bluuebunny/crossref_metadata_2025 huggingface dataset:
Importin-α7 Is Required for Enhanced Influenza A Virus Replication in the Alveolar Epithelium and Severe Lung Damage in Mice
Authors: Resa-Infante Patricia , Thieme René , Ernst Thomas , Arck Petra C. , Ittrich Harald , Reimer Rudolph , Gabriel Gülsah
Published: May 2014

Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-α7 in H1N1 influenza virus pulmonary tropism by using various
ex vivo
imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-α7 gene-deficient (α7
−/−
) mice with a recombinant H1N1 influenza virus and compared the
in vivo
viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in α7
−/−
mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and α7
−/−
mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-α7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice.

IMPORTANCE
Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-α7, for efficient virus replication in the alveolar epithelium of mice. Using complementary
ex vivo
imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-α7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-α7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.
Expression of Mosquito MicroRNA Aae-miR-2940-5p Is Downregulated in Response to West Nile Virus Infection To Restrict Viral Replication
Authors: Slonchak Andrii , Hussain Mazhar , Torres Shessy , Asgari Sassan , Khromykh Alexander A.
Published: May 2014

West Nile virus (WNV) is an enveloped virus with a single-stranded positive-sense RNA genome from the
Flaviviridae
family. WNV is spread by mosquitoes and able to infect humans, causing encephalitis and meningitis that can be fatal; it therefore presents a significant risk for human health. In insects, innate response to RNA virus infection mostly relies on RNA interference and JAK/SAT pathways; however, some evidence indicates that it can also involve microRNAs (miRNAs). miRNAs are small noncoding RNAs that regulate gene expression at posttranscriptional level and play an important role in a number of processes, including immunity and antiviral response. In this study, we focus on the miRNA-mediated response to WNV in mosquito cells. We demonstrate that in response to WNV infection the expression of a mosquito-specific miRNA, aae-miR-2940, is selectively downregulated in
Aedes albopictus
cells. This miRNA is known to upregulate the metalloprotease m41 FtsH gene, which we have also shown to be required for efficient WNV replication. Correspondingly, downregulation of aae-miR-2940 reduced the metalloprotease level and restricted WNV replication. Thus, we have identified a novel miRNA-dependent mechanism of antiviral response to WNV in mosquitoes.

IMPORTANCE
A detailed understanding of vector-pathogen interactions is essential to address the problems posed by vector-borne diseases. Host and viral miRNAs play an important role in regulating expression of viral and host genes involved in endogenous processes, including antiviral response. There has been no evidence to date for the role of mosquito miRNAs in response to flaviviruses. In this study, we show that downregulation of aae-miR-2940 in mosquito cells acts as a potential antiviral mechanism in the mosquito host to inhibit WNV replication by repressing the expression of the metalloprotease m41 FtsH gene, which is required for efficient WNV replication. This is the first identification of an miRNA-dependent antiviral mechanism in mosquitoes, which inhibits replication of WNV. Our findings should facilitate identification of targets in the mosquito genome that can be utilized to suppress vector population and/or limit WNV replication.
Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses
Authors: Opperman Pamela A. , Rotherham Lia S. , Esterhuysen Jan , Charleston Bryan , Juleff Nicholas , Capozzo Alejandra V. , Theron Jacques , Maree Francois F.
Published: May 2014

Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV.

IMPORTANCE
Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.
N
-Glycolylneuraminic Acid on Human Epithelial Cells Prevents Entry of Influenza A Viruses That Possess
N
-Glycolylneuraminic Acid Binding Ability
Authors: Takahashi Tadanobu , Takano Maiko , Kurebayashi Yuuki , Masuda Midori , Kawagishi Sawako , Takaguchi Masahiro , Yamanaka Takashi , Minami Akira , Otsubo Tadamune , Ikeda Kiyoshi , Suzuki Takashi
Published: May 2014

Some animal influenza A viruses (IAVs) bind not only to
N
-acetylneuraminic acid (Neu5Ac) but also to
N
-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of
N
-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection.

IMPORTANCE
Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to
N
-acetylneuraminic acid (Neu5Ac) as a receptor but also to
N
-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a “decoy receptor” of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-
N
-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.
Interleukin-6 (IL-6) and IL-17 Synergistically Promote Viral Persistence by Inhibiting Cellular Apoptosis and Cytotoxic T Cell Function
Authors: Hou Wanqiu , Jin Young-Hee , Kang Hyun Seok , Kim Byung S.
Published: May 2014

Interleukin-6 (IL-6) plays an important role in the development and progression of inflammatory responses, autoimmune diseases, and cancers. Many viral infections, including Theiler's murine encephalomyelitis virus (TMEV), result in the vigorous production of IL-6. However, the role of IL-6 in the development of virus-induced inflammatory responses is unclear. The infection of susceptible mice with TMEV induces the development of chronic demyelinating disease, which is considered a relevant infectious model for multiple sclerosis. In this study, we demonstrate that resistant C57BL/6 mice carrying an IL-6 transgene (IL-6 Tg) develop a TMEV-induced demyelinating disease accompanied by an increase in viral persistence and an elevated Th17 cell response in the central nervous system. Either IL-6 or IL-17 induced the expression of Bcl-2 and Bcl-xL at a high concentration. The upregulated expression of prosurvival molecules in turn inhibited target cell destruction by virus-specific CD8+T cells. More interestingly, IL-6 and IL-17 synergistically promoted the expression of these prosurvival molecules, preventing cellular apoptosis at a much lower (<5-fold) concentration. The signals involved in the synergy appear to include the activation of both STAT3 and NF-κB via distinct cytokine-dependent pathways. Thus, the excessive IL-6 promotes the generation of Th17 cells, and the resulting IL-6 and IL-17 synergistically promote viral persistence by protecting virus-infected cells from apoptosis and CD8+T cell-mediated target destruction. These results suggest that blocking both IL-6 and IL-17 functions are important considerations for therapies of chronic viral diseases, autoimmune diseases, and cancers.

IMPORTANCEThis study indicates that an excessive level of IL-6 cytokine produced following viral infection promotes the development of IL-17-producing pathogenic helper T cells. We demonstrate here for the first time that IL-6 together with IL-17 synergistically enhances the expression of survival molecules to hinder critical host defense mechanisms removing virus-infected cells. This finding has an important implication in controlling not only chronic viral infections but also autoimmune diseases and cancers, which are associated with prolonged cell survival.
Identification of Properties of the Kaposi's Sarcoma-Associated Herpesvirus Latent Origin of Replication That Are Essential for the Efficient Establishment and Maintenance of Intact Plasmids
Authors: Shrestha Prabha , Sugden Bill
Published: May 2014

The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in
cis
by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in
cis
to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis.

IMPORTANCE
KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.
Blocking Herpes Simplex Virus 2 Glycoprotein E Immune Evasion as an Approach To Enhance Efficacy of a Trivalent Subunit Antigen Vaccine for Genital Herpes
Authors: Awasthi Sita , Huang Jialing , Shaw Carolyn , Friedman Harvey M.
Published: May 2014

Herpes simplex virus 2 (HSV-2) subunit antigen vaccines targeting virus entry molecules have failed to prevent genital herpes in human trials. Our approach is to include a virus entry molecule and add antigens that block HSV-2 immune evasion. HSV-2 glycoprotein C (gC2) is an immune evasion molecule that inhibits complement. We previously reported that adding gC2 to gD2 improved vaccine efficacy compared to the efficacy of either antigen alone in mice and guinea pigs. Here we demonstrate that HSV-2 glycoprotein E (gE2) functions as an immune evasion molecule by binding the IgG Fc domain. HSV-2 gE2 is synergistic with gC2 in protecting the virus from antibody and complement neutralization. Antibodies produced by immunization with gE2 blocked gE2-mediated IgG Fc binding and cell-to-cell spread. Mice immunized with gE2 were only partially protected against HSV-2 vaginal challenge in mice; however, when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone. Importantly, the trivalent vaccine protected the dorsal root ganglia (DRG) of 32/33 (97%) mice between days 2 and 7 postchallenge, compared with 27/33 (82%) in the gD2 group. The HSV-2 DNA copy number was significantly lower in mice immunized with the trivalent vaccine than in those immunized with gD2 alone. The extent of DRG protection using the trivalent vaccine was better than what we previously reported for gC2/gD2 immunization. Therefore, gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion.

IMPORTANCEHerpes simplex virus is the most common cause of genital ulcer disease worldwide. Infection results in emotional distress for infected individuals and their partners, is life threatening for infants exposed to herpes during childbirth, and greatly increases the risk of individuals acquiring and transmitting HIV infection. A vaccine that prevents genital herpes infection will have major public health benefits. Our vaccine approach includes strategies to prevent the virus from evading immune attack. Mice were immunized with a trivalent vaccine containing an antigen that induces antibodies to block virus entry and two antigens that induce antibodies that block immune evasion from antibody and complement. Immunized mice demonstrated no genital disease, and 32/33 (97%) animals had no evidence of infection of dorsal root ganglia, suggesting that the vaccine may prevent the establishment of latency and recurrent infections.
Highly Diverse Morbillivirus-Related Paramyxoviruses in Wild Fauna of the Southwestern Indian Ocean Islands: Evidence of Exchange between Introduced and Endemic Small Mammals
Authors: Wilkinson David A. , Mélade Julien , Dietrich Muriel , Ramasindrazana Beza , Soarimalala Voahangy , Lagadec Erwan , le Minter Gildas , Tortosa Pablo , Heraud Jean-Michel , de Lamballerie Xavier , Goodman Steven M. , Dellagi Koussay , Pascalis Herve
Published: May 2014

The
Paramyxoviridae
form an increasingly diverse viral family, infecting a wide variety of different hosts. In recent years, they have been linked to disease emergence in many different animal populations and in humans. Bats and rodents have been identified as major animal populations capable of harboring paramyxoviruses, and host shifting between these animals is likely to be an important driving factor in the underlying evolutionary processes that eventually lead to disease emergence. Here, we have studied paramyxovirus circulation within populations of endemic and introduced wild small mammals of the southwestern Indian Ocean region and belonging to four taxonomic orders: Rodentia, Afrosoricida, Soricomorpha, and Chiroptera. We report elevated infection levels as well as widespread paramyxovirus dispersal and frequent host exchange of a newly emerging genus of the
Paramyxoviridae
, currently referred to as the unclassified morbillivirus-related viruses (UMRVs). In contrast to other genera of the
Paramyxoviridae
, where bats have been shown to be a key host species, we show that rodents (and, in particular,
Rattus rattus
) are significant spreaders of UMRVs. We predict that the ecological particularities of the southwestern Indian Ocean, where small mammal species often live in densely packed, multispecies communities, in combination with the increasing invasion of
R. rattus
and perturbations of endemic animal communities by active anthropological development, will have a major influence on the dynamics of UMRV infection.

IMPORTANCE
Identification of the infectious agents that circulate within wild animal reservoirs is essential for several reasons: (i) infectious disease outbreaks often originate from wild fauna; (ii) anthropological expansion increases the risk of contact between human and animal populations and, as a result, the risk of disease emergence; (iii) evaluation of pathogen reservoirs helps in elaborating preventive measures to limit the risk of disease emergence. Many paramyxoviruses for which bats and rodents serve as major reservoirs have demonstrated their potential to cause disease in humans and animals. In the context of the biodiversity hot spot of southwestern Indian Ocean islands and their rich endemic fauna, we show that highly diverse UMRVs exchange between various endemic animal species, and their dissemination likely is facilitated by the introduced
Rattus rattus
. Hence, many members of the
Paramyxoviridae
appear well adapted for the study of the viral phylodynamics that may be associated with disease emergence.
Newcastle Disease Virus (NDV) Recombinants Expressing Infectious Laryngotracheitis Virus (ILTV) Glycoproteins gB and gD Protect Chickens against ILTV and NDV Challenges
Authors: Zhao Wei , Spatz Stephen , Zhang Zhenyu , Wen Guoyuan , Garcia Maricarmen , Zsak Laszlo , Yu Qingzhong
Published: May 2014

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of ILTV and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV). The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence, whereas HVT- and FPV-vectored ILTV vaccines are less efficacious than live attenuated vaccines. Therefore, there is a pressing need to develop safer and more efficacious ILTV vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants, based on the LaSota vaccine strain, expressing glycoproteins B (gB) and D (gD) of ILTV using reverse genetics technology. These recombinant viruses, rLS/ILTV-gB and rLS/ILTV-gD, were slightly attenuated
in vivo
yet retained growth dynamics, stability, and virus titers
in vitro
that were similar to those of the parental LaSota virus. Expression of ILTV gB and gD proteins in the recombinant virus-infected cells was detected by immunofluorescence assay. Vaccination of specific-pathogen-free chickens with these recombinant viruses conferred significant protection against virulent ILTV and velogenic NDV challenges. Immunization of commercial broilers with rLS/ILTV-gB provided a level of protection against clinical disease similar to that provided by the live attenuated commercial vaccines, with no decrease in body weight gains. The results of the study suggested that the rLS/ILTV-gB and -gD viruses are safe, stable, and effective bivalent vaccines that can be mass administered via aerosol or drinking water to large chicken populations.

IMPORTANCE
This paper describes the development and evaluation of novel bivalent vaccines against chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND), two of the most economically important infectious diseases of poultry. The current commercial ILT vaccines are either not safe or less effective. Therefore, there is a pressing need to develop safer and more efficacious ILT vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants expressing glycoproteins B (gB) and D (gD) of infectious laryngotracheitis virus (ILTV) using reverse genetics technology. These recombinant viruses were safe, stable, and immunogenic and replicated efficiently in birds. Vaccination of chickens with these recombinant viruses conferred complete protection against ILTV and NDV challenge. These novel bivalent vaccines can be mass administered via aerosol or drinking water to large chicken populations at low cost, which will have a direct impact on poultry health, fitness, and performance.
Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge
Authors: Cardoso Francisco Miguel , Ibañez Lorena Itatí , Van den Hoecke Silvie , De Baets Sarah , Smet Anouk , Roose Kenny , Schepens Bert , Descamps Francis J. , Fiers Walter , Muyldermans Serge , Depicker Ann , Saelens Xavier
Published: May 2014

Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their
in vitro
and
in vivo
antiviral potential. Two of them inhibited the NA activity and
in vitro
replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The
in vitro
antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without
in vitro
NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.

IMPORTANCE
Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses
in vitro
and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect
in vivo
even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.
Traction apophysitis of the fifth metatarsal base in a child: Iselin's disease
Authors: Deniz Gokmen , Kose Ozkan , Guneri Bulent , Duygun Fatih
Published: May 2014

Although Iselin's disease, apophysitis of the fifth metatarsal base, is not infrequent in clinical practice, it is accepted as a rare cause of lateral foot pain in young adolescents. Usually a simple clinical examination and radiographs are sufficient for diagnosis. We present a patient with Iselin's disease and discuss its clinical and radiographic characteristics, differential diagnosis, aetiopathogenesis and treatment.
Analysis of occupational radiation exposure during cerebral angiography utilizing a new real time radiation dose monitoring system
Authors: James Robert F , Wainwright Kristin JoAnn , Kanaan Hilal A , Hudson Suzanne , Wainwright Mark Edison , Hightower Jerome H , Delaney John J
Published: May 2014

Background

Thermoluminescent dosimeter badges currently utilized to monitor occupational radiation exposures are limited in their ability to provide timely feedback, restricting workers' ability to identify unnecessary exposure. New real time radiation monitoring systems provide an opportunity for workers to immediately identify and alter problematic behaviors in the neuroangiography suite, decreasing unnecessary exposures, lowering risk, and maximizing safety efforts.

Methods

Real time radiation monitoring was performed for 120 diagnostic cerebral angiography procedures. Data were collected in two phases, for procedures performed by two physician participants (30 procedures per physician per phase). Workers were blinded to their real time dose in phase I, and unblinded in phase II. Individual exposures (Sv) and the incidence of red events (exposure rates ≥2.0 mSv/h) were collected for each assigned participating role (physician A, physician B, nurse, scrubbed technologist, and circulating technologist). The dose area product was collected for each procedure to standardize against variations in procedure duration or intensity.

Results

In phase II, significant decreased radiation exposure was observed for all roles except physician A. Physician B decreased most from 24.3×10−8to 6.9×10−8Sv/Gy-cm2(p<0.0001). Rates of red events decreased similarly for all roles except physician A, and were significant for all roles except the nurse role.

Conclusions

Real time radiation dose monitoring during diagnostic cerebral angiography may help to reduce occupational radiation exposures for healthcare workers.
Dorsal vertebral column abnormalities in dogs with disseminated idiopathic skeletal hyperostosis (DISH)
Authors: De Decker S. , Volk H. A.
Published: May 2014

Although disseminated idiopathic skeletal hyperostosis (DISH) most often affects the ventral aspect of the vertebral column, this study evaluated the occurrence, nature and clinical relevance of dorsal vertebral column abnormalities in 10 dogs with DISH for which CT or MRI and a complete neurological examination were available. Dorsal vertebral column abnormalities were present in eight dogs and included articular process hypertrophy (n=7 dogs), periarticular new bone formation (n=1), pseudoarthrosis between spinous processes (n=4) and thickening of the dorsal lamina (n=4). These dorsal vertebral abnormalities caused clinically relevant vertebral canal stenosis in six dogs and were the only cause of clinical signs in four of these dogs. Although the lumbosacral joint was not affected by DISH, these six dogs demonstrated lumbosacral vertebral canal stenosis and clinical signs of cauda equina compression, which included paraparesis (n=5 dogs), lumbosacral pain (n=4), urinary incontinence (n=4), faecal incontinence (n=1) and urinary and faecal incontinence (n=1). There is a possible association between DISH and hypertrophy of dorsal vertebral structures, potentially resulting in vertebral canal stenosis. Although these changes occurred at segments fused by DISH, they predominantly affected adjacent non‐affected segments.
Urinary ACE2 in healthy adults and patients with uncomplicated type 1 diabetes
Authors: Cherney David Z.I. , Xiao Fengxia , Zimpelmann Joseph , Har Ronnie L.H. , Lai Vesta , Scholey James W. , Reich Heather N. , Burns Kevin D.
Published: May 2014

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be renoprotective. We determined whether urinary ACE2 enzyme activity and protein levels (ELISA), as well as angiotensinogen and ACE, are elevated during clamped euglycemia (4–6 mmol·L–1) in patients with uncomplicated type 1 diabetes (T1D, n = 58) compared with normoglycemic controls (n = 21). We also measured the effect of clamped hyperglycemia (9–11 mmol·L–1) on each urinary factor in T1D patients. Urinary ACE2 activity and protein levels were higher during clamped euglycemia in T1D compared with the controls (p < 0.0001). In contrast, urinary angiotensinogen levels (p = 0.27) and ACE excretion (p = 0.68) did not differ. In response to clamped hyperglycemia in T1D, urinary ACE2 protein decreased (p < 0.0001), whereas urinary ACE2 activity as well as angiotensinogen and ACE levels remained unchanged. Urinary ACE2 activity and protein expression are increased in T1D patients prior to the onset of clinical complications. Further work is required to determine the functional role of urinary ACE2 in early T1D.
VERSATILE TRANSADMITTANCE-MODE OTA-C UNIVERSAL BIQUAD FILTER USING MINIMUM COMPONENTS WITH INDEPENDENTLY ELECTRONIC TUNABILITY
Authors: LEE CHEN-NONG
Published: May 2014

This paper presents a transadmittance-mode (TAM) universal biquad filter with independently electronic tunability. The proposed biquad filter only employs three operational transconductance amplifiers (OTAs) and two grounded capacitors which are the minimum components count necessary for realizing independently electronic tunability of the parameters ω0 and ω0/Q without the need of control factors matching conditions. Moreover, the proposed circuit still achieves nearly all of the main advantages: (i) simultaneous realizations of universal filtering responses (low-pass, high-pass, band-pass, band-reject and all-pass) from the same topology, (ii) versatile input/output functions, (iii) orthogonally electronic tunability of the parameters ω0 and Q without the need of control factors matching conditions, (iv) no need of any resistors, (v) cascadable feature for all input and output terminals, (vi) no need of extra inverting or non-inverting amplifiers, (vii) the employment of only grounded capacitors, (viii) no component-value constraints (except for allpass filter function) and (ix) low active and passive sensitivity performances. H-spice simulations with TSMC 0.35 μm 2P4M CMOS process technology validate theoretical predictions.
RECODE: SOFTWARE PACKAGE REFACTORING VIA COMMUNITY DETECTION IN BIPARTITE SOFTWARE NETWORKS
Authors: PAN WEIFENG , LI BING , JIANG BO , LIU KUN
Published: May 2014

It is an intrinsic property of real-world software to evolve, which is usually accompanied by the increase of software complexity and deterioration of software quality. So successful software has to be reconditioned from time to time. Though many refactoring approaches have been proposed, only a few of them are performed at the package level. In this paper, we present a novel approach to refactor the package structure of object-oriented (OO) software. It uses weighted bipartite software networks to represent classes, packages, and their dependencies; it proposes a guidance community detection algorithm (GUIDA) to obtain the optimized package structure; and it finally provides a list of classes as refactoring candidates by comparing the optimized package structure with the real package structure. Through a set of experiments we have shown that the proposed approach is able to identify a majority of classes that experts recognize as refactoring candidates, and the benefits of our approach are illustrated in comparison with other two approaches.
Mutation of a single threonine in the cytoplasmic NH2terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress
Authors: Schweikhard Eva S. , Kempson Stephen A. , Ziegler Christine , Burckhardt Birgitta C.
Published: May 2014

Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl- sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl- sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [3H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.
Semaphorin 3A inactivation suppresses ischemia-reperfusion-induced inflammation and acute kidney injury
Authors: Ranganathan Punithavathi , Jayakumar Calpurnia , Mohamed Riyaz , Weintraub Neal L. , Ramesh Ganesan
Published: May 2014

Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia-reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia-reperfusion-induced AKI. In vitro, sema3A enhanced toll-like receptor 4-mediated inflammation in epithelial cells, macrophages, and dendritic cells. Moreover, administration of sema3A-treated, bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates AKI via promoting inflammation and epithelial cell apoptosis.
Postinhibitory rebound neurons and networks are disrupted in retrovirus-induced spongiform neurodegeneration
Authors: Li Ying , Davey Robert A. , Sivaramakrishnan Shobhana , Lynch William P.
Published: May 2014

Certain retroviruses induce progressive spongiform motor neuron disease with features resembling prion diseases and amyotrophic lateral sclerosis. With the neurovirulent murine leukemia virus (MLV) FrCasE, Env protein expression within glia leads to postsynaptic vacuolation, cellular effacement, and neuronal loss in the absence of neuroinflammation. To understand the physiological changes associated with MLV-induced spongiosis, and its neuronal specificity, we employed patch-clamp recordings and voltage-sensitive dye imaging in brain slices of the mouse inferior colliculus (IC), a midbrain nucleus that undergoes extensive spongiosis. IC neurons characterized by postinhibitory rebound firing (PIR) were selectively affected in FrCasE-infected mice. Coincident with Env expression in microglia and in glia characterized by NG2 proteoglycan expression (NG2 cells), rebound neurons (RNs) lost PIR, became hyperexcitable, and were reduced in number. PIR loss and hyperexcitability were reversed by raising internal calcium buffer concentrations in RNs. PIR-initiated rhythmic circuits were disrupted, and spontaneous synchronized bursting and prolonged depolarizations were widespread. Other IC neuron cell types and circuits within the same degenerative environment were unaffected. Antagonists of NMDA and/or AMPA receptors reduced burst firing in the IC but did not affect prolonged depolarizations. Antagonists of L-type calcium channels abolished both bursts and slow depolarizations. IC infection by the nonneurovirulent isogenic virus Friend 57E (Fr57E), whose Env protein is structurally similar to FrCasE, showed no RN hyperactivity or cell loss; however, PIR latency increased. These findings suggest that spongiform neurodegeneration arises from the unique excitability of RNs, their local regulation by glia, and the disruption of this relationship by glial expression of abnormal protein.
Influence of optic flow on the control of heading and target egocentric direction during steering toward a goal
Authors: Li Li , Niehorster Diederick C.
Published: May 2014

Although previous studies have shown that people use both optic flow and target egocentric direction to walk or steer toward a goal, it remains in question how enriching the optic flow field affects the control of heading specified by optic flow and the control of target egocentric direction during goal-oriented locomotion. In the current study, we used a control-theoretic approach to separate the control response specific to these two cues in the visual control of steering toward a goal. The results showed that the addition of optic flow information (such as foreground motion and global flow) in the display improved the overall control precision, the amplitude, and the response delay of the control of heading. The amplitude and the response delay of the control of target egocentric direction were, however, not affected. The improvement in the control of heading with enriched optic flow displays was mirrored by an increase in the accuracy of heading perception. The findings provide direct support for the claim that people use the heading specified by optic flow as well as target egocentric direction to walk or steer toward a goal and suggest that the visual system does not internally weigh these two cues for goal-oriented locomotion control.
The Distance between Points of a Solution of a Second Order Linear Differential Equation Satisfying General Boundary Conditions
Authors: Almenar Pedro , Jódar Lucas
Published: May 2014

This paper presents a method to obtain lower and upper bounds for the minimum distance between pointsaandbof the solution of the second order linear differential equationy′′+q(x)y=0satisfying general separated boundary conditions of the typea11y(a)+a12y′(a)=0anda21y(b)+a22y′(b)=0. The method is based on the recursive application of a linear operator to certain functions, a recursive application that makes these bounds converge to the exact distance betweenaandbas the recursivity index grows. The method covers conjugacy and disfocality as particular cases.
Exp-Function Method for a Generalized MKdV Equation
Authors: Chai Yuzhen , Jia Tingting , Hao Huiqin , Zhang Jianwen
Published: May 2014

Under investigation in this paper is a generalized MKdV equation, which describes the propagation of shallow water in fluid mechanics. In this paper, we have derived the exact solutions for the generalized MKdV equation including the bright soliton, dark soliton, two-peak bright soliton, two-peak dark soliton, shock soliton and periodic wave solution via Exp-function method. By figures and symbolic computations, we have discussed the propagation characteristics of those solitons under different values of those coefficients in the generalized MKdV equation. The method constructing soliton solutions in this paper may be useful for the investigations on the other nonlinear mathematical physics model and the conclusions of this paper can give theory support for the study of dynamic features of models in the shallow water.
Evaluation of theIn VitroandIn VivoAntioxidant Potentials ofAframomum meleguetaMethanolic Seed Extract
Authors: Onoja Samuel Okwudili , Omeh Yusuf Ndukaku , Ezeja Maxwell Ikechukwu , Chukwu Martins Ndubuisi
Published: May 2014

Aframomum meleguetaSchum (Zingiberaceae) is a perennial herb widely cultivated for its valuable seeds in the tropical region of Africa. The present study evaluated the antioxidant effects of methanolic seed extract ofA. melegueta. The antioxidant effects were evaluated usingin vitro, 2, 2-diphenylpicrylhydrazine photometric assay andin vivoserum catalase, superoxide dismutase and thiobarbituric acid reactive substance assay method. The extract (25–400 μg/mL concentration) produced concentration dependent increase in antioxidant activity in 2, 2-diphenylpicrylhydrazine photometric assay. The extract (400 mg/kg) showed a significant (P<0.05) increase in serum catalase and superoxide dismutase activity when compared with the control group. The extract (400 mg/kg) showed a significant (P<0.05) decrease in the serum level of thiobarbituric acid reactive substance when compared with the control group. These findings suggest that the seed ofA. meleguetahas potent antioxidant activity which may be responsible for some of its reported pharmacological activities and can be used as antioxidant supplement.
Fungal Laccases and Their Applications in Bioremediation
Authors: Viswanath Buddolla , Rajesh Bandi , Janardhan Avilala , Kumar Arthala Praveen , Narasimha Golla
Published: May 2014

Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection.
Core Flow Distribution from Coupled Supercritical Water Reactor Analysis
Authors: Hu Po , Wilson Paul P. H.
Published: May 2014

This paper introduces an extended code package PARCS/RELAP5 to analyze steady state of SCWR US reference design. An 8 × 8 quarter core model in PARCS and a reactor core model in RELAP5 are used to study the core flow distribution under various steady state conditions. The possibility of moderator flow reversal is found in some hot moderator channels. Different moderator flow orifice strategies, both uniform across the core and nonuniform based on the power distribution, are explored with the goal of preventing the reversal.