bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of Reptile Pathogens Currently Known as Members of the Chrysosporium Anamorph of Nannizziopsis vriesii Complex and Relationship with Some Human-Associated Isolates
Authors: Sigler Lynne , Hambleton Sarah , Paré Jean A.
Published: August 2013

In recent years, theChrysosporiumanamorph ofNannizziopsis vriesii(CANV),Chrysosporium guarroi,Chrysosporium ophiodiicola, andChrysosporiumspecies have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared withN. vriesiibased on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the familyOnygenaceaeof the orderOnygenales. One lineage represents the genusNannizziopsisand comprisesN. vriesii,N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genusOphidiomyces, with the speciesOphidiomyces ophiodiicolaoccurring only in snakes, andParanannizziopsisgen. nov., with three new species infecting squamates and tuataras. The newly described species areNannizziopsis dermatitidis,Nannizziopsis crocodili,Nannizziopsis barbata,Nannizziopsis infrequens,Nannizziopsis hominis,Nannizziopsis obscura,Paranannizziopsis australasiensis,Paranannizziopsis californiensis, andParanannizziopsis crustacea.Chrysosporium longisporumhas been reclassified asParanannizziopsis longispora.N. guarroicauses yellow fungus disease, a common infection in bearded dragons and green iguanas, andO. ophiodiicolais an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.
Long-Term Carriage of Vancomycin-Resistant Enterococci in Patients Discharged from Hospitals: a 12-Year Retrospective Cohort Study
Authors: Karki Surendra , Land Gillian , Aitchison Stacey , Kennon Jacqueline , Johnson Paul D. R. , Ballard Susan A. , Leder Karin , Cheng Allen C.
Published: August 2013

Contact precautions are recommended in hospitals to prevent the transmission of vancomycin-resistant enterococci (VRE); however, there is no clear policy for how long patients should be under contact precautions due to a lack of information on the duration of carriage of these organisms. We conducted a retrospective cohort study to understand the duration of carriage of VRE (by screening of a single stool culture) and associated factors among patients who had been identified with VRE infection and/or colonization since the year 2000 at our health facilities. Of the 345 eligible participants, 136 did not respond, 90 declined to participate, and 16 did not send in the required specimens. Of the 103 remaining participants, 13 were found to have current VRE fecal carriage. The proportion of colonized patients fell from 40% (2/5) in the first year to 23.3% (7/30) in year 4. None of the 40 patients who had VRE detected >4 years prior were found to be colonized at the time of the study. The longest duration of detected VRE positivity was 46.5 months. Univariate analysis revealed that recent exposure to any antibiotics (
P
= 0.016), multiple antibiotics (
P
= 0.001), amoxicillin-clavulanic acid (
P
= 0.021), piperacillin-tazobactam (
P
= 0.007), glycopeptides (
P
< 0.001), meropenem (
P
= 0.007), aminoglycosides (
P
= 0.021), or fluoroquinolones (
P
= 0.021), being the index case in a clinical specimen (
P
= 0.016), and recent hospitalization (
P
< 0.001) were significantly associated with continued carriage on follow-up. In the surviving outpatients, a significant proportion appeared to clear VRE carriage. Our results suggest that in the absence of recent risk factors, such as hospitalization or antibiotic use, patients with a remote history of colonization (>4 years) may no longer require contact isolation precautions.
Four Main Virotypes among Extended-Spectrum-β-Lactamase-Producing Isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, Epidemiological, and Clinical Characteristics
Authors: Blanco Jorge , Mora Azucena , Mamani Rosalia , López Cecilia , Blanco Miguel , Dahbi Ghizlane , Herrera Alexandra , Marzoa Juan , Fernández Val , de la Cruz Fernando , Martínez-Martínez Luis , Alonso María Pilar , Nicolas-Chanoine Marie-Hélène , Johnson James R. , Johnston Brian , López-Cerero Lorena , Pascual Álvaro , Rodríguez-Baño Jesús
Published: August 2013

A total of 1,021 extended-spectrum-β-lactamase-producing
Escherichia coli
(ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (
afa
FM955459 positive,
iroN
negative,
ibeA
negative,
sat
positive or negative), B (31%) (
afa
FM955459 negative,
iroN
positive,
ibeA
negative,
sat
positive or negative), C (32%) (
afa
FM955459 negative,
iroN
negative,
ibeA
negative, sat positive), and D (13%) (
afa
FM955459 negative,
iroN
positive or negative,
ibeA
positive,
sat
positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.
Structural Insights into Adeno-Associated Virus Serotype 5
Authors: Govindasamy Lakshmanan , DiMattia Michael A. , Gurda Brittney L. , Halder Sujata , McKenna Robert , Chiorini John A. , Muzyczka Nicholas , Zolotukhin Sergei , Agbandje-McKenna Mavis
Published: August 2013

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.
Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease
Authors: Hetzel Udo , Sironen Tarja , Laurinmäki Pasi , Liljeroos Lassi , Patjas Aino , Henttonen Heikki , Vaheri Antti , Artelt Annette , Kipar Anja , Butcher Sarah J. , Vapalahti Olli , Hepojoki Jussi
Published: August 2013

Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates
in vitro
. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family
Arenaviridae
.
Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation
Authors: La Frazia Simone , Ciucci Alessandra , Arnoldi Francesca , Coira Maurizio , Gianferretti Patrizia , Angelini Mara , Belardo Giuseppe , Burrone Oscar R. , Rossignol Jean-Francois , Santoro M. Gabriella
Published: August 2013

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC
50
s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC
50
s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.
Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice
Authors: Durant Lydia R. , Makris Spyridon , Voorburg Cornelia Maaike , Loebbermann Jens , Johansson Cecilia , Openshaw Peter J. M.
Published: August 2013

During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the
Foxp3
DTR
mice (which allow specific conditional depletion of Foxp3
+
T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4
+
and CD8
+
T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4
+
T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.
Morphogenesis of Mimivirus and Its Viral Factories: an Atomic Force Microscopy Study of Infected Cells
Authors: Kuznetsov Yuri G. , Klose Thomas , Rossmann Michael , McPherson Alexander
Published: August 2013

Amoebas infected with mimivirus were disrupted at sequential stages of virus production and were visualized by atomic force microscopy. The development of virus factories proceeded over 3 to 4 h postinfection and resulted from the coalescence of 0.5- to 2-μm vesicles, possibly bearing nucleic acid, derived from either the nuclear membrane or the closely associated rough endoplasmic reticulum. Virus factories actively producing virus capsids on their surfaces were imaged, and this allowed the morphogenesis of the capsids to be delineated. The first feature to appear on a virus factory surface when a new capsid is born is the center of a stargate, which is a pentameric protein oligomer. As the arms of the stargate grow from the pentamer, a rough disk the diameter of a capsid thickens around it. This marks the initial emergence of a protein-coated membrane vesicle. The capsid self-assembles on the vesicle. Hillocks capped by different pentameric proteins spontaneously appear on the emerging vesicle at positions that are ultimately occupied by 5-fold icosahedral vertices. A lattice of coat protein nucleates at each of the 5-fold vertices, but not at the stargate, and then spreads outward from the vertices over the surface, merging seamlessly to complete the icosahedral capsid. Filling with DNA and associated proteins occurs by the transfer of nucleic acid from the interior of the virus factory into the nearly completed capsids. The portal, through which the DNA enters, is sealed by a plug of protein having a diameter of about 40 nm. A layer of integument protein that anchors the surface fibers is acquired by the passage of capsids through a membrane enriched in the protein. The coating of surface fibers is similarly acquired when the integument protein-coated capsids pass through a second membrane that has a forest of surface fibers embedded on one side.
Divergent MicroRNA Targetomes of Closely Related Circulating Strains of a Polyomavirus
Authors: Chen Chun Jung , Cox Jennifer E. , Kincaid Rodney P. , Martinez Angel , Sullivan Christopher S.
Published: August 2013

Hundreds of virus-encoded microRNAs (miRNAs) have been uncovered, but an in-depth functional understanding is lacking for most. A major challenge for the field is separating those miRNA targets that are biologically relevant from those that are not advantageous to the virus. Here, we show that miRNAs from related variants of the polyomavirus simian vacuolating virus 40 (SV40) have differing host target repertoires (targetomes) while their direct autoregulatory activity on virus-encoded early gene products is completely preserved. These results underscore the importance of miRNA-mediated viral gene autoregulation in some polyomavirus life cycles. More broadly, these findings imply that some host targets of virus-encoded miRNAs are likely to be of little selective advantage to the virus, and our approach provides a strategy for prioritizing relevant targets.
Bovine Herpesvirus 1 Regulatory Proteins bICP0 and VP16 Are Readily Detected in Trigeminal Ganglionic Neurons Expressing the Glucocorticoid Receptor during the Early Stages of Reactivation from Latency
Authors: Frizzo da Silva Leticia , Kook Insun , Doster Alan , Jones Clinton
Published: August 2013

Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viraltrans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons.
Rotavirus NSP4 Triggers Secretion of Proinflammatory Cytokines from Macrophages via Toll-Like Receptor 2
Authors: Ge Yi , Mansell Ashley , Ussher James E. , Brooks Anna E. S. , Manning Kristy , Wang Carol J. H. , Taylor John A.
Published: August 2013

Nonstructural protein 4 (NSP4), encoded by rotavirus, exhibits various properties linked to viral pathogenesis, including enterotoxic activity. A recent study (O. V. Kavanagh et al., Vaccine 28:3106-3111, 2010) indicated that NSP4 also has adjuvant properties, suggesting a possible role in the innate immune response to rotavirus infection. We report here that NSP4 purified from the medium of rotavirus-infected Caco-2 cells triggers the secretion of proinflammatory cytokines from macrophage-like THP-1 cells and nitric oxide from murine RAW 264.7 cells. Secretion is accompanied by the stimulation of p38 and JNK mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB. NSP4 triggered the secretion of cytokines from murine macrophages derived from wild-type but not MyD88
−/−
or Toll-like receptor 2 (TLR2
−/−
) mice and induced secretion of interleukin-8 (IL-8) from human embryonic kidney cells transfected with TLR2 but not TLR4. Our studies identify NSP4 as a pathogen-associated molecular pattern (PAMP) encoded by rotavirus and provide a mechanism for the production of proinflammatory cytokines associated with the clinical symptoms of infection in humans and animals.
Immeasurable glycosylated haemoglobin: a marker for severe haemolysis
Authors: Aggarwal Nidhi , Rai Anand Kumar , Kupfer Yizhak , Tessler Sidney
Published: August 2013

Glycosylated haemoglobin (HbA1c) is a measurement commonly performed in patients with diabetes. Factors causing a change in the life span of the red blood cell (RBC) can affect the measurement of HbA1c. Thus haemolysis is an important factor that may affect the HbA1c level determination. Haemolysis has been shown to cause a falsely low HbA1c. A 62-year-old man with a history of autoimmune haemolytic anaemia was admitted for severe haemolytic anaemia and an Hb of 2.9 g/dL. HbA1c tested during hospitalisation was unrecordable due to the extremely low Hb. The patient was treated with intravenous steroids, immunoglobulin, fluids and RBC transfusions but continued to haemolyse and eventually expired. We emphasise that an extremely low HbA1c level can serve as a marker of haemolysis and an unrecordable HbA1c level may point towards fatal haemolysis.
Evaluating the reproductive performance of British beef and dairy herds using national cattle movement records
Authors: Gates M. C.
Published: August 2013

National cattle movement databases provide a valuable opportunity to monitor the reproductive performance of breeding cattle on an industry‐wide scale. In this analysis, records from the Cattle Tracing System database were used to derive key measures of reproductive efficiency for British beef and dairy herds, including calving spread, age at first calving, calving interval, culling rate and calf mortality rate. At the animal level, only 8.5 per cent of beef heifers and 6.9 per cent of dairy heifers calved by the target age of 24 months. The average calving interval was 394 days for beef dams (median: 371) and 426 days for dairy dams (median: 400). Differences in performance were noted between cattle breeds. An estimated 43.9 per cent calves born in dairy herds were crossbreed beef animals, which may limit the availability of replacement dairy heifers. At the herd level, calving spread and calf mortality rates increased with herd size, while average age at first calving, calving interval, and crossbreeding generally decreased with herd size. Dam age, calving month, breed and twinning were significant risk factors for culling and calf mortality at the animal level. Wide variation in performance between individual herds highlights the potential for improving the efficiency of British cattle production.
Magma sources and tectonic setting of Central Andean andesites (25.5–28°S) related to crustal thickening, forearc subduction erosion and delamination
Authors: Kay Suzanne Mahlburg , Mpodozis Constantino , Gardeweg Moyra
Published: August 2013

The Central Andean margin, where the name andesite originated, is the type locality for arc andesites erupted through thick continental crust. The <25 Ma mid to high K
2
O andesites erupted from 25.5°S to 28.2°S exhibit a large variation in trace element and isotopic ratios, reflecting formation over an evolving slab, a crust thickening to 65–75 km and a frontal arc that migrated
c.
45 km eastward at 8–3 Ma. Andesites at 28–26.8°S have the most variable and extreme heavy rare earth element (REE), high field strength element (HFSE) and Ba/La ratios and wt% Na
2
O, with the highest values in those erupted as the frontal arc migrated and the slab shallowed to the south. The required garnet-bearing, feldspar-free residue is generated in both the thick crust and the mantle wedge, into which crust was injected in a peak of forearc subduction erosion as the arc migrated. Andesites at 25.5–26.8°S, east of the Puna plateau under which the slab shallowed at 18–7 Ma and then steepened as lithospheric delamination occurred, generally lack extreme REE and HFSE ratios. Their upper crust-like features reflect eruption in a mixed stress regime and incorporation of westward-flowing radiogenic crust from a region of extensive deep crustal melting to the east.
Surface-based reservoir modelling for flow simulation
Authors: Jackson M. D. , Hampson G. J. , Saunders J. H. , El-Sheikh A. , Graham G. H. , Massart B. Y. G.
Published: August 2013

We present a surface-based approach to reservoir model construction in which all geological heterogeneity, whether structural, stratigraphic, sedimentological or diagenetic, that impacts on the spatial distribution of petrophysical properties is modelled as one or more discrete volumes bounded by surfaces. The modelled surfaces can be deterministically interpolated between control lines or points, or incorporate a stochastic element where control data are sparse. Models constructed from surfaces are not constrained by an underlying grid; indeed, the model is generated without reference to a grid. The only difference between ‘geological’ and ‘simulation’ models is that the latter incorporates a grid or mesh to allow numerical solution of the governing flow equations, the architecture of which is driven by the architecture of the modelled surfaces. This approach to gridding (or meshing) is directly compatible with the next generation of unstructured-mesh simulators, and allows the capabilities of the latter to be utilized fully in the modelling of complex reservoir architectures. A surface-based approach to model construction may facilitate a step change in reservoir modelling capabilities: once the requirement to upscale geological models to a structured simulation grid is removed, there is no need to build geological models that are restricted by grid resolution.
A naturally occurring truncated Cav1.2 α1-subunit inhibits Ca2+ current in A7r5 cells
Authors: Cox Robert H. , Fromme Samantha J.
Published: August 2013

Alternative splicing of the voltage-gated Ca2+ (CaV) α1-subunit adds to the functional diversity of Ca2+ channels. A variant with a 73-nt deletion in exon 15 of the Cav1.2 α1-subunit (Cav1.2Δ73) produced by alternative splicing that predicts a truncated protein has been described, but its function, if any, is unknown. We sought to determine if, by analogy to other truncated CaV α1-subunits, Cav1.2Δ73 acts as an inhibitor of wild-type Cav1.2 currents. HEK-293 cells were transfected with Cav1.2Δ73 in a pIRES vector with CD8 or in pcDNA3.1 with a V5/ his COOH-terminal tag plus β2 and α2δ1 accessory subunits and pEGFP. Production of Cav1.2Δ73 protein was confirmed by Western blotting and immunofluorescence. Voltage-clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full-length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca2+ currents. A7r5 cells exhibited endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage-dependent activation when transfected with Cav1.2Δ73-IRES-CD8 compared with empty vector or pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.3Δ73-IRES-CD8 had no effect on Ca2+ currents. Immunofluorescence showed intracellular, but not plasma membrane, localization of Cav1.2Δ73-V5/ his, as well as colocalization with an endoplasmic reticulum marker, ER Organelle Lights. Expression of Cav1.2Δ73 α1-subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
Neuropeptide W increases mean arterial pressure as a result of behavioral arousal
Authors: Pate Alicia T. , Yosten Gina L. C. , Samson Willis K.
Published: August 2013

Neuropeptide W (NPW), an endogenous ligand for G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8), has been detected in neurons in limbic and reticular activating system areas known to be important in arousal, as well as hypothalamic nuclei known to be important in food and water intake and the neuroendocrine response to stress. In rat, central administration of NPW increased mean arterial pressure (MAP) and behaviors associated with locomotion and grooming. We hypothesized that the NPW-induced increase in MAP was secondary to those increases in physical activity. Since peptides that stimulate arousal have been shown to increase sympathetic activity (e.g., orexin), we tested the ability of the mixed α1- and α2-adrenergic antagonist, phentolamine, to block the NPW-23-induced rise in MAP. Phentolamine pretreatment abrogated the NPW-induced MAP increase. However, we noticed the animals no longer exhibited NPW-associated behavioral arousal when pretreated with phentolamine. Anesthesia also blocked the NPW-induced increase in MAP, although the animals still were able to respond with an increase in MAP to centrally administered ANG II. Additionally, pretreatment with an orexin type 1 receptor antagonist significantly reduced the behavioral action of NPW-23 and completely blocked the peptide's action to increase MAP, suggesting that orexin neurons are downstream targets of NPW. Our results suggest that NPW increased MAP secondary to increased behavioral arousal.
Nonuniform, age-related decrements in regional sweating and skin blood flow
Authors: Smith Caroline J. , Alexander Lacy M. , Kenney W. Larry
Published: August 2013

Aging is associated with attenuated thermoregulatory function that varies regionally over the body. Decrements in vasodilation and sweating are well documented with age, yet limited data are available concerning the regional relation between these responses. We aimed to examine age-related alterations in the relation between regional sweating (RSR) and skin blood flow (SkBF) to thermal and pharmacological stimuli. Four microdialysis fibers were inserted in the ventral forearm, abdomen, thigh, and lower back of eight healthy aged subjects (64 ± 7 yr) and nine young (23 ± 3 yr) during 1) ACh dose response (1 × 10−7 to 0.1 M, mean skin temperature 34°C) and 2) passive whole body heating to Δ1°C rise in oral temperature (Tor). RSR and SkBF were measured over each microdialysis membrane using ventilated capsules and laser-Doppler flowmetry. Maximal SkBF was measured at the end of both protocols (50 mM SNP). Regional sweating thresholds and RSR were attenuated in aged vs. young at all sites ( P < 0.0001) during whole body heating. Vasodilation thresholds were similar between groups ( P > 0.05). Attenuated SkBF were observed at the arm and back in the aged, representing 56 and 82% of those in the young at these sites, respectively (0.5 ΔTor). During ACh perfusion, SkBF ( P = 0.137) and RSR were similar between groups ( P = 0.326). Together these findings suggest regional age-related decrements in heat-activated sweat gland function but not cholinergic sensitivity. Functional consequences of such thermoregulatory impairment include the compromised ability of older individuals to defend core temperature during heat exposure and a subsequently greater susceptibility to heat-related illness and injury.
Post-oral appetite stimulation by sugars and nonmetabolizable sugar analogs
Authors: Zukerman Steven , Ackroff Karen , Sclafani Anthony
Published: August 2013

Post-oral sugar actions enhance the intake of and preference for sugar-rich foods, a process referred to as appetition. Here, we investigated the role of intestinal sodium glucose cotransporters (SGLTs) in sugar appetition in C57BL/6J mice using sugars and nonmetabolizable sugar analogs that differ in their affinity for SGLT1 and SGLT3. In experiments 1 and 2, food-restricted mice were trained (1 h/day) to consume a flavored saccharin solution [conditioned stimulus (CS−)] paired with intragastric (IG) self-infusions of water and a different flavored solution (CS+) paired with infusions of 8 or 12% sugars (glucose, fructose, and galactose) or sugar analogs (α-methyl-d-glucopyranoside, MDG; 3-O-methyl-d-glucopyranoside, OMG). Subsequent two-bottle CS+ vs. CS− choice tests were conducted without coinfusions. Infusions of the SGLT1 ligands glucose, galactose, MDG, and OMG stimulated CS+ licking above CS− levels. However, only glucose, MDG, and galactose conditioned significant CS+ preferences, with the SGLT3 ligands (glucose, MDG) producing the strongest preferences. Fructose, which is not a ligand for SGLTs, failed to stimulate CS+ intake or preference. Experiment 3 revealed that IG infusion of MDG+phloridzin (an SGLT1/3 antagonist) blocked MDG appetition, whereas phloridzin had minimal effects on glucose-induced appetition. However, adding phloretin (a GLUT2 antagonist) to the glucose+phloridzin infusion blocked glucose appetition. Taken together, these findings suggest that humoral signals generated by intestinal SGLT1 and SGLT3, and to a lesser degree, GLUT2, mediate post-oral sugar appetition in mice. The MDG results indicate that sugar metabolism is not essential for the post-oral intake-stimulating and preference-conditioning actions of sugars in mice.
Hypertension in an experimental model of systemic lupus erythematosus occurs independently of the renal nerves
Authors: Mathis Keisa W. , Venegas-Pont Marcia , Flynn Elizabeth R. , Williams Jan Michael , Maric-Bilkan Christine , Dwyer Terry M. , Ryan Michael J.
Published: August 2013

Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder with prevalent hypertension and renal injury. In this study, we tested whether the renal nerves contribute to the development of hypertension in an established mouse model of SLE ( NZBWF1). Female SLE and control ( NZW/LacJ) mice were subjected to either bilateral renal denervation or a sham procedure at 32 wk of age. Two weeks later, blood pressure was assessed in conscious mice using carotid artery catheters. Blood pressure was higher in SLE mice compared with controls, as previously reported; however, blood pressure was not altered in the denervated SLE or control mice. The development of albuminuria was markedly blunted in denervated SLE mice; however, glomerulosclerosis was increased. Renal denervation reduced renal cortical expression of monocyte-chemoattractant protein in SLE mice but did not significantly alter renal monocyte/macrophage infiltration. Renal cortical TNF-α expression was also increased in sham SLE mice, but this was not impacted by denervation. This study suggests that the renal nerves do not have a significant role in the pathogenesis of hypertension, but have a complex effect on the associated renal inflammation and renal injury.
Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct
Authors: Yu Zhiyuan , Kong Qun , Kone Bruce C.
Published: August 2013

Aldosterone increases tubular Na+ absorption largely by increasing α-epithelial Na+ channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.
Accounting for direction and speed of eye motion in planning visually guided manual tracking
Authors: Leclercq Guillaume , Blohm Gunnar , Lefèvre Philippe
Published: August 2013

Accurate motor planning in a dynamic environment is a critical skill for humans because we are often required to react quickly and adequately to the visual motion of objects. Moreover, we are often in motion ourselves, and this complicates motor planning. Indeed, the retinal and spatial motions of an object are different because of the retinal motion component induced by self-motion. Many studies have investigated motion perception during smooth pursuit and concluded that eye velocity is partially taken into account by the brain. Here we investigate whether the eye velocity during ongoing smooth pursuit is taken into account for the planning of visually guided manual tracking. We had 10 human participants manually track a target while in steady-state smooth pursuit toward another target such that the difference between the retinal and spatial target motion directions could be large, depending on both the direction and the speed of the eye. We used a measure of initial arm movement direction to quantify whether motor planning occurred in retinal coordinates (not accounting for eye motion) or was spatially correct (incorporating eye velocity). Results showed that the eye velocity was nearly fully taken into account by the neuronal areas involved in the visuomotor velocity transformation (between 75% and 102%). In particular, these neuronal pathways accounted for the nonlinear effects due to the relative velocity between the target and the eye. In conclusion, the brain network transforming visual motion into a motor plan for manual tracking adequately uses extraretinal signals about eye velocity.
A place for time: the spatiotemporal structure of neural dynamics during natural audition
Authors: Stephens Greg J. , Honey Christopher J. , Hasson Uri
Published: August 2013

We use functional magnetic resonance imaging (fMRI) to analyze neural responses to natural auditory stimuli. We characterize the fMRI time series through the shape of the voxel power spectrum and find that the timescales of neural dynamics vary along a spatial gradient, with faster dynamics in early auditory cortex and slower dynamics in higher order brain regions. The timescale gradient is observed through the unsupervised clustering of the power spectra of individual brains, both in the presence and absence of a stimulus, and is enhanced in the stimulus-locked component that is shared across listeners. Moreover, intrinsically faster dynamics occur in areas that respond preferentially to momentary stimulus features, while the intrinsically slower dynamics occur in areas that integrate stimulus information over longer timescales. These observations connect the timescales of intrinsic neural dynamics to the timescales of information processing, suggesting a temporal organizing principle for neural computation across the cerebral cortex.
Convergence and cross talk in urogenital neural circuitries
Authors: Hubscher C. H. , Gupta D. S. , Brink T. S.
Published: August 2013

Despite common comorbidity of sexual and urinary dysfunctions, the interrelationships between the neural control of these functions are poorly understood. The medullary reticular formation (MRF) contributes to both mating/arousal functions and micturition, making it a good site to test circuitry interactions. Urethane-anesthetized adult Wistar rats were used to examine the impact of electrically stimulating different nerve targets [dorsal nerve of the penis (DNP) or clitoris (DNC); L6/S1 trunk] on responses of individual extracellularly recorded MRF neurons. The effect of bladder filling on MRF neurons was also examined, as was stimulation of DNP on bladder reflexes via cystometry. In total, 236 MRF neurons responded to neurostimulation: 102 to DNP stimulation (12 males), 64 to DNC stimulation (12 females), and 70 to L6/S1 trunk stimulation (12 males). Amplitude thresholds were significantly different at DNP (15.0 ± 0.6 μA), DNC (10.5 ± 0.7 μA), and L6/S1 trunk (54.2 ± 4.6 μA), whereas similar frequency responses were found (max responses near 30–40 Hz). In five males, filling/voiding cycles were lengthened with DNP stimulation (11.0 ± 0.9 μA), with a maximal effective frequency plateau beginning at 30 Hz. Bladder effects lasted ∼2 min after DNP stimulus offset. Many MRF neurons receiving DNP/DNC input responded to bladder filling (35.0% and 68.3%, respectively), either just before (43%) or simultaneously with (57%) the voiding reflex. Taken together, MRF-evoked responses with neurostimulation of multiple nerve targets along with different responses to bladder infusion have implications for the role of MRF in multiple aspects of urogenital functions.
Pathological and physiological high-frequency oscillations in focal human epilepsy
Authors: Matsumoto Andrew , Brinkmann Benjamin H. , Matthew Stead S. , Matsumoto Joseph , Kucewicz Michal T. , Marsh W. Richard , Meyer Frederic , Worrell Gregory
Published: August 2013

High-frequency oscillations (HFO; gamma: 40–100 Hz, ripples: 100–200 Hz, and fast ripples: 250–500 Hz) have been widely studied in health and disease. These phenomena may serve as biomarkers for epileptic brain; however, a means of differentiating between pathological and normal physiological HFO is essential. We categorized task-induced physiological HFO during periods of HFO induced by a visual or motor task by measuring frequency, duration, and spectral amplitude of each event in single trial time-frequency spectra and compared them to pathological HFO similarly measured. Pathological HFO had higher mean spectral amplitude, longer mean duration, and lower mean frequency than physiological-induced HFO. In individual patients, support vector machine analysis correctly classified pathological HFO with sensitivities ranging from 70–98% and specificities >90% in all but one patient. In this patient, infrequent high-amplitude HFO were observed in the motor cortex just before movement onset in the motor task. This finding raises the possibility that in epileptic brain physiological-induced gamma can assume higher spectral amplitudes similar to those seen in pathologic HFO. This method if automated and validated could provide a step towards differentiating physiological HFO from pathological HFO and improving localization of epileptogenic brain.
Innate Immune Cells in Inflammation and Cancer
Authors: Nowarski Roni , Gagliani Nicola , Huber Samuel , Flavell Richard A.
Published: August 2013

The innate immune system has evolved in multicellular organisms to detect and respond to situations that compromise tissue homeostasis. It comprises a set of tissue-resident and circulating leukocytes primarily designed to sense pathogens and tissue damage through hardwired receptors and eliminate noxious sources by mediating inflammatory processes. While indispensable to immunity, the inflammatory mediators produced in situ by activated innate cells during injury or infection are also associated with increased cancer risk and tumorigenesis. Here, we outline basic principles of innate immune cell functions in inflammation and discuss how these functions converge upon cancer development. Cancer Immunol Res; 1(2); 77–84. ©2013 AACR.
Individual Music Therapy for Mental Health Care Clients with Low Therapy Motivation: Multicentre Randomised Controlled Trial
Authors: Gold Christian , Mössler Karin , Grocke Denise , Heldal Tor Olav , Tjemsland Lars , Aarre Trond , Aarø Leif Edvard , Rittmannsberger Hans , Stige Brynjulf , Assmus Jörg , Rolvsjord Randi
Published: August 2013

Background: Music therapy (MT) has been shown to be efficacious for mental health care clients with various disorders such as schizophrenia, depression and substance abuse. Referral to MT in clinical practice is often based on other factors than diagnosis. We aimed to examine the effectiveness of resource-oriented MT for mental health care clients with low motivation for other therapies. Method: This was a pragmatic parallel trial. In specialised centres in Norway, Austria and Australia, 144 adults with non-organic mental disorders and low therapy motivation were randomised to 3 months of biweekly individual, resource-oriented MT plus treatment as usual (TAU) or TAU alone. TAU was typically intensive (71% were inpatients) and included the best combination of therapies available for each participant, excluding MT. Blinded assessments of the Scale for the Assessment of Negative Symptoms (SANS) and 15 secondary outcomes were collected before randomisation and after 1, 3 and 9 months. Changes were analysed on an intention-to-treat basis using generalised estimating equations in longitudinal linear models, controlling for diagnosis, site and time point. Results: MT was superior to TAU for total negative symptoms (SANS, d = 0.54, p < 0.001) as well as functioning, clinical global impressions, social avoidance through music, and vitality (all p < 0.01). Conclusion: Individual MT as conducted in routine practice is an effective addition to usual care for mental health care clients with low motivation.