bluuebunny/crossref_metadata_2025 huggingface dataset:
Multilocus Sequence Typing Analysis of Staphylococcus lugdunensis Implies a Clonal Population Structure
Authors: Chassain Benoît , Lemée Ludovic , Didi Jennifer , Thiberge Jean-Michel , Brisse Sylvain , Pons Jean-Louis , Pestel-Caron Martine
Published: July 2012

Staphylococcus lugdunensis
is recognized as one of the major pathogenic species within the genus
Staphylococcus
, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87
S. lugdunensis
isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (
aroE
,
dat
,
ddl
,
gmk
,
ldh
,
recA
, and
yqiL
). The number of alleles ranged from four (
gmk
and
ldh
) to nine (
yqiL
). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within
S. lugdunensis
. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for
S. lugdunensis
. This first MLST scheme for
S. lugdunensis
provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative
Staphylococcus
.
Resolution of a Meningococcal Disease Outbreak from Whole-Genome Sequence Data with Rapid Web-Based Analysis Methods
Authors: Jolley Keith A. , Hill Dorothea M. C. , Bratcher Holly B. , Harrison Odile B. , Feavers Ian M. , Parkhill Julian , Maiden Martin C. J.
Published: July 2012

The increase in the capacity and reduction in cost of whole-genome sequencing methods present the imminent prospect of such data being used routinely in real time for investigations of bacterial disease outbreaks. For this to be realized, however, it is necessary that generic, portable, and robust analysis frameworks be available, which can be readily interpreted and used in real time by microbiologists, clinicians, and public health epidemiologists. We have achieved this with a set of analysis tools integrated into the PubMLST.org website, which can in principle be used for the analysis of any pathogen. The approach is demonstrated with genomic data from isolates obtained during a well-characterized meningococcal disease outbreak at the University of Southampton, United Kingdom, that occurred in 1997. Whole-genome sequence data were collected,
de novo
assembled, and deposited into the PubMLST
Neisseria
BIGSdb database, which automatically annotated the sequences. This enabled the immediate and backwards-compatible classification of the isolates with a number of schemes, including the following: conventional, extended, and ribosomal multilocus sequence typing (MLST, eMLST, and rMLST); antigen gene sequence typing (AGST); analysis based on genes conferring antibiotic susceptibility. The isolates were also compared to a reference isolate belonging to the same clonal complex (ST-11) at 1,975 loci. Visualization of the data with the NeighborNet algorithm, implemented in SplitsTree 4 within the PubMLST website, permitted complete resolution of the outbreak and related isolates, demonstrating that multiple closely related but distinct strains were simultaneously present in asymptomatic carriage and disease, with two causing disease and one responsible for the outbreak itself.
Improved Multiplex PCR Strategy for Rapid Assignment of the Four Major Escherichia coli Phylogenetic Groups
Authors: Doumith M. , Day M. J. , Hope R. , Wain J. , Woodford N.
Published: July 2012

Using data from whole-genome projects, an updated multiplex PCR strategy was developed to assign
Escherichia coli
isolates rapidly to major phylogenetic groups. This assay accommodates sequence variations detected within target sequences, thereby increasing sensitivity and reliability. It was validated using 185 isolates of known sequence types and showed improved congruence with multilocus sequence typing data.
Transmission of Mycobacterium orygis (M. tuberculosis Complex Species) from a Tuberculosis Patient to a Dairy Cow in New Zealand
Authors: Dawson Kara L. , Bell Anita , Kawakami R. Pamela , Coley Kathryn , Yates Gary , Collins Desmond M.
Published: July 2012

Mycobacterium orygis
, previously called the oryx bacillus, is a member of the
Mycobacterium tuberculosis
complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.
Heparan Sulfate Is an Attachment Factor for Foamy Virus Entry
Authors: Plochmann Kathrin , Horn Anne , Gschmack Eva , Armbruster Nicole , Krieg Jennifer , Wiktorowicz Tatiana , Weber Conrad , Stirnnagel Kristin , Lindemann Dirk , Rethwilm Axel , Scheller Carsten
Published: July 2012

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID
50
s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (
P
< 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.
Characterization of Nucleosome Positioning in Hepadnaviral Covalently Closed Circular DNA Minichromosomes
Authors: Shi Liping , Li Shaohua , Shen Fang , Li Haodong , Qian Shuiming , Lee Daniel H. S. , Wu Jim Z. , Yang Wengang
Published: July 2012

Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined thein vivonucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor variouscistranscription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.
5′-Triphosphate-Short Interfering RNA: Potent Inhibition of Influenza A Virus Infection by Gene Silencing and RIG-I Activation
Authors: Lin Li , Liu Qiang , Berube Nathalie , Detmer Susan , Zhou Yan
Published: July 2012

Limited protection of current vaccines and antiviral drugs against influenza A virus infection underscores the urgent need for development of novel anti-influenza virus interventions. While short interfering RNA (siRNA) has been shown to be able to inhibit influenza virus infection in a gene-specific manner, activation of the retinoic acid-inducible gene I protein (RIG-I) pathway has an antiviral effect in a non-gene-specific mode. In this study, we designed and tested the anti-influenza virus effect of a short double-stranded RNA, designated 3p-mNP1496-siRNA, that possesses dual functions: an siRNA-targeting influenza NP gene and an agonist for RIG-I activation. This double-stranded siRNA possesses a triphosphate group at the 5′ end of the sense strand and is blunt ended. Our study showed that 3p-mNP1496-siRNA could potently inhibit influenza A virus infection both in cell culture and in mice. The strong inhibition effect was attributed to its siRNA function as well as its ability to activate the RIG-I pathway. To the best of our knowledge, this is the first report that the combination of siRNA and RIG-I pathway activation can synergistically inhibit influenza A virus infection. The development of such dual functional RNA molecules will greatly contribute to the arsenal of tools to combat not only influenza viruses but also other important viral pathogens.
Roles of the Phosphorylation of Specific Serines and Threonines in the NS1 Protein of Human Influenza A Viruses
Authors: Hsiang Tien-Ying , Zhou Ligang , Krug Robert M.
Published: July 2012

We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C α (PKCα) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKCα. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus.
Vaccination with Adenovirus Serotypes 35, 26, and 48 Elicits Higher Levels of Innate Cytokine Responses than Adenovirus Serotype 5 in Rhesus Monkeys
Authors: Teigler Jeffrey E. , Iampietro M. Justin , Barouch Dan H.
Published: July 2012

Adenovirus (Ad) vaccine vectors have proven highly immunogenic in multiple experimental models, but the innate immune responses induced by these vectors remain poorly characterized. Here we report innate cytokine responses to 5 different Ad vectors in 26 rhesus monkeys. Vaccination with adenovirus serotype 35 (Ad35), Ad26, and Ad48 induced substantially higher levels of antiviral (gamma interferon [IFN-γ], 10-kDa gamma interferon-induced protein [IP-10]) and proinflammatory (interleukin 1 receptor antagonist [IL-1RA], IL-6) cytokines than vaccination with Ad5 on day 1 following immunization.In vitrostudies with capsid chimeric vectors and receptor-blocking monoclonal antibodies suggested that fiber-receptor interactions, as well as other capsid components, were critical for triggering these innate responses. Moreover, multiple cell populations, including dendritic cells, monocytes/macrophages, and T lymphocytes, contributed to these innate cytokine profiles. These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their primary cellular receptor, induce significantly greater innate cytokine responses than Ad5, which uses the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors.
Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential
Authors: El-Andaloussi Nazim , Bonifati Serena , Kaufmann Johanna K. , Mailly Laurent , Daeffler Laurent , Deryckère François , Nettelbeck Dirk M. , Rommelaere Jean , Marchini Antonio
Published: July 2012

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral
NS
expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs
NS
expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.
Glycan Binding Avidity Determines the Systemic Fate of Adeno-Associated Virus Type 9
Authors: Shen Shen , Bryant Kelli D. , Sun Junjiang , Brown Sarah M. , Troupes Andrew , Pulicherla Nagesh , Asokan Aravind
Published: July 2012

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors
in vitro
; however, their contributions
in vivo
are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.
Anguillid Herpesvirus 1 Transcriptome
Authors: van Beurden Steven J. , Gatherer Derek , Kerr Karen , Galbraith Julie , Herzyk Pawel , Peeters Ben P. H. , Rottier Peter J. M. , Engelsma Marc Y. , Davison Andrew J.
Published: July 2012

We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5′ untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5′ and 3′ ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5′ untranslated exons or, because of alternative splicing, 5′ untranslated and 5′ translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome.
Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice
Authors: Krammer Florian , Pica Natalie , Hai Rong , Tan Gene S. , Palese Peter
Published: July 2012

Previously, it has been shown that infection in humans with the pandemic swine influenza virus induces antibodies with specificity to the stalk domain of the viral hemagglutinin. Following the generation of these data, we sought to recapitulate these findings in the mouse model by sequential influenza virus infection. Mice that were inoculated with a seasonal influenza H1N1 virus followed by infection with a pandemic H1N1 strain produced higher antihemagglutinin stalk antibody titers than mice sequentially infected with drifted seasonal strains. In order to achieve antibody titers of comparable magnitude using sequential infection, mice had to be infected with 100- to 1,000-fold more of the drifted seasonal virus. The antistalk antibodies produced by these infections were influenza virus neutralizing, which illustrates the utility of the mouse model in which to study this interaction between virus and host.
Kinetics of Immune Responses in Deer Mice Experimentally Infected with Sin Nombre Virus
Authors: Schountz Tony , Acuña-Retamar Mariana , Feinstein Shira , Prescott Joseph , Torres-Perez Fernando , Podell Brendan , Peters Staci , Ye Chunyan , Black William C. , Hjelle Brian
Published: July 2012

Deer mice are the principal reservoir hosts of Sin Nombre virus, the etiologic agent of most hantavirus cardiopulmonary syndrome cases in North America. Infection of deer mice results in persistence without conspicuous pathology, and most, if not all, infected mice remain infected for life, with periods of viral shedding. The kinetics of viral load, histopathology, virus distribution, and immune gene expression in deer mice were examined. Viral antigen was detected as early as 5 days postinfection and peaked on day 15 in the lungs, hearts, kidneys, and livers. Viral RNA levels varied substantially but peaked on day 15 in the lungs and heart, and antinucleocapsid IgG antibodies appeared in some animals on day 10, but a strong neutralizing antibody response failed to develop during the 20-day experiment. No clinical signs of disease were observed in any of the infected deer mice. Most genes were repressed on day 2, suggesting a typical early downregulation of gene expression often observed in viral infections. Several chemokine and cytokine genes were elevated, and markers of a T cell response occurred but then declined days later. Splenic transforming growth factor beta (TGF-β) expression was elevated early in infection, declined, and then was elevated again late in infection. Together, these data suggest that a subtle immune response that fails to clear the virus occurs in deer mice.
Attitudes to animal euthanasia do not correlate with acceptance of human euthanasia or suicide
Authors: Ogden U. , Kinnison T. , May S. A.
Published: July 2012

Several reasons have been suggested for the elevated risk of suicide experienced by those in the veterinary profession. The current study aimed to investigate possible links between veterinarians' attitudes to ‘convenience’ or non‐justified animal euthanasia and attitudes towards human euthanasia and suicide. Veterinary students and graduates had a negative attitude towards convenience animal euthanasia, but their attitudes changed over time (pre‐clinical studies, clinical studies and recently graduated). A greater tolerance to euthanasia was displayed in the later years of study and post qualification – primarily by males. Attitudes towards both human euthanasia and suicide, however, remained stable over time and indicated on average a neutral stance. No correlations were found between attitudes to convenience euthanasia and either human euthanasia or suicide, suggesting a tolerance to convenience euthanasia of animals does not lead to desensitisation in valuing human life and a changed attitude to human euthanasia or suicide, or vice versa. Attitudes to human euthanasia and suicide were predictably correlated, perhaps suggesting an overarching attitude towards control over human death. The results of the current study throw into question the argument that it is the changes in attitudes to animal life that affect veterinarian's attitudes to human life and contribute to the high suicide rate.
Anaesthetic and cardiorespiratory effects of a constant‐rate infusion of alfaxalone in desflurane‐anaesthetised sheep
Authors: del Mar Granados M. , Domínguez J. Manuel , Fernández‐Sarmiento A. , Funes F. Javier , Morgaz J. , Navarrete R. , Ma Carrillo J. , Rubio M. , Muñoz‐Rascón P. , Gómez de Segura I. A. , Gómez‐Villamandos R. J.
Published: July 2012

A prospective, randomised, blinded controlled study was performed to determine the anaesthetic and cardiorespiratory effects of a constant‐rate infusion (CRI) of alfaxalone in 12 sheep anaesthetised with desflurane, and undergoing experimental orthopaedic surgery. Sheep were sedated with dexmedetomidine (4 μg/kg, intravenously) and butorphanol (0.3 mg/kg, intravenously). Anaesthesia was induced with alfaxalone (1 mg/kg/minute to effect, intravenously) and maintained with desflurane in oxygen and alfaxalone 0.07 mg/kg/minute or saline for 150 minutes (range 150–166 minutes). The anaesthetic induction dose of alfaxalone, the desflurane expiratory fraction required for anaesthetic maintenance, cardiorespiratory measurements and blood‐gases were recorded at predetermined intervals. Quality of sedation, anaesthetic induction and recovery were assessed. The alfaxalone induction dose was 1.7 mg/kg (1.2 to 2.6 mg/kg). The desflurane expiratory fraction was lower (22 per cent) in sheep receiving alfaxalone CRI (P = 0). Also, heart rate (P = 0), cardiac index (P = 0.002), stroke index (P = 0) and contractility (P = 0) were higher, and systemic vascular resistance (P = 0.002) was lower. Although respiratory rate tended to be higher with alfaxalone, there was no difference in PCO2 between the groups. Recovery times were significantly longer in sheep given alfaxalone (25.4 v 9.5 minutes) but recovery quality was similar. Alfaxalone reduced requirements of desflurane and maintained similar cardiorespiratory function, but recovery time was more prolonged.
Perilimbal pocket technique for surgical repositioning of prolapsed nictitans gland in dogs
Authors: Prémont J. E. , Monclin S. , Farnir F. , Grauwels M.
Published: July 2012

The objective of this prospective study was to investigate the success rate, practicality and complications of a new perilimbal pocket technique for the replacement of prolapsed nictitans gland in 30 dogs (44 eyes). A first incision was made in the bulbar conjunctiva, 2–3 mm from and parallel to the infero‐nasal limbus, a second incision on the bulbar aspect of the nictitating membrane (NM), 2–3 mm parallel to the free edge. The gland was returned to its normal position by suturing the subconjunctival tissues of the NM to the episcleral tissues, using four to six interrupted horizontal mattress sutures. The English bulldog, Neapolitan mastiff, great dane and American cocker spaniel were commonly presented. Nictitans gland prolapse occurred prior to one year of age in 83.3 per cent of dogs, and unilaterally in 15 patients. The procedure was easy to perform, and had a 90.9 per cent success rate, with minimal complications. The median duration of follow‐up, conducted by ophthalmic examination or telephone contact with the owners, was 21.5 months. Tear production and ocular health were not affected in 17 eyes with at least six months follow‐up. There was a statistically significant increase between preoperative and postoperative Schirmer tear test‐1 measurements.
Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle
Authors: Cully Tanya R. , Edwards Joshua N. , Friedrich Oliver , Stephenson D. George , Murphy Robyn M. , Launikonis Bradley S.
Published: July 2012

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca2+ from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca2+. The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca2+ entry (SOCE) can also be activated upon release of SR Ca2+. In muscle, the SR Ca2+ sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca2+ channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca2+ influx in mdx compared with WT muscle for similar levels of SR Ca2+ release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca2+ via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca2+ extrusion properties as in the WT muscle.
Hypoglycemic effects of brassinosteroid in diet-induced obese mice
Authors: Esposito Debora , Kizelsztein Pablo , Komarnytsky Slavko , Raskin Ilya
Published: July 2012

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50–300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phospho enolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1–15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.
Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β
Authors: Punia Sohan , Rumery Kyle K. , Yu Elizabeth A. , Lambert Christopher M. , Notkins Abner L. , Weaver David R.
Published: July 2012

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.
Physiological responses to food deprivation in the house sparrow, a species not adapted to prolonged fasting
Authors: Khalilieh Anton , McCue Marshall D. , Pinshow Berry
Published: July 2012

Many wild birds fast during reproduction, molting, migration, or because of limited food availability. Species that are adapted to fasting sequentially oxidize endogenous fuels in three discrete phases. We hypothesized that species not adapted to long fasts have truncated, but otherwise similar, phases of fasting, sequential changes in fuel oxidization, and similar changes in blood metabolites to fasting-adapted species. We tested salient predictions in house sparrows ( Passer domesticus biblicus), a subspecies that is unable to tolerate more than ∼32 h of fasting. Our main hypothesis was that fasting sparrows sequentially oxidize substrates in the order carbohydrates, lipids, and protein. We dosed 24 house sparrows with [13C]glucose, palmitic acid, or glycine and measured13CO2in their breath while they fasted for 24 h. To ascertain whether blood metabolite levels reflect fasting-induced changes in metabolic fuels, we also measured glucose, triacylglycerides, and β-hydroxybutyrate in the birds' blood. The results of both breath13CO2and plasma metabolite analyses did not support our hypothesis; i.e., that sparrows have the same metabolic responses characteristic of fasting-adapted species, but on a shorter time scale. Contrary to our main prediction, we found that recently assimilated13C-tracers were oxidized continuously in different patterns with no definite peaks corresponding to the three phases of fasting and also that changes in plasma metabolite levels accurately tracked the changes found by breath analysis. Notably, the rate of recently assimilated [13C]glycine oxidization was significantly higher ( P < 0.001) than that of the other metabolic tracers at all postdosing intervals. We conclude that the inability of house sparrows to fast for longer than 32 h is likely related to their inability to accrue large lipid stores, separately oxidize different fuels, and/or spare protein during fasting.
Endotoxemia alters tight junction gene and protein expression in the kidney
Authors: Eadon Michael T. , Hack Bradley K. , Xu Chang , Ko Benjamin , Toback F. Gary , Cunningham Patrick N.
Published: July 2012

Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.
Loss of vitamin D receptor in chronic kidney disease: a potential mechanism linking inflammation to epithelial-to-mesenchymal transition
Authors: Xiong Min , Gong Junbo , Liu Youhua , Xiang Rong , Tan Xiaoyue
Published: July 2012

Both peritubular inflammation and tubular epithelial-to-mesenchymal transition (EMT) are critical events during the pathogenesis of renal fibrosis. However, the relationship between these two processes is unclear. Here, we investigated the potential role of the vitamin D receptor (VDR) in coupling peritubular inflammation and EMT. In a mouse model of unilateral ureteral obstruction (UUO), loss of VDR was observed as early as 1 day after surgery. In cultured proximal tubular epithelial HK-2 cells, proinflammatory TNF-α inhibited the expression of VDR in a dose- and time-dependant manner. Treatment with TNF-α sensitized HK-2 cells to EMT stimulated by transforming growth factor (TGF)-β1. Ectopic expression of VDR counteracted the synergistic effect of TNF-α and TGF-β1 on EMT. Furthermore, knockdown of VDR using a small interfering RNA strategy mimicked the effect of TNF-α on facilitating EMT. Either TNF-α treatment or a loss of VDR induced β-catenin activation and its nuclear translocation. The VDR ligand calcitriol reversed the VDR loss and inhibited EMT in the mouse UUO model, and late administration of active vitamin D was effective in restoring VDR expression as well, and reduced collagen accumulation and deposition compared with the vehicle control. β-Catenin expression induced by UUO was also significantly inhibited after the late administration of vitamin D. These results indicate that the early loss of VDR in chronic kidney diseases was likely mediated by proinflammatory TNF-α, which renders tubular cells susceptible to EMT. Our data suggest that loss of VDR couples peritubular inflammation and EMT, two key events in renal fibrogenesis.
Cholinergic suppression of visual responses in primate V1 is mediated by GABAergic inhibition
Authors: Disney Anita A. , Aoki Chiye , Hawken Michael J.
Published: July 2012

Acetylcholine (ACh) has been implicated in selective attention. To understand the local circuit action of ACh, we iontophoresed cholinergic agonists into the primate primary visual cortex (V1) while presenting optimal visual stimuli. Consistent with our previous anatomical studies showing that GABAergic neurons in V1 express ACh receptors to a greater extent than do excitatory neurons, we observed suppressed visual responses in 36% of recorded neurons outside V1's primary thalamorecipient layer (4c). This suppression is blocked by the GABAA receptor antagonist gabazine. Within layer 4c, ACh release produces a response gain enhancement (Disney AA, Aoki C, Hawken MJ. Neuron 56: 701–713, 2007); elsewhere, ACh suppresses response gain by strengthening inhibition. Our finding contrasts with the observation that the dominant mechanism of suppression in the neocortex of rats is reduced glutamate release. We propose that in primates, distinct cholinergic receptor subtypes are recruited on specific cell types and in specific lamina to yield opposing modulatory effects that together increase neurons' responsiveness to optimal stimuli without changing tuning width.
Kainate receptor-mediated synaptic transmissions in the adult rodent insular cortex
Authors: Koga Kohei , Sim Su-Eon , Chen Tao , Wu Long-Jun , Kaang Bong-Kiun , Zhuo Min
Published: July 2012

Kainate (KA) receptors are expressed widely in the central nervous system and regulate both excitatory and inhibitory synaptic transmission. KA receptors play important roles in fear memory, anxiety, and pain. However, little is known about their function in synaptic transmission in the insular cortex (IC), a critical region for taste, memory, and pain. Using whole cell patch-clamp recordings, we have shown that KA receptors contribute to fast synaptic transmission in neurons in all layers of the IC. In the presence of the GABAA receptor antagonist picrotoxin, the NMDA receptor antagonist AP-5, and the selective AMPA receptor antagonist GYKI 53655, KA receptor-mediated excitatory postsynaptic currents (KA EPSCs) were revealed. We found that KA EPSCs are ∼5–10% of AMPA/KA EPSCs in all layers of the adult mouse IC. Similar results were found in adult rat IC. KA EPSCs had a significantly slower rise time course and decay time constant compared with AMPA receptor-mediated EPSCs. High-frequency repetitive stimulations at 200 Hz significantly facilitated the summation of KA EPSCs. In addition, genetic deletion of GluK1 or GluK2 subunit partially reduced postsynaptic KA EPSCs, and exposure of GluK2 knockout mice to the selective GluK1 antagonist UBP 302 could significantly reduce the KA EPSCs. These data suggest that both GluK1 and GluK2 play functional roles in the IC. Our study may provide the synaptic basis for the physiology and pathology of KA receptors in the IC-related functions.
Real-time Monitoring ofIn VivoAcute Necrotic Cancer Cell Death Induced by Near Infrared Photoimmunotherapy Using Fluorescence Lifetime Imaging
Authors: Nakajima Takahito , Sano Kohei , Mitsunaga Makoto , Choyke Peter L. , Kobayashi Hisataka
Published: July 2012

A new type of monoclonal antibody (mAb)-based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with a mAb, has recently been described. NIR light exposure leads to immediate, target-selective necrotic cell death in vitro. Detecting immediate in vivo cell death is more difficult because it takes at least 3 days for the tumor to begin to shrink in size. In this study, fluorescence lifetime (FLT) was evaluated before and after PIT for monitoring the immediate cytotoxic effects of NIR mediated mAb-IR700 PIT. Anti-epidermal growth factor receptor (EGFR) panitumumab-IR700 was used for targeting EGFR-expressing A431 tumor cells. PIT with various doses of NIR light was conducted in cell pellets in vitro and in subcutaneously xenografted tumors in mice in vivo. FLT measurements were obtained before and 0, 6, 24, and 48 hours after PIT. In vitro, PIT at higher doses of NIR light immediately led to FLT shortening in A431 cells. In vivo PIT induced immediate shortening of FLT in treated tumors after a threshold NIR dose of 30 J/cm2 or greater. In contrast, lower levels of NIR light (10 J/cm2 or smaller) did not induce shortening of FLT. Prolongation of FLT in tissue surrounding the tumor site was noted 6 hours after PIT, likely reflecting phagocytosis by macrophages. In conclusion, FLT imaging can be used to monitor the acute cytotoxic effects of mAb-IR700-induced PIT even before morphological changes can be seen in the targeted tumors. Cancer Res; 72(18); 4622–8. ©2012 AACR.
Predictors of Sustained Ventricular Arrhythmias in Cardiac Resynchronization Therapy
Authors: Friedman Daniel J. , Altman Robert K. , Orencole Mary , Picard Michael H. , Ruskin Jeremy N. , Singh Jagmeet P. , Heist E. Kevin
Published: July 2012

Background—

Patients undergoing cardiac resynchronization therapy (CRT) are at high risk for ventricular arrhythmias (VAs), and risk stratification in this population remains poor.

Methods and Results—

This study followed 269 patients (left ventricular ejection fraction <35%; QRS >120 ms; New York Heart Association class III/IV) undergoing CRT with a defibrillator for 553±464 days after CRT with defibrillator implantation to assess for independent predictors of appropriate device therapy for VAs. Baseline medication use, medical comorbidities, and echocardiographic parameters were considered. The 4-year incidence of appropriate device therapy was 36%. A Cox proportional hazard model identified left ventricular end-systolic diameter >61 mm as an independent predictor in the entire population (hazard ratio [HR], 2.66;
P
=0.001). Those with left ventricular end-systolic diameter >61 mm had a 51% 3-year incidence of VA compared with a 26% incidence among those with a less dilated ventricle (
P
=0.001). Among patients with left ventricular end-systolic diameter ≤61 mm, multivariate predictors of appropriate therapy were absence of β-blocker therapy (HR, 6.34;
P
<0.001), left ventricular ejection fraction <20% (HR, 4.22;
P
<0.001), and history of sustained VA (HR, 2.97;
P
=0.013). Early (<180 days after implant) shock therapy was found to be a robust predictor of hospitalization for heart failure (HR, 3.41;
P
<0.004) and mortality (HR, 5.16;
P
<0.001.)

Conclusions—

Among patients with CRT and a defibrillator, left ventricular end-systolic diameter >61 mm is a powerful predictor of VAs, and further risk stratification of those with less dilated ventricles can be achieved based on assessment of ejection fraction, history of sustained VA, and absence of β-blocker therapy.
Comparison of Family History of Sudden Cardiac Death in Nonischemic and Ischemic Heart Disease
Authors: Hookana Eeva , Junttila M. Juhani , Kaikkonen Kari S. , Ukkola Olavi , Kesäniemi Y. Antero , Kortelainen Marja-Leena , Huikuri Heikki V.
Published: July 2012

Background—

Recent studies have identified the presence of familial clustering of ischemic sudden cardiac death (SCD) as a clinical expression of coronary artery disease. The purpose of this study was to determine whether nonischemic SCD has a similar familial background, which would be evidence of a genetic predisposition.

Methods and Results—

The retrospective case-control study included (1) consecutive victims of nonischemic SCD (n=223), (2) consecutive victims of ischemic SCD (n=596), whose deaths and diagnosis were verified at medicolegal autopsy, and (3) control subjects without heart disease (n=475). In each study group, the family history of SCD among the first-degree relatives was determined and verified from death certificates. The prevalence of SCD in ≥1 first-degree relative was significantly higher in victims of ischemic (34.2%) than nonischemic SCD (13.4%;
P
<0.001) or controls (17.6%;
P
<0.001). The history of SCD in first-degree relatives did not differ from controls in nonischemic SCD victims (
P
=0.155). In a subgroup analysis of victims of ischemic SCD, the prevalence of family history of SCD in first-degree relatives did not differ between those with or without a prior infarct scar at autopsy (33.1% versus 29.9%, respectively;
P
=0.222).

Conclusions—

Ischemic SCD has a strong familial background both in cases with and without a prior myocardial infarction. The family history of SCD is not significantly increased in victims of nonischemic SCD, suggesting a larger role of sporadic occurrence than inherited traits as the cause of nonischemic SCD.