bluuebunny/crossref_metadata_2025 huggingface dataset:
Directed Evolution of Ionizing Radiation Resistance in
Escherichia coli
Authors: Harris Dennis R. , Pollock Steve V. , Wood Elizabeth A. , Goiffon Reece J. , Klingele Audrey J. , Cabot Eric L. , Schackwitz Wendy , Martin Joel , Eggington Julie , Durfee Timothy J. , Middle Christina M. , Norton Jason E. , Popelars Michael C. , Li Hao , Klugman Sarit A. , Hamilton Lindsay L. , Bane Lukas B. , Pennacchio Len A. , Albert Thomas J. , Perna Nicole T. , Cox Michael M. , Battista John R.
Published: June 2009

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species,
Escherichia coli
, by directed evolution. Four populations of
Escherichia coli
K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D
37
values for strains isolated from two of the populations approached that exhibited by
Deinococcus radiodurans
. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the
recA
gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.
An Excretory Function for theEscherichia coliOuter Membrane Pore TolC: Upregulation ofmarAandsoxSTranscription and Rob Activity Due to Metabolites Accumulated intolCMutants
Authors: Rosner Judah L. , Martin Robert G.
Published: June 2009

Efflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps inEscherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. BecausetolCtranscription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators intolCmutants. Using transcriptional fusions, we detected significant upregulation ofmarRABandsoxStranscription and Rob protein activity intolCmutants. Three mechanisms could be distinguished: (i) activation ofmarRABtranscription was independent ofmarRAB,soxR, androbfunctions; (ii) activation ofsoxStranscription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation ofmarRAB,soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of othermarA/soxS/robregulon promoters, includingtolCitself, was also elevated intolCmutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulatetolCand help rid the bacteria of these metabolites, thereby restoring homeostasis.
Shiga Toxin as a Bacterial Defense against a Eukaryotic Predator,
Tetrahymena thermophila
Authors: Lainhart William , Stolfa Gino , Koudelka Gerald B.
Published: June 2009

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian “targets,” suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that
Tetrahymena thermophila
, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with
Tetrahymena
, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx.
Tetrahymena
is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by
Tetrahymena
. Bacterially mediated
Tetrahymena
killing was blocked by mutations that prevented the bacterial SOS response (
recA
mutations) or by enzymes that breakdown H
2
O
2
(catalase), suggesting that the production of H
2
O
2
by
Tetrahymena
signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.
A Critical Process Controlled by MalT and OmpR Is Revealed through Synthetic Lethality
Authors: Reimann Sylvia A. , Wolfe Alan J.
Published: June 2009

The death of cells harboring defects in two distinct pathways implicates these pathways in the control of an essential process. Here we report that cells lacking OmpR and harboring constitutively active MalT undergo premature death that involves increased expression of the outer membrane porin LamB.
Specific Interactions between Four Molybdenum-Binding Proteins Contribute to Mo-Dependent Gene Regulation in
Rhodobacter capsulatus
Authors: Wiethaus Jessica , Müller Alexandra , Neumann Meina , Neumann Sandra , Leimkühler Silke , Narberhaus Franz , Masepohl Bernd
Published: June 2009

The phototrophic purple bacterium
Rhodobacter capsulatus
encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the
R. capsulatus
Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent
mop
-
lacZ
fusion in mutant strains defective for either
mopA
,
mopB
, or both suggested that MopB negatively modulates expression of the
mop
promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling
mop
gene expression.
Comparative Genomic Analysis of Ten
Streptococcus pneumoniae
Temperate Bacteriophages
Authors: Romero Patricia , Croucher Nicholas J. , Hiller N. Luisa , Hu Fen Z. , Ehrlich Garth D. , Bentley Stephen D. , García Ernesto , Mitchell Tim J.
Published: June 2009

Streptococcus pneumoniae
is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a
Siphoviridae
morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the
pblB
gene of
Streptococcus mitis
and the
vapE
gene of
Dichelobacter nodosus
, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage φSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them.
Implementation of the Thin Layer Agar Method for Diagnosis of Smear-Negative Pulmonary Tuberculosis in a Setting with a High Prevalence of Human Immunodeficiency Virus Infection in Homa Bay, Kenya
Authors: Martin Anandi , Munga Waweru Peter , Babu Okatch Fred , Amondi Ouma Naureen , Bonte Laurence , Varaine Francis , Portaels Françoise
Published: June 2009

The objective of this study was to evaluate the performance of a low-cost method, the thin layer agar (TLA) method, for the diagnosis of smear-negative patients. This prospective study was performed in Homa Bay District Hospital in Kenya. Out of 1,584 smear-negative sputum samples, 212 (13.5%) were positive by culture in Löwenstein-Jensen medium (LJ) and 220 (14%) were positive by the TLA method. The sensitivities of LJ and TLA were 71% and 74%, respectively. TLA could become an affordable method for the diagnosis of smear-negative tuberculosis in resource-limited settings, with results available within 2 weeks.
Human Papillomavirus Genotype Specificity of Hybrid Capture 2 Low-Risk Probe Cocktail
Authors: Poljak Mario , Kocjan Boštjan J. , Kovanda Anja , Lunar Maja M. , Lepej Snježana Židovec , Planinić Ana , Seme Katja , Vince Adriana
Published: June 2009

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.
First Case of Bloodstream Infection Caused by
Rhodococcus erythropolis
Authors: Baba Hisashi , Nada Toshi , Ohkusu Kiyofumi , Ezaki Takayuki , Hasegawa Yoshinori , Paterson David L.
Published: June 2009

We describe the first case of bloodstream infection caused by
Rhodococcus erythropolis
. The identification was performed using 16S rRNA sequencing. This case illustrates that non-
equi Rhodococcus
infections may be underdiagnosed due to difficulties in identification in the routine clinical microbiology laboratory.
Cephalosporin MIC Distribution of Extended-Spectrum-β-Lactamase- and pAmpC-Producing
Escherichia coli
and
Klebsiella
Species
Authors: Kohner Peggy C. , Robberts Frans J. L. , Cockerill Franklin R. , Patel Robin
Published: June 2009

The acquisition of β-lactamases in members of the
Enterobacteriaceae
family poses a challenge to antimicrobial susceptibility testing in the clinical laboratory. We correlated the distribution of the MICs for
Klebsiella
spp. and
Escherichia coli
with the presence of extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pAmpC) genes. A total of 264 isolates were subjected to cefazolin, ceftriaxone, cefotaxime, ceftazidime, cefepime, and aztreonam agar dilution MIC determination; ESBL screening and confirmatory testing by the methods of the Clinical and Laboratory Standards Institute (CLSI); and for isolates for which the MICs of extended-spectrum cephalosporins were ≥1 μg/ml or the MICs of cefpodoxime were >4 μg/ml, PCR amplification and sequencing of the ESBL and pAmpC genes. PCR was positive for 73/81 isolates (45 isolates with an ESBL gene alone, 24 isolates with a pAmpC gene alone, with 4 isolates with both genes). Compared to PCR, confirmatory testing by the CLSI method yielded a sensitivity and a specificity of 98.0 and 96.3%, respectively; there were six false-positive results and one false-negative result. No distinction in the MIC distribution was apparent between isolates with the ESBL gene and isolates with the pAmpC gene. A substantial percentage of the isolates with PCR-confirmed ESBL and/or pAmpC genes fell within the current CLSI susceptible category. For a ceftazidime, ceftriaxone, or cefotaxime MIC of ≥2 μg/ml, a dichotomy existed between isolates with and without ESBL and pAmpC genes in most cases. This suggests that the presence of the ESBL and the pAmpC enzymes may yield similar MICs of extended-spectrum cephalosporins, many of which fall within the current nonresistant categories. Lowering of the current CLSI breakpoints for cephalosporins appears to be warranted.
Genes Related to Long Polar Fimbriae of Pathogenic
Escherichia coli
Strains as Reliable Markers To Identify Virulent Isolates
Authors: Torres Alfredo G. , Blanco Miguel , Valenzuela Patricio , Slater Terry M. , Patel Shilpa D. , Dahbi Ghizlane , López Cecilia , Barriga Ximena Fernández , Blanco Jesús E. , Gomes Tânia A. T. , Vidal Roberto , Blanco Jorge
Published: June 2009

Lpf (stands for long polar fimbriae) is one of the few adhesive factors of enterohemorrhagic
Escherichia coli
O157:H7 associated with colonization of the intestine.
E. coli
O157:H7 strains possess two
lpf
loci encoding highly regulated fimbrial structures. Database analysis of the genes encoding the major fimbrial subunits demonstrated that they are present in commensal as well as pathogenic (both intestinal and extraintestinal)
E. coli
strains and in
Salmonella
strains and that the
lpfA1
and
lpfA2
genes are highly prevalent among LEE (locus of enterocyte effacement)-positive
E. coli
strains associated with severe and/or epidemic disease. Further DNA sequence analysis of the
lpfA1
and
lpfA2
genes from different attaching-and-effacing
E. coli
strains has led us to the identification of several polymorphisms and the classification of the major fimbrial subunits into distinct variants. Using collections of pathogenic
E. coli
isolates from Europe and Latin America, we demonstrated that the different
lpfA
types are associated with the presence of specific intimin (
eae
) adhesin variants and, most importantly, that they are found in specific
E. coli
pathotypes. Our results showed that the use of these fimbrial genes as markers, in combination with the different intimin types, resulted in a specific test for the identification of
E. coli
O157:H7, distinguishing it from other pathogenic
E. coli
strains.
Risk Factors for Campylobacteriosis: an Epidemiological Surveillance Study of Patients and Retail Poultry
Authors: Lindmark H. , Boqvist S. , Ljungström M. , Ågren P. , Björkholm B. , Engstrand L.
Published: June 2009

Isolates from
Campylobacter jejuni-
infected patients were collected and fresh poultry meat from retail sources was sampled during the same time period and within the same geographical area. The patients were interviewed about exposure to known risk factors, and a significant correlation between the presence of a poultry subtype in patients and the consumption of fresh poultry meat was observed.
Mycobacterium tuberculosis
in Ontario, Canada: Insights from IS
6110
Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping
Authors: Alexander David C. , Guthrie Jennifer L. , Pyskir Daria , Maki Anne , Kurepina Natalia , Kreiswirth Barry N. , Chedore Pamela , Drews Steven J. , Jamieson Frances
Published: June 2009

A collection of 1,308 clinical
Mycobacterium tuberculosis
isolates from Ontario, Canada, was genotyped by IS
6110
restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.
Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep
Authors: Maddison B. C. , Baker C. A. , Rees H. C. , Terry L. A. , Thorne L. , Bellworthy S. J. , Whitelam G. C. , Gough K. C.
Published: June 2009

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP
Sc
within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.
Combinations of the First and Next Generations of Human Immunodeficiency Virus (HIV) Fusion Inhibitors Exhibit a Highly Potent Synergistic Effect against Enfuvirtide- Sensitive and -Resistant HIV Type 1 Strains
Authors: Pan Chungen , Cai Lifeng , Lu Hong , Qi Zhi , Jiang Shibo
Published: June 2009

T20 (generic name, enfuvirtide; brand name, Fuzeon) is a first-generation human immunodeficiency virus (HIV) fusion inhibitor approved for salvage therapy of HIV-infected patients refractory to current antiretroviral drugs. However, its clinical use is limited because of rapid emergence of T20-resistant viruses in T20-treated patients. Therefore, T1249 and T1144 are being developed as the second- and third-generation HIV fusion inhibitors, respectively, with improved efficacy and drug resistance profiles. Here, we found that combinations of T20 with T1249 and/or T1144 resulted in exceptionally potent synergism (combination index, <0.01) against HIV-1-mediated membrane fusion by 2 to 3 orders of magnitude in dose reduction. Highly potent synergistic antiviral efficacy was also achieved against infection by laboratory-adapted and primary HIV-1 strains, including T20-resistant variants. The mechanism underlying the synergistic effect could be attributed to the fact that T20, T1249, and T1144 all contain different functional domains and have different primary binding sites in gp41. As such, they may work cooperatively to inhibit gp41 six-helix bundle core formation, thereby suppressing virus-cell fusion. Therefore, these findings strongly imply that, rather than replacing T20, combining it with HIV fusion inhibitors of different generations might produce synergistic activity against both T20-sensitive and -resistant HIV-1 strains, suggesting a new therapeutic strategy for the treatment of HIV-1 infection/AIDS.
Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment
Authors: Jackson Sara , Van Hoeven Neal , Chen Li-Mei , Maines Taronna R. , Cox Nancy J. , Katz Jacqueline M. , Donis Ruben O.
Published: June 2009

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.
Nuclear Factor 90 Negatively Regulates Influenza Virus Replication by Interacting with Viral Nucleoprotein
Authors: Wang Pui , Song Wenjun , Mok Bobo Wing-Yee , Zhao Pengxi , Qin Kun , Lai Alexander , Smith Gavin J. D. , Zhang Jinxia , Lin Tianwei , Guan Yi , Chen Honglin
Published: June 2009

Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.
Time-Dependent Transformation of the Herpesvirus Tegument
Authors: Newcomb William W. , Brown Jay C.
Published: June 2009

All herpesviruses have a layer of protein called the tegument that lies between the virion membrane and the capsid. The tegument consists of multiple, virus-encoded protein species that together can account for nearly half the total virus protein. To clarify the structure of the tegument and its attachment to the capsid, we used electron microscopy and protein analysis to examine the tegument of herpes simplex virus type 1 (HSV-1). Electron microscopic examination of intact virions revealed that whereas the tegument was asymmetrically distributed around the capsid in extracellular virions, it was symmetrically arranged in cell-associated virus. Examination of virions after treatment with nonionic detergent demonstrated that: (i) in extracellular virus the tegument was resistant to removal with Triton X-100 (TX-100), whereas it was lost nearly completely when cell-associated virus was treated in the same way; (ii) the tegument in TX-100-treated extracellular virions was asymmetrically distributed around the capsid as it is in unextracted virus; and (iii) in some images, tegument was seen to be linked to the capsid by short, regularly spaced connectors. Further analysis was carried out with extracellular virus harvested from cells at different times after infection. It was observed that while the amount of tegument present in virions was not affected by time of harvest, the amount remaining after TX-100 treatment increased markedly as the time of harvest was increased from 24 h to 64 h postinfection. The results support the view that HSV-1 virions undergo a time-dependent change in which the tegument is transformed from a state in which it is symmetrically organized around the capsid and extractable with TX-100 to a state where it is asymmetrically arranged and resistant to extraction.
Dynamics of Biologically Active Subpopulations of Influenza Virus: Plaque-Forming, Noninfectious Cell-Killing, and Defective Interfering Particles
Authors: Marcus Philip I. , Ngunjiri John M. , Sekellick Margaret J.
Published: June 2009

The dynamic changes in the temporal appearance and quantity of a new class of influenza virus, noninfectious cell-killing particles (niCKP), were compared to defective interfering particles (DIP). After a single high-multiplicity passage in MDCK cells of an egg-derived stock that lacked detectable niCKP or DIP, both classes of particles appeared in large numbers (>5 × 108/ml), and the plaque-forming particle (PFP) titer dropped ∼60-fold. After two additional serial high-multiplicity passages the DIP remained relatively constant, the DIP/niCKP ratio reached 10:1, and the PFP had declined by about 10,000-fold. Together, the niCKP and DIP subpopulations constituted ca. 20% of the total hemagglutinating particle population in which these noninfectious biologically active particles (niBAP) were subsumed. DIP neither killed cells nor interfered with the cell-killing (apoptosis-inducing) activity of niCKP or PFP (infectious CKP), even though they blocked the replication of PFP. Relative to the UV-target of ∼13,600 nucleotides (nt) for inactivation of PFP, the UV target for niCKP was ∼2,400 nt, consistent with one of the polymerase subunit genes, and that for DIP was ∼350 nt, consistent with the small DI-RNA responsible for DIP-mediated interference. Thus, niCKP and DIP are viewed as distinct particles with a propensity to form during infection at high multiplicities. These conditions are postulated to cause aberrations in the temporally regulated replication of virus and its packaging, leading to the production of niBAP. DIP have been implicated in the virulence of influenza virus, but the role of niCKP is yet unknown.
Circulation and Dispersion on Browns Bank
Authors: Smith Peter C.
Published: June 2009

Year-long current measurements reveal a permanent clockwise gyre over the western cap of Browns Bank. Maximum mean currents (0.25 m∙s−1) over the steep northern flank are uniform in the upper water column, except when pulses of low salinity water arrive from the Scotian Shelf producing stratification and vertical shear. Significant off-bank Eulerian mean currents, found near the bottom on the 100-m isobath, are consistent with the models for topographic interactions of the semidiurnal tides. A multiple regression analysis in the spectral domain reveals most low-frequency energy is concentrated in the 14–28-d bands where wind- and tidally-forced currents make roughly equal contributions (30–40%) to the total variance. In the synoptic bands (2–10-d), wind forcing alone contributes 60–80% of the variance. The temporal and spatial variability of the gyre is supported by satellite-tracked drogue studies on the Bank in three seasons. April and November 1983 observations show that the gyre circulation may be broken down by wind-forced currents. In July/September 1983, drogues consistently exited the western cap of the Bank from the northern flank after a residence time of 14 d, comparable with the near-surface recirculation time. Isotropic dispersion coefficients [Formula: see text] are found to follow Okubo's (1971) empirical formulae.
Copepod Production on the Scotian Shelf based on Life-History Analyses and Laboratory Bearings
Authors: McLaren Ian A. , Tremblay M. John , Corkett C. J. , Roff John C.
Published: June 2009

We estimated monthly and annual production of 10 copepod species (> 99% of copepod biomass) from Emerald Bank, Scotian Shelf, by use of numerical and weight changes between samples for Calanus spp., and by applying daily growth rates based on stage durations and weight increments between stages for others. Of these others, stage durations were estimated from generation intervals inferred from samples of Metridia lucens, Pseudocalanus newmani, and Oithona similis. For species without evident cohorts, notably Centropages typicus and Paracalanus parvus, we used published temperature-dependent durations. Production in dry weight was estimated as ca. 0.44 g∙m−3∙yr−1 (39 g∙m−2∙yr−1), with peaks in May (largely Calanus spp.) and September (largely C. typicus, P. parvus, and other warm-water forms). All species except C. finmarchicus and O. similis had annual rates 2–4 times those predicted by the Banse-Mosher allometric relationship between production/biomass ratios and adult body mass. This deviation is not attributable to assumptions in our analyses. Annual copepod production is broadly similar to recent estimates by others using similar methods for Georges Bank, Passamaquoddy Bay, N.B., and the North Sea.
Origin and Dispersion of Larval Herring (Clupea harengus) in Coastal Waters of Eastern Maine and Southwestern New Brunswick
Authors: Chenoweth S. B. , Libby D. A. , Stephenson R. L. , Power M. J.
Published: June 2009

Repetitive surveys of larval herring (Clupea harengus) were undertaken in coastal waters between Mt. Desert, Maine and Saint John, New Brunswick to define the location, timing, and extent of spawning and the distribution of larvae. Three surveys during September and October 1986 showed two separate areas of larval dispersal. The easternmost stations contained low densities of large larvae which were assumed to be part of the annually occurring larval aggregation off Nova Scotia. High densities of small larvae west of Grand Manan indicated that spawning in this area was confined to a small area southwest of Grand Manan and along the eastern Maine coast. This unique larval distribution confirms the autonomous nature of the eastern Maine–Grand Manan spawning group. Back calculation indicated hatching times between late July and the middle of October, but most of the larvae taken in the survey hatched in August and September. The larvae west of Grand Manan occurred within a well mixed water mass containing a characteristic neritic zooplankton community. Some larval herring remained near the spawning ground over a 2-mo period, while others dispersed west along the coast.
Density Dependence in Spiny Lobster, Panulirus marginatus, in the Northwestern Hawaiian Islands
Authors: Polovina Jeffrey J.
Published: June 2009

A positive linear relationship between the ratio of natural mortality to von Bertalanffy growth and catch per unit effort (CPUE) and a negative relationship between asymptotic length and CPUE were observed for the spiny lobster, Panulirus marginatus, among seven banks in the Northwestern Hawaiian Islands prior to the development of a fishery (1977–81). Resampling two of these banks in 1986–87 after heavy fishing indicated an increase in the asymptotic length and a decrease in the length at onset of egg production.
Age Determination of the Spider Crab Maja squinado and the European Lobster Homarus gammarus by 228Th/228Ra Chronology: Possible Extension to Other Crustaceans
Authors: Foll Didier Le , Brichet Evelyne , Reyss Jean Louis , Lalou Claude , Latrouite Daniel
Published: June 2009

A method of age determination was developed on carapaces of the spider crab, Maja squinado Herbst and European lobster Homarus gammarus L., by measuring the natural radionuclides activity ratio, 228Th/228Ra in the exoskeleton. This method allows the determination of the time elapsed since the preceding molt of the animal. It was successfully tested on five spider crabs and four lobsters which had molted in captivity and therefore had a carapace of known age. It is probable that the method could, with some reservations, be extended to all marine Decapod Crustacea bearing a well calcified exoskeleton.
Networks and Economic Behavior
Authors: Jackson Matthew O.
Published: June 2009

Recent analyses of social networks, both empirical and theoretical, are discussed, with a focus on how social networks influence economic behavior, as well as how social networks form. Some challenges of such research are discussed as are some of the important considerations for the future.
Vascular regression and survival are differentially regulated by MT1-MMP and TIMPs in the aortic ring model of angiogenesis
Authors: Aplin A. C. , Zhu W. H. , Fogel E. , Nicosia R. F.
Published: June 2009

This study was designed to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in the reabsorption of neovessels in collagen gel cultures of rat and mouse aortic rings. Aortic angiogenesis was associated with collagen lysis and production of the matrix-degrading enzymes MMP-2, MMP-9, and membrane-type MMP (MT1-MMP, or MMP-14). Vascular growth and regression were not affected by disruption of MMP-2 or MMP-9. In addition, no effect on vascular regression was observed by blocking plasmin, a protease implicated in the activation of MMPs, with ε-aminocaproic acid or by adding plasminogen, which caused a modest increase in vascular proliferation. Conversely, angiogenesis was blocked and vessels stabilized by inhibiting MT1-MMP with neutralizing antibodies, TIMP-2, TIMP-3, or TIMP-4. TIMP-1, which blocks MMP-2 and MMP-9 but is a poor inhibitor of MT1-MMP, had no antiangiogenic effect. However, TIMP-1 prolonged the survival of neovessels following angiogenesis. Vascular regression was accelerated in aortic cultures from TIMP-1- and TIMP-2-deficient mice. The vascular survival effect of anti-MT1-MMP antibodies and TIMPs with MT1-MMP inhibitory activity was associated with complete inhibition of collagen lysis. In contrast, TIMP-1 had no anticollagenolytic effect. These results indicate that MT1-MMP plays a critical role not only in angiogenesis but also in vascular regression and demonstrate that TIMPs with anti-MT1-MMP activity have opposite effects on angiogenic outcomes depending on the stage of the angiogenic process. This study also suggests the existence of a TIMP-1-mediated alternate pathway of vascular survival that is unrelated to MT1-MMP inhibitory activity.
Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level
Authors: McSpadden Luke C. , Kirkton Robert D. , Bursac Nenad
Published: June 2009

Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle α-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from −71.6 ± 4.9 mV in controls to −65.0 ± 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.
Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties
Authors: Cheng Xiaoyang , Pachuau Judith , Blaskova Eva , Asuncion-Chin Maria , Liu Jianxi , Dopico Alejandro M. , Jaggar Jonathan H.
Published: June 2009

Voltage-dependent calcium (Ca2+, CaV1.2) channels are the primary Ca2+ entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified CaV1.2 α1-subunit splice variation in myocytes of rat resistance-size (100–200 μm diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and ± 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major CaV1.2 variant contained exons 1c, 8, 21, and 32+33, with ∼57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ ± 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the CaV1.2 α1-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell CaV1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range.
Decreased upright cerebral blood flow and cerebral autoregulation in normocapnic postural tachycardia syndrome
Authors: Ocon Anthony J. , Medow Marvin S. , Taneja Indu , Clarke Debbie , Stewart Julian M.
Published: June 2009

Postural tachycardia syndrome (POTS), a chronic form of orthostatic intolerance, has signs and symptoms of lightheadedness, loss of vision, headache, fatigue, and neurocognitive deficits consistent with reductions in cerebrovascular perfusion. We hypothesized that young, normocapnic POTS patients exhibit abnormal cerebral autoregulation (CA) that results in decreased static and dynamic cerebral blood flow (CBF) autoregulation. All subjects had continuous recordings of mean arterial pressure (MAP) and CBF velocity (CBFV) using transcranial Doppler sonography in both the supine supine position and during a 70° head-up tilt. During tilt, POTS patients ( n = 9) demonstrated a higher heart rate than controls ( n = 7) (109 ± 6 vs. 80 ± 2 beats/min, P < 0.05), whereas controls demonstrated a higher MAP than POTS (87 ± 2 vs. 77 ± 3 mmHg, P < 0.05). Also during tilt, mean CBFV decreased 19.5 ± 2.6% in POTS patients versus 10.3 ± 2.0% in controls ( P < 0.05). We then used a transfer function analysis of MAP and CFBV in the frequency domain to quantify these changes. The low-frequency (LF; 0.04–0.15 Hz) component of CBFV variability increased during tilt in POTS patients (supine: 3 ± 0.9 vs. tilt: 9 ± 2, P < 0.02). In POTS patients, there was an increase in LF and high-frequency coherence between MAP and CBFV, an increase in LF gain, and a lack of significant change in phase. Static CA may be less effective in POTS patients compared with controls, since immediately after tilt CBFV decreased more in POTS patients and was highly oscillatory and autoregulation did not restore CBFV to baseline values until the subjects became supine. Dynamic CA may be less effective in POTS patients because MAP and CBFV during tilt became almost perfectly synchronous. We conclude that dynamic and static autoregulation of CBF are less effective in POTS patients compared with control subjects during orthostatic challenge.
Reduction in myocardial infarct size at 48 hours after brief intravenous infusion of ATL-146e, a highly selective adenosine A2Areceptor agonist
Authors: Patel Rajan A. G. , Glover David K. , Broisat Alexis , Kabul Hasan K. , Ruiz Mirta , Goodman N. Craig , Kramer Christopher M. , Meerdink Denis J. , Linden Joel , Beller George A.
Published: June 2009

This study was undertaken to determine whether the myocardial infarct-sparing effect of ATL-146e, a selective adenosine A2Areceptor agonist, persists without a rebound effect for at least 48 h and to determine the optimal duration of ATL-146e treatment in anesthetized dogs. Reperfusion injury after myocardial infarction (MI) is associated with inflammation lasting 24–48 h that contributes to ongoing myocyte injury. We previously showed that an ATL-146e infusion, starting just before reperfusion, decreased inflammation and infarct size in dogs examined 2 h after MI without increasing coronary blood flow. In the present study, adult dogs underwent 90 min of left anterior descending coronary artery occlusion. Thirty minutes before reperfusion, ATL-146e (0.01 μg·kg−1·min−1; n = 21) or vehicle ( n = 12) was intravenously infused and continued for 2.5 h ( protocol 1) or 24 h ( protocol 2). At 48 h after reperfusion hearts were excised and assessed for histological risk area and infarct size. Infarct size based on triphenyltetrazolium chloride (TTC) staining as a percentage of risk area was significantly smaller in ATL-146e-treated vs. control dogs (16.7 ± 3.7% vs. 33.3 ± 6.2%, P < 0.05; protocol 1). ATL-146e reduced neutrophil accumulation into infarcted myocardium of ATL-146e-treated vs. control dogs (30 ± 7 vs. 88 ± 16 cells/high-power field, P < 0.002). ATL-146e infusion for 24 h ( protocol 2) conferred no significant additional infarct size reduction compared with 2.5 h of infusion. A 2.5-h ATL-146e infusion initiated 30 min before reperfusion results in marked, persistent (48 h) reduction in infarct size as a percentage of risk area in dogs with a reduction in infarct zone neutrophil infiltration. No significant further benefit was seen with a 24-h infusion.
Activation of the nitric oxide-cGMP pathway reduces phasic contractions in neonatal rat bladder strips via protein kinase G
Authors: Artim Debra E. , Kullmann F. Aura , Daugherty Stephanie L. , Wu Hsi-Yang , de Groat William C.
Published: June 2009

Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models but do not affect normal bladder activity in the adult rat. In this study, the NO donor S-nitroso- N-acetyl-dl-penicillamine (SNAP; 100 μM) decreased the amplitude and frequency of spontaneous and carbachol-enhanced contractions in neonatal rat bladder strips, which are intrinsically hyperactive. This effect was blocked by inhibition of sGC and mimicked by application of a membrane-permeable cGMP analog (8-bromo-cGMP, 100 μM). Inhibition of PKG prevented or reversed the inhibitory effects of 8-bromo-cGMP. A portion of the SNAP-mediated inhibition was also dependent upon PKG; however, a short-lasting, sGC-dependent inhibitory effect of SNAP was still present after PKG inhibition. Inhibition of NO synthase with l-NAME (100 μM) did not change the amplitude or frequency of contractions. However, inhibition of endogenous phosphodiesterase (PDE)-5 with zaprinast (25 μM) reduced the amplitude and frequency of phasic contractions and increased the magnitude of inhibition produced by maximal concentrations of SNAP, suggesting that endogenous PDEs are constitutively active and regulate cGMP production. These results suggest that the NO-cGMP-PKG pathway may be involved in inhibitory control of the neonatal rat bladder.
Cardiac output and systemic transit time dispersion as determinants of circulatory mixing time: a simulation study
Authors: Weiss Michael
Published: June 2009

A new approach to characterize the kinetics of intravascular mixing process is presented. The mixing time, defined as the time required for achieving 95% homogeneity, is calculated by numerical simulations using a circulatory model applied to the intravascular marker indocyanine green (ICG). The results suggest that the mixing time is determined by cardiac output and the relative dispersion of transit time distribution across the systemic circulation, whereby the rate of mixing increases with increasing cardiac output and decreasing transit time dispersion, and vice versa. The estimation of plasma volume from simulated ICG dilution data using the backextrapolation method shows that slow mixing is accompanied by an overestimation of blood volume. This error may be negligible for mixing times of less than ∼3 min but high in disease states characterized by low cardiac output and/or high transit time dispersion. In view of the role of transit time dispersion as determinant of intravascular mixing, it would be interesting to know more about the effect of disease states on systemic transit time dispersion.