bluuebunny/crossref_metadata_2025 huggingface dataset:
A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin
Authors: Zhu Xueyong , Guo Yong-Hui , Jiang Tao , Wang Ya-Di , Chan Kwok-Hung , Li Xiao-Feng , Yu Wenli , McBride Ryan , Paulson James C. , Yuen Kwok-Yung , Qin Cheng-Feng , Che Xiao-Yan , Wilson Ian A.
Published: September 2013

Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.
Displacement of the C Terminus of Herpes Simplex Virus gD Is Sufficient To Expose the Fusion-Activating Interfaces on gD
Authors: Gallagher John R. , Saw Wan Ting , Atanasiu Doina , Lou Huan , Eisenberg Roselyn J. , Cohen Gary H.
Published: September 2013

Viral entry by herpes simplex virus (HSV) is executed and tightly regulated by four glycoproteins. While several viral glycoproteins can mediate viral adhesion to host cells, only binding of gD to cellular receptor can activate core fusion proteins gB and gH/gL to execute membrane fusion and viral entry. Atomic structures of gD bound to receptor indicate that the C terminus of the gD ectodomain must be displaced before receptor can bind to gD, but it is unclear which conformational changes in gD activate membrane fusion. We rationally designed mutations in gD to displace the C terminus and observe if fusion could be activated without receptor binding. Using a cell-based fusion assay, we found that gD V231W induced cell-cell fusion in the absence of receptor. Using recombinant gD V231W protein, we observed binding to conformationally sensitive antibodies or HSV receptor and concluded that there were changes proximal to the receptor binding interface, while the tertiary structure of gD V231W was similar to that of wild-type gD. We used a biosensor to analyze the kinetics of receptor binding and the extent to which the C terminus blocks binding to receptor. We found that the C terminus of gD V231W was enriched in the open or displaced conformation, indicating a mechanism for its function. We conclude that gD V231W triggers fusion through displacement of its C terminus and that this motion is indicative of how gD links receptor binding to exposure of interfaces on gD that activate fusion via gH/gL and gB.
Retrovirus Glycoprotein Functionality Requires Proper Alignment of the Ectodomain and the Membrane-Proximal Cytoplasmic Tail
Authors: Janaka Sanath Kumar , Gregory Devon A. , Johnson Marc C.
Published: September 2013

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the −1L, −2L, −3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.
A Mouse Monoclonal Antibody against Dengue Virus Type 1 Mochizuki Strain Targeting Envelope Protein Domain II and Displaying Strongly Neutralizing but Not Enhancing Activity
Authors: Yamanaka Atsushi , Kotaki Tomohiro , Konishi Eiji
Published: September 2013

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.
An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate
Authors: Trinité Benjamin , Ohlson Eric C. , Voznesensky Igor , Rana Shashank P. , Chan Chi N. , Mahajan Saurabh , Alster Jason , Burke Sean A. , Wodarz Dominik , Levy David N.
Published: September 2013

Integration is a central event in the replication of retroviruses, yet ≥90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4+T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4+T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4+T cells. We found that infection of cytokine-treated resting CD4+T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yieldedde novovirus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression andde novovirus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.
Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration
Authors: Gupta Saumya Shree , Maetzig Tobias , Maertens Goedele N. , Sharif Azar , Rothe Michael , Weidner-Glunde Magdalena , Galla Melanie , Schambach Axel , Cherepanov Peter , Schulz Thomas F.
Published: September 2013

Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV IN
in vitro
. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integration
in vitro
and, in the case of BRD2, also
in vivo
. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.
The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm
Authors: Xiao Han , Killip Marian J. , Staeheli Peter , Randall Richard E. , Jackson David
Published: September 2013

The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.
Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication
Authors: Luo Yong , Kleiboeker Steve , Deng Xuefeng , Qiu Jianming
Published: September 2013

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be “G
2
/M arrest.” However, a B19V mutant infectious DNA (M20
mTAD2
) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G
2
/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol.
86:
10748–10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2′-deoxyuridine (BrdU) pulse-labeling and DAPI (4′,6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20
mTAD2
mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.
European Public Assessment Report (EPAR) summaries for the public: are they fit for purpose? A user-testing study
Authors: Raynor David K , Bryant David
Published: September 2013

Objectives

Apply ‘user testing’ methodology to test the readability of a European Public Assessment Report (EPAR) summary—which describes how the decision was made by the European Medicines Agency to approve a medicine.

Design

User testing uses mixed methods (questionnaire and semistructured interview), applied iteratively, to assess document performance—can people find and understand key points of information.

Setting and participants

Testing was undertaken with 40 members of the public in four consecutive rounds of 10. Inclusion criteria, matched across rounds, included range of ages and educational attainment.

Tested documents

In round 1 we tested 19 key points of information in a printed version of the EPAR summary for Bondronat (a cancer medicine). This was then revised to address the findings, and tested in round 2. In round 3 we tested the summary on-screen, and in round 4, tested a revised on-screen version, after addressing findings from both rounds 1 and 3.

Primary outcome measure

The target followed European guidance for medicine leaflets: for each point of information 90% of participants should be able to find, and of those, 90% able to show understanding of the point.

Results

For the original EPAR summary, 6 of the 19 points of information reached the target (both paper-based and on-screen). After revisions to format and content, using good practice in information writing and design, 14 and 16 points, respectively, met the target. The problems related to both finding (dependent on layout, headings and design) and understanding (words and sentences used, as well as design). We devised a new heading structure, increased use of bullet points, replaced difficult and technical words and divided long sentences.

Conclusions

People had difficulty finding and understanding key messages in the summary, but user testing identified the problems, and application of good practice resulted in a revised format which performed well.
An economic evaluation of anonymised information sharing in a partnership between health services, police and local government for preventing violence-related injury
Authors: Florence Curtis , Shepherd Jonathan , Brennan Iain , Simon Thomas R
Published: September 2013

Objective

To assess the costs and benefits of a partnership between health services, police and local government shown to reduce violence-related injury.

Methods

Benefit-cost analysis.

Results

Anonymised information sharing and use led to a reduction in wounding recorded by the police that reduced the economic and social costs of violence by £6.9 million in 2007 compared with the costs the intervention city, Cardiff UK, would have experienced in the absence of the programme. This includes a gross cost reduction of £1.25 million to the health service and £1.62 million to the criminal justice system in 2007. By contrast, the costs associated with the programme were modest: setup costs of software modifications and prevention strategies were £107 769, while the annual operating costs of the system were estimated as £210 433 (2003 UK pound). The cumulative social benefit-cost ratio of the programme from 2003 to 2007 was £82 in benefits for each pound spent on the programme, including a benefit-cost ratio of 14.80 for the health service and 19.1 for the criminal justice system. Each of these benefit-cost ratios is above 1 across a wide range of sensitivity analyses.

Conclusions

An effective information-sharing partnership between health services, police and local government in Cardiff, UK, led to substantial cost savings for the health service and the criminal justice system compared with 14 other cities in England and Wales designated as similar by the UK government where this intervention was not implemented.
Successful treatment of extensive cerebral venous sinus thrombosis using a combined approach with Penumbra aspiration system and Solitaire FR retrieval device
Authors: Raychev Radoslav , Tateshima Satoshi , Rastogi Sachin , Balgude Amit , Yafeh Banafsheh , Saver Jeffrey L , Vespa Paul M , Buitrago Manuel , Duckwiler Gary
Published: September 2013

We present a young woman with rapidly progressive neurologic decline in the setting of malignant cerebral edema due to extensive superior sagittal sinus thrombosis and cortical venous thrombosis despite intravenous heparin administration. Complete revascularization of the occluded sinus was achieved using suction thrombectomy with the 5 max Penumbra catheter in combination with the Solitaire FR clot retrieval device. The successful endovascular treatment halted the progression of her cerebral edema, and the patient eventually had an excellent recovery after prolonged intensive medical therapy. To our knowledge, this is the first reported case describing such a combined mechanical approach for cerebral venous sinus thrombosis. The clot retrieval properties of the Solitaire device combined with direct aspiration via the newest generation Penumbra catheters may allow more rapid, safe and efficient revascularization than all previously reported endovascular treatments for this potentially devastating condition.
N-methyl-d-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation
Authors: Makhro Asya , Hänggi Pascal , Goede Jeroen S. , Wang Jue , Brüggemann Andrea , Gassmann Max , Schmugge Markus , Kaestner Lars , Speer Oliver , Bogdanova Anna
Published: September 2013

The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca2+ uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca2+ accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca2+ homeostasis and modulate oxygen delivery to peripheral tissues.
Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli
Authors: Ilatovskaya Daria V. , Palygin Oleg , Levchenko Vladislav , Staruschenko Alexander
Published: September 2013

Calcium flux in the podocytes is critical for normal and pathophysiological regulation of these types of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine the P2 receptor profile in podocytes of the intact Sprague-Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving and loaded with Fluo-4/Fura Red cell permeable calcium indicators, and the purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared with the effect of ATP, specifically, UDP, MRS 2365, bzATP, αβ-methylene, 2-meSADP, MRS 4062, and MRS 2768, were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079 , and NF 449) were tested when 10 μM ATP was applied as the EC50 for ATP activation of the calcium influx in the podocytes was determined to be 10.7 ± 1.5 μM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only the P2Y1-specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y1 receptors are highly expressed in the podocytes. We conclude that P2Y1 receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y1 might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases.
ENaC-expressing neurons in the sensory circumventricular organs become c-Fos activated following systemic sodium changes
Authors: Miller Rebecca L. , Wang Michelle H. , Gray Paul A. , Salkoff Lawrence B. , Loewy Arthur D.
Published: September 2013

The sensory circumventricular organs (CVOs) are specialized collections of neurons and glia that lie in the midline of the third and fourth ventricles of the brain, lack a blood-brain barrier, and function as chemosensors, sampling both the cerebrospinal fluid and plasma. These structures, which include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), are sensitive to changes in sodium concentration but the cellular mechanisms involved remain unknown. Epithelial sodium channel (ENaC)-expressing neurons of the CVOs may be involved in this process. Here we demonstrate with immunohistochemical and in situ hybridization methods that ENaC-expressing neurons are densely concentrated in the sensory CVOs. These neurons become c-Fos activated, a marker for neuronal activity, after various manipulations of peripheral levels of sodium including systemic injections with hypertonic saline, dietary sodium deprivation, and sodium repletion after prolonged sodium deprivation. The increases seen c-Fos activity in the CVOs were correlated with parallel increases in plasma sodium levels. Since ENaCs play a central role in sodium reabsorption in kidney and other epithelia, we present a hypothesis here suggesting that these channels may also serve a related function in the CVOs. ENaCs could be a significant factor in modulating CVO neuronal activity by controlling the magnitude of sodium permeability in neurons. Hence, some of the same circulating hormones controlling ENaC expression in kidney, such as angiotensin II and atrial natriuretic peptide, may coordinate ENaC expression in sensory CVO neurons and could potentially orchestrate sodium appetite, osmoregulation, and vasomotor sympathetic drive.
Acute systemic hypoxia activates hypothalamic paraventricular nucleus-projecting catecholaminergic neurons in the caudal ventrolateral medulla
Authors: King T. Luise , Kline David D. , Ruyle Brian C. , Heesch Cheryl M. , Hasser Eileen M.
Published: September 2013

Hypoxia activates catecholamine neurons in the caudal ventrolateral medulla (CVLM). The hypothalamic paraventricular nucleus (PVN) modulates arterial chemoreflex responses and receives catecholaminergic projections from the CVLM, but it is not known whether the CVLM-PVN projection is activated by chemoreflex stimulation. We hypothesized that acute hypoxia (AH) activates PVN-projecting catecholaminergic neurons in the CVLM. Fluoro-Gold (2%, 60–90 nl) was microinjected into the PVN of rats to retrogradely label CVLM neurons. After recovery, conscious rats underwent 3 h of normoxia (21% O2, n = 4) or AH (12, 10, or 8% O2; n = 5 each group). We used Fos immunoreactivity as an index of CVLM neuronal activation and tyrosine hydroxylase (TH) immunoreactivity to identify catecholaminergic neurons. Positively labeled neurons were counted in six caudal-rostral sections containing CVLM. Hypoxia progressively increased the number of Fos-immunoreactive CVLM cells (21%, 19 ± 6; 12%, 49 ± 2; 10%, 117 ± 8; 8%, 179 ± 7; P < 0.001). Catecholaminergic cells colabeled with Fos immunoreactivity in the CVLM were observed following 12% O2, and further increases in hypoxia severity caused markedly more activation. PVN-projecting CVLM cells were activated following more severe hypoxia (10% and 8% O2). A large proportion (89 ± 3%) of all activated PVN-projecting CVLM neurons were catecholaminergic, regardless of hypoxia intensity. Data suggest that catecholaminergic, PVN-projecting CVLM neurons are particularly hypoxia-sensitive, and these neurons may be important in the cardiorespiratory and/or neuroendocrine responses elicited by the chemoreflex.
Phylogenetic, expression, and functional analyses of anoctamin homologs inCaenorhabditis elegans
Authors: Wang Ying , Alam Tashrique , Hill-Harfe Katherine , Lopez Alejandro J. , Leung Chi K. , Iribarne Daniela , Bruggeman Bradley , Miyamoto Michael M. , Harfe Brian D. , Choe Keith P.
Published: September 2013

Ca2+-activated Cl−channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.
Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp,Litopenaeus vannamei
Authors: Hardy Kristin M. , Burnett Karen G. , Burnett Louis E.
Published: September 2013

Estuarine species frequently encounter areas of simultaneously low dissolved O2(hypoxia) and high CO2(hypercapnia). Organisms exposed to hypoxia experience a metabolic depression that serves to decrease ATP utilization and O2demand during stress. This downregulation is typically facilitated by a reduction in protein synthesis, a process that can be responsible for up to 60% of basal metabolism. The added effects of hypercapnia, however, are unclear. Certain decapods also exhibit a metabolic depression in response to bacterial challenges, leading us to hypothesize that protein synthesis may also be reduced during infection. In the present study, we examined the effects of hypoxia (H), hypercapnic hypoxia (HH), and bacterial infection ( Vibrio campbellii) on tissue-specific (muscle and hepatopancreas) fractional protein synthesis rates ( ks) in Litopenaeus vannamei. We observed a significant decrease in ksin muscle after 24 h exposure to both H and HH, and in hepatopancreas after 24 h exposure to HH. Thus ksis responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ksis more severe than hypoxia alone. These reductions in ksappear to be driven by changes in RNA translational efficiency ( kRNA), and not RNA capacity ( Cs). Bacterial infection, however, had no significant effect on ksin either tissue. These results suggest that crustaceans reduce metabolic demand during environmental hypoxia by reducing global protein synthesis, and that this effect is magnified when hypercapnia is concomitantly present. Conversely, an immune-mediated metabolic depression is not associated with a decrease in overall protein production.
TGF-β1 stimulates mitochondrial oxidative phosphorylation and generation of reactive oxygen species in cultured mouse podocytes, mediated in part by the mTOR pathway
Authors: Abe Yoshifusa , Sakairi Toru , Beeson Craig , Kopp Jeffrey B.
Published: September 2013

Transforming growth factor (TGF)-β has been associated with podocyte injury; we have examined its effect on podocyte bioenergetics. We studied transformed mouse podocytes, exposed to TGF-β1, using a label-free assay system, Seahorse XF24, which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Both basal OCR and ATP generation-coupled OCR were significantly higher in podocytes exposed to 0.3–10 ng/ml of TGF-β1 for 24, 48, and 72 h. TGF-β1 (3 ng/ml) increased oxidative capacity 75%, and 96% relative to control after 48 and 72 h, respectively. ATP content was increased 19% and 30% relative to control after a 48- and 72-h exposure, respectively. Under conditions of maximal mitochondrial function, TGF-β1 increased palmitate-driven OCR by 49%. Thus, TGF-β1 increases mitochondrial oxygen consumption and ATP generation in the presence of diverse energy substrates. TGF-β1 did not increase cell number or mitochondrial DNA copy number but did increase mitochondrial membrane potential (MMP), which could explain the OCR increase. Reactive oxygen species (ROS) increased by 32% after TGF-β1 exposure for 48 h. TGF-β activated the mammalian target of rapamycin (mTOR) pathway, and rapamycin reduced the TGF-β1-stimulated increases in OCR, ECAR, ATP generation, cellular metabolic activity, and protein generation. Our data suggest that TGF-β1, acting, in part, via mTOR, increases mitochondrial MMP and OCR, resulting in increased ROS generation and that this may contribute to podocyte injury.
Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture
Authors: Girard Beatrice M. , Merriam Laura A. , Tompkins John D. , Vizzard Margaret A. , Parsons Rodney L.
Published: September 2013

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.
Functional significance of cortical NMDA receptors in somatosensory information processing
Authors: Lo Fu-Sun , Akkentli Fatih , Tsytsarev Vassiliy , Erzurumlu Reha S.
Published: September 2013

N-methyl-d-aspartate receptor (NMDAR)-mediated activity is required for whisker-related neural patterning in the rodent brain. Deletion of the essential NMDAR subunit NR1 gene in excitatory cortical neurons prevents whisker-specific barrel formation and impairs thalamocortical afferent patterning. We used electrophysiological and voltage-sensitive dye imaging methods to assess synaptic and sensory evoked cortical activity and immunohistochemistry to examine immediate early gene expression following whisker stimulation in cortex-specific NR1 knockout (Cx NR1KO) mice. In mutant mice, layer IV neurons lacked NMDAR-mediated excitatory postsynaptic currents, and temporal summation of excitatory postsynaptic potentials (EPSPs) was impaired. Barrel neurons showed both phasic and tonic responses to whisker deflection. The averaged tonic response in Cx NR1KO mice was significantly less than that in control mice due to impaired EPSP temporal summation. Electrophysiological estimation of the number of thalamic neurons innervating single barrel neurons indicated a significant increase in Cx NR1KO mice. Similarly, voltage-sensitive dye optical signals in response to whisker stimulation were widespread. Immediate early gene expression following whisker stimulation also showed a diffuse expression pattern in the Cx NR1KO cortex compared with whisker-specific expression patterns in controls. Thus, when NMDAR function is impaired, spatial discrimination of whisker inputs is severely compromised, and sensory stimulation evokes diffuse, topographically misaligned activity in the barrel cortex.
Medullary serotonin neurons are CO2 sensitive in situ
Authors: Iceman Kimberly E. , Richerson George B. , Harris Michael B.
Published: September 2013

Brainstem central chemoreceptors are critical to the hypercapnic ventilatory response, but their location and identity are poorly understood. When studied in vitro, serotonin-synthesizing (5-HT) neurons within the rat medullary raphé are intrinsically stimulated by CO2/acidosis. The contributions of these neurons to central chemosensitivity in vivo, however, are controversial. Lacking is documentation of CO2-sensitive 5-HT neurons in intact experimental preparations and understanding of their spatial and proportional distribution. Here we test the hypothesis that 5-HT neurons in the rat medullary raphé are sensitive to arterial hypercapnia. We use extracellular recording and hypercapnic challenge of spontaneously active medullary raphé neurons in the unanesthetized in situ perfused decerebrate brainstem preparation to assess chemosensitivity of individual cells. Juxtacellular labeling of a subset of recorded neurons and subsequent immunohistochemistry for the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) identify or exclude this neurotransmitter phenotype in electrophysiologically characterized chemosensitive and insensitive cells. We show that the medullary raphé houses a heterogeneous population, including chemosensitive and insensitive 5-HT neurons. Of 124 recorded cells, 16 cells were juxtacellularly filled, visualized, and immunohistochemically identified as 5-HT synthesizing, based on TPH-immunoreactivity. Forty-four percent of 5-HT cells were CO2 stimulated (increased firing rate with hypercapnia), while 56% were unstimulated. Our results demonstrate that medullary raphé neurons are heterogeneous and clearly include a subset of 5-HT neurons that are excited by arterial hypercapnia. Together with data identifying intrinsically CO2-sensitive 5-HT neurons in vitro, these results support a role for such cells as central chemoreceptors in the intact system.
The specificity of stimulus-specific adaptation in human auditory cortex increases with repeated exposure to the adapting stimulus
Authors: Briley Paul M. , Krumbholz Katrin
Published: September 2013

The neural response to a sensory stimulus tends to be more strongly reduced when the stimulus is preceded by the same, rather than a different, stimulus. This stimulus-specific adaptation (SSA) is ubiquitous across the senses. In hearing, SSA has been suggested to play a role in change detection as indexed by the mismatch negativity. This study sought to test whether SSA, measured in human auditory cortex, is caused by neural fatigue (reduction in neural responsiveness) or by sharpening of neural tuning to the adapting stimulus. For that, we measured event-related cortical potentials to pairs of pure tones with varying frequency separation and stimulus onset asynchrony (SOA). This enabled us to examine the relationship between the degree of specificity of adaptation as a function of frequency separation and the rate of decay of adaptation with increasing SOA. Using simulations of tonotopic neuron populations, we demonstrate that the fatigue model predicts independence of adaptation specificity and decay rate, whereas the sharpening model predicts interdependence. The data showed independence and thus supported the fatigue model. In a second experiment, we measured adaptation specificity after multiple presentations of the adapting stimulus. The multiple adapters produced more adaptation overall, but the effect was more specific to the adapting frequency. Within the context of the fatigue model, the observed increase in adaptation specificity could be explained by assuming a 2.5-fold increase in neural frequency selectivity. We discuss possible bottom-up and top-down mechanisms of this effect.
Calcium Unresponsive Hypocalcemic Tetany: Gitelman Syndrome with Hypocalcemia
Authors: Desai Madhav , Kolla Praveen Kumar , Reddy P. L. Venkata Pakki
Published: September 2013

Introduction. Gitelman’s syndrome (GS) is autosomal recessive renal tubular disorder characterized by hypokalemia, hypomagnesemia, hypocalciuria, metabolic alkalosis, and hyperreninemic hyperaldosteronism. It is usually associated with normal serum calcium. We report a patient presented with hypocalcemic tetany, and evaluation showed Gitelman’s syndrome with hypocalcemia.Case Report. A 28-year-old woman presented with cramps of the arms, legs, fatigue, and carpal spasms of one week duration. She has history of similar episodes on and off for the past two years. Her blood pressure was 98/66 mmHg. Chvostek’s sign and Trousseau’s sign were positive. Evaluation showed hypokalemia, hypocalcemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. Self-medication, diuretic use, laxative abuse, persistent vomiting, and diarrhoea were ruled out. Urinary prostaglandins and genetic testing could not be done because of nonavailability. To differentiate Gitelman syndrome from Bartter’s syndrome (BS), thiazide loading test was done. It showed blunted fractional chloride excretion. GS was confirmed and patient was treated with spironolactone along with magnesium, calcium, and potassium supplementation. Symptomatically, she improved and did not develop episodes of tetany again.Conclusion. In tetany patient along with serum calcium measurement, serum magnesium, serum potassium, and arterial blood gases should be measured. Even though hypocalcemia in Gitelman syndrome is rare, it still can occur.
An Integral Equations Method for the Cauchy Problem Connected with the Helmholtz Equation
Authors: Sun Yao , Zhang Deyue
Published: September 2013

We are concerned with the Cauchy problem connected with the Helmholtz equation. We propose a numerical method, which is based on the Helmholtz representation, for obtaining an approximate solution to the problem, and then we analyze the convergence and stability with a suitable choice of regularization method. Numerical experiments are also presented to show the effectiveness of our method.
Leptin Increases Blood Pressure and Markers of Endothelial Activation during Pregnancy in Rats
Authors: Ibrahim Hisham Saleh , Omar Effat , Froemming Gabrielle Ruth Anisah , Singh Harbindar Jeet
Published: September 2013

Raised leptin levels have been reported in the placentae and serum of women with elevated blood pressure and proteinuria during pregnancy. The role of leptin in this however remains unknown. This study investigates the effect of leptin administration on systolic blood pressure (SBP) and proteinuria and serum markers of endothelial activation during pregnancy inSprague Dawley rats. From day 1 of pregnancy, 24 rats were randomised into those given either saline (group 1) or leptin at 60 or 120 μg/kg/body weight/day (groups 2 and 3 resp.). SBP was measured every 5 days and 24-h urinary protein was measured at days 0 and 20 of pregnancy. Animals were euthanised on day 20 of pregnancy, and serum was collected for estimation of E-selectin and ICAM-1. Compared to group 1, SBP during the latter part of the pregnancy was significantly higher in the leptin-treated group (P<0.01). Urinary protein excretion, serum E-selectin, and ICAM-1 were significantly higher in leptin-treated rats (P<0.05). It seems that leptin administration to normotensiveSprague Dawley ratsduring pregnancy significantly increases SBP, urinary protein excretion, and markers of endothelial activation. However, further studies are required to examine the underlying mechanism responsible for this and its relevance to preeclampsia in humans.
A Kinetic Model for Predicting the Relative Humidity in Modified Atmosphere Packaging and Its Application inLentinula edodesPackages
Authors: Lu Li-xin , Tang Ya-li , Lu Su-yue
Published: September 2013

Adjusting and controlling the relative humidity (RH) inside package is crucial for ensuring the quality of modified atmosphere packaging (MAP) of fresh produce. In this paper, an improved kinetic model for predicting the RH in MAP was developed. The model was based on heat exchange and gases mass transport phenomena across the package, gases heat convection inside the package, and mass and heat balances accounting for the respiration and transpiration behavior of fresh produce. Then the model was applied to predict the RH in MAP of freshLentinula edodes(one kind of Chinese mushroom). The model equations were solved numerically using Adams-Moulton method to predict the RH in model packages. In general, the model predictions agreed well with the experimental data, except that the model predictions were slightly high in the initial period. The effect of the initial gas composition on the RH in packages was notable. In MAP of lower oxygen and higher carbon dioxide concentrations, the ascending rate of the RH was reduced, and the RH inside packages was saturated slowly during storage. The influence of the initial gas composition on the temperature inside package was not much notable.
A Rare Case with a Solitary Fibrous Tumour of the Colon and an Epithelioid Angiomyolipoma of the Kidney
Authors: Pham Thong Quang , Aida Shinsuke , Mori Ichiro , Koshida Takeo , Ohigashi Takashi , Katase Katsuyoshi , Murakami Fumiyoshi , Tran Thong Minh , Osamura Robert Y.
Published: September 2013

Solitary fibrous tumour is a soft tissue tumour composed of a subset of fibroblast-like cells and frequently needs immunohistochemical staining for final diagnosis. Epithelioid angiomyolipoma is a variant of angiomyolipoma but characterized by the absence of both adipocytes and abnormal blood vessels. We introduce a very rare case with the combination of these two tumours. A Japanese female patient without significant symptom was hospitalized and operated due to multiple uterine leiomyomas. During the operation, the surgeons found another tumour attaching to serosa of sigmoid colon. This tumour was resected and interpreted as solitary fibrous tumour, suspicious of malignancy. After 13 months of treatment, she was hospitalized again due to hematuria. The doctors detected a tumour in her right kidney. After consultation, laparoscopic right nephrectomy was done. The pathological result of this tumour was epithelioid angiomyolipoma. This is the first report on this very rare combination of tumours with extensive immunohistochemical demonstration of both tumours. Hereby, we review clinical information and histopathological findings together with discussion on each tumour.
Morphologic Changes in the Foveal Photoreceptor Layer before and after Laser Treatment in Acute and Chronic Central Serous Chorioretinopathy Documented in Spectral-Domain Optical Coherence Tomography
Authors: Odrobina Dominik , Laudańska-Olszewska Iwona , Gozdek Piotr , Maroszyński Mariusz , Amon Michael
Published: September 2013

Purpose. To analyze microstructural changes in the external limiting membrane (ELM) and photoreceptor layer before and after early and late conventional laser treatment in central serous chorioretinopathy (CSC) in 12 months follow-up study.Methods. A retrospective observational study included Group A: 19 patients (19 eyes) with symptomatic acute CSC and Group B: 16 patients (16 eyes) with symptomatic chronic CSC. Retinal microstructural changes were analyzed with SD-OCT paying a particular role in examining the photoreceptor layer and ELM.Results. The length of the photoreceptors, prior to treatment, was approximately 84 μm in Group A and 82,5 μm in Group B. Twelve months after laser treatment, photoreceptor length was approximately 49 μm in Group A and 43 μm (range 20–55 μm) in Group B. No patients in Group A had noticeable photoreceptor defects nor ELM defects, but in 15 eyes in Group B photoreceptor and ELM defects were detected ().Conclusions. When analyzing the photoreceptor layer and ELM during active CSC, it is not possible to evaluate any irreversible changes which have already occurred in this layer. Damage to the photoreceptor layer and ELM in patients with chronic CSC was only found after laser treatment and the absorption of subretinal fluid.