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1.  Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels 
Channels  2014;8(1):62-75.
Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.
doi:10.4161/chan.27470
PMCID: PMC4048344  PMID: 24394769
SK channel; nAChR; sensory hair cell; neuron; calcium; calmodulin; olivocochlear; synapse
2.  Determinants of hospital fall rate trajectory groups: A longitudinal assessment of nurse staffing and organizational characteristics 
Health care management review  2014;39(4):352-360.
Background
Patient falls in acute care hospitals represent a significant patient safety concern. Although cross-sectional studies have shown that fall rates vary widely between acute care hospitals, it is not clear whether hospital fall rates remain consistent over time.
Purpose
The aim of this study was to determine whether hospitals can be categorized into fall rate trajectory groups over time and to identify nurse staffing and hospital characteristics associated with hospital fall rate trajectory groups.
Methodology/Approach
We conducted a 54-month (July 2006–December 2010) longitudinal study of U.S. acute care general hospitals participating in the National Database for Nursing Quality Indicators® (2007). We used latent class growth modeling to categorize hospitals into groups based on their long-term fall rates. Nurse staffing and hospital characteristics associated with membership in the highest hospital fall rate group were identified using logistic regression.
Findings
A sample of 1,529 hospitals (mean fall rate of 3.65 per 1,000 patient days) contributed data to the analysis. Latent class growth modeling findings classified hospital into three groups based on fall rate trajectories: consistently high (mean fall rate of 4.96 per 1,000 patient days), consistently medium (mean fall rate of 3.63 per 1,000 patient days), and consistently low (mean fall rate of 2.50 per 1,000 patient days). Hospitals with higher total nurse staffing (odds ratio [OR] = 0.92, 95% confidence interval [CI] [0.85, 0.99]), Magnet status (OR = 0.49, 95% CI [0.35, 0.70]), and bed size greater than 300 beds (OR = 0.70, 95% CI [0.51, 0.94]) were significantly less likely to be categorized in the “consistently high” fall rate group.
Practice Implications
Over this 54-month period, hospitals were categorized into three groups based on long-term fall rates. Hospital-level factors differed among these three groups. This suggests that there may be hospitals in which “best practices” for fall prevention might be identified. In addition, administrators may be able to reduce fall rates by maintaining greater nurse staffing ratios as well as fostering an environment consistent with that of Magnet hospitals.
doi:10.1097/HMR.0000000000000013
PMCID: PMC4277236  PMID: 24566249
Hospitals; magnet status; nursing staffing; patient falls
3.  Proteomic Analysis of Serum and Urine of HIV-Monoinfected and HIV/HCV-Coinfected Patients Undergoing Long Term Treatment with Nevirapine 
Disease Markers  2014;2014:315824.
Nevirapine (NVP) is an effective nonnucleoside reverse transcriptase inhibitor (NNRTI) of particular interest as it is often used in resource limited countries. However, one of the main concerns with the use of NVP is hepatotoxicity and elevation of liver enzymes as a consequence of highly active antiretroviral therapy (HAART) containing NVP is more often reported in HIV patients coinfected with hepatitis C virus than in HIV-monoinfected patients. To discover possible markers of NVP induced hepatotoxicity, serum and urine samples from twenty-five HIV or HIV/HCV patients, all of whom had received NVP continuously for at least four months, and healthy controls were subjected to in-solution or in-gel proteomic analysis. A total of 83 differentially regulated proteins consisted of 34 proteins identified in serum by in-solution analysis, 2 proteins identified from serum in a 2D gel electrophoresis analysis, and 47 proteins identified in urine in an in-solution analysis. Three proteins, namely, haptoglobin, Rho-related BTB domain containing protein 3, and death-associated protein kinase 3, were selected for further validation by Western blot analysis and results showed that haptoglobin has potential for further development as an additional marker of NVP induced hepatotoxicity.
doi:10.1155/2014/315824
PMCID: PMC4280805  PMID: 25580050
4.  CRYO-EM STRUCTURES OF THE ACTIN:TROPOMYOSIN FILAMENT REVEAL THE MECHANISM FOR THE TRANSITION FROM C- TO M-STATE 
Journal of molecular biology  2013;425(22):10.1016/j.jmb.2013.08.020.
Tropomyosin is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of tropomyosin along the actin:tropomyosin:troponin thin filament to block or expose myosin binding sites on actin. In striated muscle, including involuntary cardiac muscle, tropomyosin regulates muscle contraction by coupling Ca2+ binding to troponin with myosin binding to the thin filament. In smooth muscle, the switch is the post-translational modification of the myosin. Depending on the activation state of troponin and the binding state of myosin, tropomyosin can occupy the blocked, closed, or open position on actin. Using native cryogenic 3DEM, we have directly resolved and visualized cardiac and gizzard muscle tropomyosin on filamentous actin in the position that corresponds to the closed state. From the 8-Å resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed state position.
doi:10.1016/j.jmb.2013.08.020
PMCID: PMC3845445  PMID: 24021812
Actin; Tropomyosin; cryogenic 3DEM; electron microscopy; thin filament; cytoskeleton
5.  SAMSN1 Is a Tumor Suppressor Gene in Multiple Myeloma12 
Neoplasia (New York, N.Y.)  2014;16(7):572-585.
Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138 + PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell–PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.
doi:10.1016/j.neo.2014.07.002
PMCID: PMC4198825  PMID: 25117979
6.  Voltage Dependent Anion Channel Is Redistributed during Japanese Encephalitis Virus Infection of Insect Cells 
The Scientific World Journal  2014;2014:976015.
Despite the availability of an effective vaccine, Japanese encephalitis remains a significant cause of morbidity and mortality in many parts of Asia. Japanese encephalitis is caused by the Japanese encephalitis virus (JEV), a mosquito transmitted flavivirus. Many of the details of the virus replication cycle in mosquito cells remain unknown. This study sought to determine whether GRP78, a well-characterized flavivirus E protein interacting protein, interacted with JEV E protein in insect cells, and whether this interaction was mediated at the cell surface. GRP78 was shown to interact with JEV E protein by coimmunoprecipitation, and was additionally shown to interact with voltage dependent anion protein (VDAC) through the same methodology. Antibody inhibition experiments showed that neither GRP78 nor VDAC played a role in JEV internalization to insect cells. Interestingly, VDAC was shown to be significantly relocalized in response to JEV infection, and significant levels of colocalization between VDAC and GRP78 and VDAC and ribosomal L28 protein were seen in JEV infected but not uninfected cells. This is the first report of relocalization of VDAC in response to JEV infection and suggests that this may be a part of the JEV replication strategy in insect cells.
doi:10.1155/2014/976015
PMCID: PMC4121193  PMID: 25126612
7.  Comparative Plasma Protein Profiling of Hemoglobin H Disease 
Disease Markers  2014;2014:340214.
HbH and HbH-constant spring (HbH-CS) are the most common forms of α-thalassemia detected in the Thai population. The accumulation of excess β globin chains in these diseases results in increased red cell hemolysis, and patients with HbH-CS normally have a more severe clinical presentation than patients with HbH disease. This study aimed to detect alterations in the expression of plasma proteins of HbH and HbH-CS patients as compared to normal plasma. Platelet poor plasma was separated from HbH and HbH-CS and normal subjects and differential plasma proteins were detected using two-dimensional gel electrophoresis and identified using LC/MS/MS. A total of 14 differentially expressed proteins were detected of which 5 proteins were upregulated and 9 were downregulated. Most of the differentially expressed proteins are liver secreted proteins involved in hemolysis, oxidative stress response, and hemoglobin degradation. Seven proteins were found to be differentially expressed between HbH and HbH-CS. Levels of haptoglobin, a hemoglobin scavenging protein, were significantly increased in HbH patients as compared to HbH-CS patients. The identification of differentially expressed proteins may lead to a better understanding of the biological events underlying the clinical presentation of HbH and HbH-CS patients and can have application as hemolytic markers or severity predictors.
doi:10.1155/2014/340214
PMCID: PMC4082903  PMID: 25024506
8.  Comparing short to standard duration of antibiotic therapy for patients hospitalized with cellulitis (DANCE): study protocol for a randomized controlled trial 
BMC Infectious Diseases  2014;14:235.
Background
Recommended therapy duration for patients hospitalized with cellulitis is 10–14 days. Unnecessary use of antibiotics is one of the key factors driving resistance. Recent studies have shown that antibiotic therapy for cellulitis in outpatients can safely be shortened, despite residual inflammation. This study will compare in hospitalized patients the safety and effectiveness of shortening antibiotic therapy for cellulitis from 12 to 6 days.
Methods/design
In a multicenter, randomized, double-blind, non-inferiority trial, adult patients admitted with cellulitis will be included. Cellulitis is defined as warmth, erythema, and induration of the skin and/or subcutaneous tissue, with or without pain (including erysipelas). All patients will initially be treated with intravenous flucloxacillin, and will be evaluated after 5–6 days. Those who have improved substantially (defined as being afebrile, and having a lower cellulitis severity score) will be randomized at day 6 between additional 6 days of oral flucloxacillin (n = 198) or placebo (n = 198). Treatment success is defined as resolution of cellulitis on day 14 (disappearance of warmth and tenderness, improvement of erythema and edema), without the need of additional antibiotics for cellulitis by day 28. Secondary endpoints are relapse rate (up to day 90), speed of recovery (using a cellulitis severity score until day 28, and VAS scores on pain and swelling until day 90), quality of life (using the SF-36 and EQ-5D questionnaires) and costs (associated with total antibiotic use and health-care resource utilization up to day 90).
Discussion
Inclusion is planned to start in Q2 2014.
Trial registration
ClinicalTrials.gov (NCT02032654) and the Netherlands Trial Register (NTR4360).
doi:10.1186/1471-2334-14-235
PMCID: PMC4016641  PMID: 24885384
Cellulitis; Erysipelas; Skin infections; Antibiotics; Flucloxacillin; Therapy duration; Hospital setting; Randomised controlled trial
9.  Combining topographical and genetic cues to promote neuronal fate specification in stem cells 
Biomacromolecules  2012;13(11):3427-3438.
There is little remedy for the devastating effects resulting from neuronal loss caused by neural injury or neurodegenerative disease. Reconstruction of damaged neural circuitry with stem cell-derived neurons is a promising approach to repair these defects, but controlling differentiation and guiding synaptic integration with existing neurons remain significant unmet challenges. Biomaterial surfaces can present nanoscale topographical cues which influence neuronal differentiation and process outgrowth. By combining these scaffolds with additional molecular biology strategies, synergistic control over cell fate can be achieved. Here, we review recent progress in promoting neuronal fate using techniques at the interface of biomaterial science and genetic engineering. New data demonstrates that combining nanofiber topography with an induced genetic program enhances neuritogenesis in a synergistic fashion. We propose combining patterned biomaterial surface cues with prescribed genetic programs to achieve neuronal cell fates with the desired sublineage specification, neurochemical profile, targeted integration and electrophysiological properties.
doi:10.1021/bm301220k
PMCID: PMC3992984  PMID: 23098293
stem cell; topography; nanofibers; neuron; gene; scaffold
10.  Comprehensive proteomic analysis of white blood cells from chikungunya fever patients of different severities 
Background
Chikungunya fever (CHIKF) is a recently re-emerged mosquito transmitted viral disease caused by the chikungunya virus (CHIKV), an Alphavirus belonging to the family Togaviridae. Infection of humans with CHIKV can result in CHIKF of variable severity, although the factors mediating disease severity remain poorly defined.
Methods
White blood cells were isolated from blood samples collected during the 2009-2010 CHIKF outbreak in Thailand. Clinical presentation and viral load data were used to classify samples into three groups, namely non chikungunya fever (non-CHIKF), mild CHIKF, and severe CHIKF. Five samples from each group were analyzed for protein expression by GeLC-MS/MS.
Results
CHIKV proteins (structural and non-structural) were found only in CHIKF samples. A total of 3505 human proteins were identified, with 68 proteins only present in non-CHIKF samples. A total of 240 proteins were found only in CHIKF samples, of which 65 and 46 were found only in mild and severe CHIKF samples respectively. Proteins with altered expression mapped predominantly to cellular signaling pathways (including toll-like receptor and PI3K-Akt signaling) although many other processes showed altered expression as a result of CHIKV infection. Expression of proteins consistent with the activation of the inflammasome was detected, and quantitation of (pro)-caspase 1 at the protein and RNA levels showed an association with disease severity.
Conclusions
This study confirms the infection of at least a component of white blood cells by CHIKV, and shows that CHIKV infection results in activation of the inflammasome in a manner that is associated with disease severity.
doi:10.1186/1479-5876-12-96
PMCID: PMC4022080  PMID: 24721947
Chikungunya; Proteome; Inflammasome; Caspase 1
11.  Camera Trapping: A Contemporary Approach to Monitoring Invasive Rodents in High Conservation Priority Ecosystems 
PLoS ONE  2014;9(3):e86592.
Invasive rodent species have established on 80% of the world's islands causing significant damage to island environments. Insular ecosystems support proportionally more biodiversity than comparative mainland areas, highlighting them as critical for global biodiversity conservation. Few techniques currently exist to adequately detect, with high confidence, species that are trap-adverse such as the black rat, Rattus rattus, in high conservation priority areas where multiple non-target species persist. This study investigates the effectiveness of camera trapping for monitoring invasive rodents in high conservation areas, and the influence of habitat features and density of colonial-nesting seabirds on rodent relative activity levels to provide insights into their potential impacts. A total of 276 camera sites were established and left in situ for 8 days. Identified species were recorded in discrete 15 min intervals, referred to as ‘events’. In total, 19 804 events were recorded. From these, 31 species were identified comprising 25 native species and six introduced. Two introduced rodent species were detected: the black rat (90% of sites), and house mouse Mus musculus (56% of sites). Rodent activity of both black rats and house mice were positively associated with the structural density of habitats. Density of seabird burrows was not strongly associated with relative activity levels of rodents, yet rodents were still present in these areas. Camera trapping enabled a large number of rodents to be detected with confidence in site-specific absences and high resolution to quantify relative activity levels. This method enables detection of multiple species simultaneously with low impact (for both target and non-target individuals); an ideal strategy for monitoring trap-adverse invasive rodents in high conservation areas.
doi:10.1371/journal.pone.0086592
PMCID: PMC3943715  PMID: 24599307
12.  A rare three part proximal ulnar shear fracture requiring innovative reduction and fixation in a paediatric elbow 
Journal of Surgical Case Reports  2014;2014(3):rju021.
Fracture of the proximal ulna in children is an uncommon injury with various classification models. An 8-year-old boy presented to our emergency department with an isolated three part intra-articular fracture of his right proximal ulna from an extension injury sustained during skiing which has not been previously described in the literature. He was taken to the operating room for open reduction and internal fixation. Two cannulated screws positioned in a posterior–anterior direction were used to hold the reduction. He was discharged from the hospital 24 h post-reduction in a complete cast. At 5 weeks follow-up, his elbow radiographs indicate no loss of reduction and at 4 months follow-up, he has regained a full range of movement at the elbow joint. The cannulated screws were left in situ as it did not cause him any harm. We propose that the described fracture pattern should be managed by open reduction and internal fixation (cannulated screws used to hold the reduction, position in a posterior–anterior direction).
doi:10.1093/jscr/rju021
PMCID: PMC4197916  PMID: 24876402
13.  Tailored Immune Responses: Novel Effector Helper T Cell Subsets in Protective Immunity 
PLoS Pathogens  2014;10(2):e1003905.
Differentiation of naïve CD4+ cells into functionally distinct effector helper T cell subsets, characterised by distinct “cytokine signatures,” is a cardinal strategy employed by the mammalian immune system to efficiently deal with the rapidly evolving array of pathogenic microorganisms encountered by the host. Since the TH1/TH2 paradigm was first described by Mosmann and Coffman, research in the field of helper T cell biology has grown exponentially with seven functionally unique subsets having now been described. In this review, recent insights into the molecular mechanisms that govern differentiation and function of effector helper T cell subsets will be discussed in the context of microbial infections, with a focus on how these different helper T cell subsets orchestrate immune responses tailored to combat the nature of the pathogenic threat encountered.
doi:10.1371/journal.ppat.1003905
PMCID: PMC3930558  PMID: 24586147
14.  The scale of repeat prescribing – time for an update 
Background
The NHS spends billions of pounds annually on repeat prescriptions in primary care, but data on their extent and use is out of date. Understanding the scale of repeat prescribing and for whom it is prescribed is important for the NHS to plan services and develop policies to improve patient care.
Method
Anonymous data on prescription numbers and practice population demographics was obtained from GP computer systems in a large urban area.
Searches were conducted in November 2011 to identify the numbers of repeat items listed on individuals’ repeat lists by sex and age.
The proportion of all prescription items issued as repeats was identified by conducting searches on items issued as repeat and acute prescriptions.
Results
In the year of study 4,453,225 items were issued of which 3,444,769 (77%) were repeats (mean 13 items per patient/annum) and 1,008,456 (23%) acute prescriptions (mean 3.9 items per patient per annum). The mean number of repeat Items per patient was 1.87 (range 0.45 ages 0-9 years; 7.1 ages 80-89 years). At least one repeat medicine was prescribed to 43% of the population (range 20% for ages 0-9; over 75% for ages 60+).
Conclusion
A significant proportion of the population receive repeat prescriptions and the proportion increases with age. Whilst the proportion of repeat items to acute items has remained unchanged over the last two decades the number of repeat prescriptions items issued has doubled (from 5.8 to 13.3 items/patient/annum). This has implications for general practice workload, patient convenience, NHS costs and risk.
doi:10.1186/1472-6963-14-76
PMCID: PMC3943802  PMID: 24552190
Repeat prescribing; Prescribing analysis; Long-term medication
15.  BDNF profoundly and specifically increases KCNQ4 expression in neurons derived from embryonic stem cells 
Stem cell research  2012;10(1):29-35.
Neurons resembling the spiral ganglion neurons (SGNs) of the auditory nerve can be generated from embryonic stem cells through induced over-expression of the transcription factor Neurogenin-1 (Neurog1). While recapitulating this developmental pathway produces glutamatergic, bipolar neurons reminiscent of SGNs, these neurons are functionally immature, being characterized by a depolarized resting potential and limited excitability. We explored the effects of two neurotrophins known to be present in the inner ear, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), on the electrophysiology of neurons following Neurog1-induction. Our data reveal a significant reduction in resting membrane potential (RMP) following neurotrophin exposure, with BDNF producing a more robust effect than NT-3. This effect was accompanied by a profound and specific upregulation of the KCNQ4 subtype, where a 9-fold increase was observed with quantitative PCR. The other neuronally-expressed KCNQ subtypes (2, 3, and 5) exhibited upregulation which was 3-fold or less in magnitude. Quantitative immunohistochemistry confirmed the increase in KCNQ4 expression at the protein level. The present data show a novel link between BDNF and KCNQ4 expression, yielding insight into the restricted expression pattern of a channel known to play a special roles in setting the resting potential of auditory cells and in the etiology of progressive high frequency hearing loss.
doi:10.1016/j.scr.2012.08.005
PMCID: PMC3525722  PMID: 23089626
16.  Exploiting Human CD34+ Stem Cell–conditioned Medium for Tissue Repair 
Molecular Therapy  2013;22(1):149-159.
Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.
doi:10.1038/mt.2013.194
PMCID: PMC3978788  PMID: 23985698
17.  Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants 
The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been utilized in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1dw), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated β-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1dw mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1dw mutant outer hair cells and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1dw mice.
doi:10.1523/JNEUROSCI.4957-08.2009
PMCID: PMC3862029  PMID: 19176829
Secondary hypothyroidism; tectorial membrane; KCNQ1; KCNQ4; KCNJ10; prestin; POU1F1
18.  Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death 
BMC Research Notes  2013;6:372.
Background
A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy.
Results
In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy.
Conclusions
These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.
doi:10.1186/1756-0500-6-372
PMCID: PMC3847886  PMID: 24034452
Apoptosis; Autophagy; Caspase; Dengue; ER stress liver
19.  EMAP/EMAPA ontology of mouse developmental anatomy: 2013 update 
Background
The Edinburgh Mouse Atlas Project (EMAP) ontology of mouse developmental anatomy provides a standard nomenclature for describing normal and mutant mouse embryo anatomy. The ontology forms the core of the EMAP atlas and is used for annotating gene expression data by the mouse Gene Expression Database (GXD), Edinburgh Mouse Atlas of Gene Expression (EMAGE) and other database resources.
Findings
The original EMAP ontology listed anatomical entities for each developmental stage separately, presented as uniparental graphs organized as a strict partonomy. An "abstract" (i.e. non-stage-specific) representation of mouse developmental anatomy has since been developed. In this version (EMAPA) all instances for a given anatomical entity are presented as a single term, together with the first and last stage at which it is considered to be present. Timed-component anatomies are now derived using staging information in the "primary" non-timed version. Anatomical entities are presented as a directed acyclic graph enabling multiple parental relationships. Subsumption classification as well as partonomic and other types of relationships can now be represented. Most concept names are unique, with compound names constructed using standardized nomenclature conventions, and alternative names associated as synonyms.
Conclusions
The ontology has been extended and refined in a collaborative effort between EMAP and GXD, with additional input from others. Efforts are also underway to improve the revision process with regards to updating and editorial control. The revised EMAPA ontology is freely available from the OBO Foundry resource, with descriptive information and other documentation presented in associated Wiki pages (http://www.obofoundry.org/wiki/index.php/EMAPA:Main_Page).
doi:10.1186/2041-1480-4-15
PMCID: PMC3851555  PMID: 23972281
Mouse development; Anatomy ontology; Developmental anatomy; OBO format
20.  Host innate immune responses to sepsis 
Virulence  2013;5(1):36-44.
The immune response to sepsis can be seen as a pattern recognition receptor-mediated dysregulation of the immune system following pathogen invasion in which a careful balance between inflammatory and anti-inflammatory responses is vital. Invasive infection triggers both pro-inflammatory and anti-inflammatory host responses, the magnitude of which depends on multiple factors, including pathogen virulence, site of infection, host genetics, and comorbidities. Toll-like receptors, the inflammasomes, and other pattern recognition receptors initiate the immune response after recognition of danger signals derived from microorganisms, so-called pathogen-associated molecular patterns or derived from the host, so-called danger-associated molecular patterns. Further dissection of the role of host–pathogen interactions, the cytokine response, the coagulation cascade, and their multidirectional interactions in sepsis should lead toward the development of new therapeutic strategies in sepsis.
doi:10.4161/viru.25436
PMCID: PMC3916381  PMID: 23774844
sepsis; innate immunity; pattern recognition receptors; pathogen-associated molecular patterns (PAMP); danger-associated molecular patterns (DAMP); neutrophil extracellular traps (NET); myeloid related protein (Mrp)-8/14; coagulation; activated protein C; protease activated receptors
21.  Acculturation and orofacial pain among Hispanic adults 
This study examined the associations between acculturation and orofacial pain and healthcare among Hispanic adults. Understanding the effects of acculturation on Hispanic oral health may improve understanding of oral health disparities in the United States. Data were collected from 911 Hispanic adults reporting tooth pain and painful oral sores who were part of a larger study of South Florida residents conducted using random-digit dialing methodology. The survey was conducted in Spanish or English by bilingual interviewers per the choice of each respondent. Greater use of the Spanish language was associated with disparities in healthcare visits for orofacial pain, not having a usual dentist, having greater pain, increased difficulty eating and sleeping, and more depression. Respondents’ and their parents’ nativity (families that had been in the United States longer) and those identifying more closely to Hispanic culture were also predictive of several of the outcomes. Gender, financial status, and age, independent of acculturation, were also associated with orofacial pain, accessing health care, and pain-related loss of functioning among Hispanics. The data support the hypothesis that Hispanics with less acculturation are less able to access needed oral health care. This study highlights the need for outreach programs targeting recent Hispanic immigrants focusing on oral health care.
doi:10.1016/j.jpain.2008.03.007
PMCID: PMC3597078  PMID: 18456564
Disparities; orofacial pain; dental pain; Hispanic; acculturation; depression; health care use; sleep disturbance
22.  LPS and PAN-induced podocyte injury in an in vitro model of minimal change disease: changes in TLR profile 
Minimal change disease (MCD), the most common idiopathic nephrotic syndrome in children, is characterized by proteinuria and loss of glomerular visceral epithelial cell (podocyte) ultrastructure. Lipopolysaccharide (LPS) and puromycin aminonucleoside (PAN) are used to study podocyte injury in models of MCD in vivo and in vitro. We hypothesized that LPS and PAN influence components of the innate immune system in podocytes such as the Toll-Like Receptor (TLRs), TLR adapter molecules, and associated cytokines. Our results show that cultured human podocytes constitutively express TLRs 1–6 and TLR-10, but not TLRs 7–9. LPS (25 μg/ml) or PAN (60 μg/ml) caused comparable derangement of the actin cytoskeleton in podocytes. Quantitative RT-PCR analysis show that LPS differentially up-regulated the expression of genes for TLRs (1 > 4 ≥ 2 > 3 > 6 > 5), the adapter molecule, MyD88, and transcription factor NF-κB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-α, IFN-α or TGF-β1 at 24 h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-κB by 6 h. In contrast, PAN-induced only small changes in the expression of TLRs 2–6 that included a persistent increase in TLRs 2 and 5, a transient increase in TLR-4, and a gradual increase in TLRs 3 and 6 between 1 and 6 h. Correspondingly, it did not alter pro-inflammatory cytokine levels in podocytes. However, PAN induced a low but significant increase in NF-κB nuclear translocation within one hour that remained unchanged up to 6 h. In summary, these novel findings show that LPS, a known TLR-4 ligand, induced the gene expression of multiple TLRs with maximum effect on the expression of TLR-1 suggesting a loss of receptor selectivity and induction of receptor interactions in podocytes. A comparable derangement of the podocyte cytoskeleton and significant increase in the nuclear translocation of NF-κB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD.
doi:10.1007/s12079-012-0184-0
PMCID: PMC3590361  PMID: 23161414
Innate immunity; Toll-like receptors; Cytokines; Lipopolysaccharide; Puromycin Aminonucleoside; Minimal Change Disease; Glomerular filtration barrier; Podocytes
23.  Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures 
Journal of neuroscience methods  2011;203(1):141-145.
Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.
doi:10.1016/j.jneumeth.2011.09.007
PMCID: PMC3221776  PMID: 21968036
Primary neuronal culture; Cytotoxicity; Calcium mobilization; Drug screening; Neuroprotection; High-throughput screening
24.  Hearing Loss and Hair Cell Death in Mice Given the Cholesterol-Chelating Agent Hydroxypropyl-β-Cyclodextrin 
PLoS ONE  2012;7(12):e53280.
Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, 2-hydroxypropyl-β-cyclodextrin (HPβCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPβCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPβCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology.
doi:10.1371/journal.pone.0053280
PMCID: PMC3532434  PMID: 23285273
25.  A Simple, Low-Cost Conductive Composite Material for 3D Printing of Electronic Sensors 
PLoS ONE  2012;7(11):e49365.
3D printing technology can produce complex objects directly from computer aided digital designs. The technology has traditionally been used by large companies to produce fit and form concept prototypes (‘rapid prototyping’) before production. In recent years however there has been a move to adopt the technology as full-scale manufacturing solution. The advent of low-cost, desktop 3D printers such as the RepRap and Fab@Home has meant a wider user base are now able to have access to desktop manufacturing platforms enabling them to produce highly customised products for personal use and sale. This uptake in usage has been coupled with a demand for printing technology and materials able to print functional elements such as electronic sensors. Here we present formulation of a simple conductive thermoplastic composite we term ‘carbomorph’ and demonstrate how it can be used in an unmodified low-cost 3D printer to print electronic sensors able to sense mechanical flexing and capacitance changes. We show how this capability can be used to produce custom sensing devices and user interface devices along with printed objects with embedded sensing capability. This advance in low-cost 3D printing with offer a new paradigm in the 3D printing field with printed sensors and electronics embedded inside 3D printed objects in a single build process without requiring complex or expensive materials incorporating additives such as carbon nanotubes.
doi:10.1371/journal.pone.0049365
PMCID: PMC3504018  PMID: 23185319

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