
Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.
PMCID: PMC4488949  PMID: 26152585
3.  Right Breast Mastectomy and Reconstruction with Tissue Expander under Thoracic Paravertebral Blocks in a 12-Week Parturient 
Case Reports in Anesthesiology  2015;2015:842725.
Paravertebral blocks are becoming increasingly utilized for breast surgery with studies showing improved postoperative pain control, decreased need for opioids, and less nausea and vomiting. We describe the anesthetic management of an otherwise healthy woman who was 12 weeks pregnant presenting for treatment of her breast cancer. For patients undergoing breast mastectomy and reconstruction with tissue expanders, paravertebral blocks offer an anesthetic alternative when general anesthesia is not desired.
PMCID: PMC4503581
4.  RASA3 is a critical inhibitor of RAP1-dependent platelet activation 
The Journal of Clinical Investigation  2015;125(4):1419-1432.
The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.
PMCID: PMC4396462  PMID: 25705885
5.  Current issues in patient safety in surgery: a review 
Current surgical safety guidelines and checklists are generic and are not specifically tailored to address patient issues and risk factors in surgical subspecialties. Patient safety in surgical subspecialties should be templated on general patient safety guidelines from other areas of medicine and mental health but include and develop specific processes dedicated for the care of the surgical patients. Safety redundant systems must be in place to decrease errors in surgery. Therefore, different surgical subspecialties should develop a specific curriculum in patient safety addressing training in academic centers and application of these guidelines in all practices. Clearly, redundant safety systems must be in place to decrease errors in surgery, in analogy to safety measures in other high-risk industries. Specific surgical subspecialties are encouraged to develop a specific patient safety curriculum that address training in academic centers and applicability to daily practice, with the goal of keeping our surgical patients safe in all disciplines. The present review article is designed to outline patient safety practices that should be adapted and followed to fit particular specialties.
PMCID: PMC4455056  PMID: 26045717
6.  Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin 
Biochimica et biophysica acta  2014;1841(6):880-887.
In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1H−/−), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression of sarco/endoplasmic reticulum calcium ATPase and phospholambin showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1H−/− mice with rapamycin. Six to eight week old Acsl1H−/− mice and their littermate controls were given i.p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10 weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1H−/− hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1H−/− hearts exhibited an 8-fold higher uptake of 2-deoxy[1-14C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-14C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1H−/− mice.
PMCID: PMC4047709  PMID: 24631848
fatty acid oxidation; mTOR; insulin resistance; gene expression; ventricular function; substrate switching
7.  Development and Validation of a Lung Transplant-Specific Disability Questionnaire 
Thorax  2013;69(5):437-442.
Lung transplant (LT) aims to extend survival and improve patient-centered outcomes (PCOs) by reducing disability and improving health-related quality of life (HRQL). Few PCO instruments have been validated in LT populations. We aimed to develop and validate a shortened version of the Valued Life Activities (VLA) disability scale specific to LT.
We used data from 140 subjects participating in an ongoing cohort study of LT. Subjects completed a survey battery, including VLA items, and physical assessments before LT. To develop a shortened lung transplant specific VLA (LT-VLA), we iteratively deleted items from a longer 32-item VLA battery, retaining the instrument’s conceptual framework, scoring and performance characteristics. We evaluated LT-VLA validity by testing correlations with a HRQL measure (Short Form-12 Physical Function [SF-12 PF] subscale), FVC% predicted, and 6-minute walk distance (6MWD). Responsiveness was evaluated in 84 subjects who completed assessments before and after LT.
The 15-item LT-VLA scoring closely matched the longer VLA (correlations ≥0.96) and had excellent internal consistency (Cronbach’s alpha: 0.92). The LT-VLA required only 3 minutes or less to administer. The LT-VLA, measured as mean difficulty in performing each of the 15 activities queried, correlated with FVC% predicted (r= −0.30), 6MWD (r= −0.38) and SF-12 PF (r= −0.47) (all p<0.01). The LT-VLA mean difficulty was responsive to change from before to after LT: (63% improvement; effect size=1.60)
The LT-VLA is a short, easy to administer, valid, and responsive disease-specific PCO instrument that may be useful in clinical and research applications for lung transplantation.
PMCID: PMC4113506  PMID: 24355825
lung transplantation; disability; patient centered outcomes; health-related quality of life
8.  Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor 
Journal of Virology  2014;88(19):11540-11555.
Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15–25, 2013, doi:10 .1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses.
IMPORTANCE Dengue viruses are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic to subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or treatment of DV infections, respectively. This study reports the characterization of the mode of action of ST-148, a small-molecule capsid inhibitor with potent antiviral activity against all DV serotypes. Our results demonstrate that ST-148 stabilizes capsid protein self-interaction, thereby likely perturbing assembly and disassembly of viral nucleocapsids by inducing structural rigidity. This, in turn, might interfere with the release of viral RNA from incoming nucleocapsids (uncoating) as well as assembly of progeny virus particles. As previously reported for other enveloped viruses, we propose the capsid as a novel tractable target for flavivirus inhibitors.
PMCID: PMC4178822  PMID: 25056895
9.  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.
PMCID: PMC4200109  PMID: 25100592
bystander effect; cell death; connexin; gap junctions; neuroprotection; retina
10.  Cardiac Energy Dependence on Glucose Increases Metabolites Related to Glutathione and Activates Metabolic Genes Controlled by Mechanistic Target of Rapamycin 
Long chain acyl‐CoA synthetases (ACSL) catalyze long‐chain fatty acids (FA) conversion to acyl‐CoAs. Temporal ACSL1 inactivation in mouse hearts (Acsl1H−/−) impaired FA oxidation and dramatically increased glucose uptake, glucose oxidation, and mTOR activation, resulting in cardiac hypertrophy. We used unbiased metabolomics and gene expression analyses to elucidate the cardiac cellular response to increased glucose use in a genetic model of inactivated FA oxidation.
Methods and Results
Metabolomics analysis identified 60 metabolites altered in Acsl1H−/− hearts, including 6 related to glucose metabolism and 11 to cysteine and glutathione pathways. Concurrently, global cardiac transcriptional analysis revealed differential expression of 568 genes in Acsl1H−/− hearts, a subset of which we hypothesized were targets of mTOR; subsequently, we measured the transcriptional response of several genes after chronic mTOR inhibition via rapamycin treatment during the period in which cardiac hypertrophy develops. Hearts from Acsl1H−/− mice increased expression of several Hif1α‐responsive glycolytic genes regulated by mTOR; additionally, expression of Scl7a5, Gsta1/2, Gdf15, and amino acid‐responsive genes, Fgf21, Asns, Trib3, Mthfd2, were strikingly increased by mTOR activation.
The switch from FA to glucose use causes mTOR‐dependent alterations in cardiac metabolism. We identified cardiac mTOR‐regulated genes not previously identified in other cellular models, suggesting heart‐specific mTOR signaling. Increased glucose use also changed glutathione‐related pathways and compensation by mTOR. The hypertrophy, oxidative stress, and metabolic changes that occur within the heart when glucose supplants FA as a major energy source suggest that substrate switching to glucose is not entirely benign.
PMCID: PMC4345858  PMID: 25713290
acyl‐CoA synthetase; fuel switching; glutathione; mTOR; oxidative stress
11.  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.
PMCID: PMC3863394  PMID: 24176257
assessment; early intervention; eligibility; developmental delay; preterm; Bayley Scales of Infant Development; Third Edition
12.  Consumer Participation in Quality Improvements for Chronic Disease Care: Development and Evaluation of an Interactive Patient-Centered Survey to Identify Preferred Service Initiatives 
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.
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.
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.
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.
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.
PMCID: PMC4285719  PMID: 25532217
ambulatory care; health care surveys; patient-centered care; consumer participation; medical oncology; chronic disease; cardiology; neurology
13.  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.
PMCID: PMC4231108  PMID: 25392992
14.  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.
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.
This literature review examined a large number of studies of implementation of tele-ICU systems 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 of tele-ICUs is initially expensive but eventually results in cost savings and better clinical outcomes.
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.
PMCID: PMC4206175  PMID: 25662529
15.  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.
PMCID: PMC3808728  PMID: 23958747
gap junction; connexin; dye transfer; serotonin
16.  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.
PMCID: PMC3606039  PMID: 23519670
VP55; Vaccinia virus poly(A) polymerase; polymerases
17.  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.
PMCID: PMC3807400  PMID: 23885072
18.  Complementary and Alternative Medicine use in women during pregnancy: do their healthcare providers know? 
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.
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.
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.
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.
PMCID: PMC3945795  PMID: 24592860
Complementary and Alternative Medicine; Pregnancy; Maternal-fetal health; Patient-physician communication; Self-care; Prevalence; Cross-sectional study
19.  Survey on Amacrine Cells Coupling to Retrograde-Identified Ganglion Cells in the Mouse Retina 
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.
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.
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.
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.
PMCID: PMC3732024  PMID: 23821205
retrograde labeling; connexin; GABA; NOS; 5-HT; ChAT
20.  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.
PMCID: PMC4215792  PMID: 25400552
cortical column; cortical development; synaptic organization; cortical response properties; neuromicrocircuitry; S1 segregates
21.  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.
PMCID: PMC3888888  PMID: 24459652
Erectile dysfunction; Pharmacology; Phosphodiesterase 5 inhibitors; Prostatic hyperplasia; Tadalafil
22.  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.
PMCID: PMC3478409  PMID: 22959928
Arsenic; folate; epigenetics; DNA methylation; transplacental exposure; CD1 mice
23.  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.
PMCID: PMC3741614  PMID: 20709095
mouse; scotopic; melanopsin; electroretinogram; connexin
24.  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.
PMCID: PMC3720567  PMID: 23936012
25.  Field Trips as a Novel Means of Experiential Learning in Ambulatory Pediatrics 
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.
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.
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.
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.
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.
PMCID: PMC3399621  PMID: 23730450

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