bluuebunny/crossref_metadata_2025 huggingface dataset:
Marriage Patterns of the Foreign-Born Population in a New Country of Immigration: The Case of Spain
Authors: Trilla Clara Cortina , Esteve Albert , Domingo Andreu
Published: November 2008

We use 2001 Spanish census microdata and multivariate logistic regression analysis to explore differences in marriage patterns between the foreign-born population in Spain, a country that has experienced a dramatic increase in international migration rates in the last decade. In particular, we examine separately the prevalence of being in a consensual and in an endogamous union for a selected and representative group of origins. Results show that after controlling for individual and union characteristics, major differences in cohabitation between groups disappear while major differences in endogamy prevail. This suggests that, when appropriate data are available, future research should take into account contextual factors.
Late-Life Migration, Work Status, and Survival: The Case of Older Immigrants from the Former Soviet Union in Israel
Authors: Litwin Howard , Leshem Elazar
Published: November 2008

This analysis examined mortality among late-life Soviet Jewish immigrants in Israel, and the contribution of post-migration work status to their survival. The study linked 1997 survey data to mortality records, seven years hence. The results revealed that mortality was associated with older age, male gender, morbidity, and having less resourceful social networks. More importantly, after controlling for these background variables work status remained a significant correlate. Late-life immigrants who had never worked in the host country had a significantly greater risk of death than their immigrant counterparts who had ever worked (or were still working).
Bivariate flood frequency analysis: Part 1. Determination of marginals by parametric and nonparametric techniques
Authors: Karmakar S. , Simonovic S.P.
Published: November 2008

In flood frequency analysis, a flood event is mainly characterized by peak flow, volume and duration. These three variables or characteristics of floods are random in nature and mutually correlated. In this article, an effort is made to find out appropriate marginal distribution of the flood characteristics considering a set of parametric and nonparametric distributions, and further mathematically model the correlated nature among them. A set of parametric distribution functions and nonparametric methods based on kernel density estimation and orthonormal series are used to determine the marginal distribution functions for peak flow, volume and duration. In conventional methods of flood frequency analysis, the marginal distribution functions of peak flow, volume and duration are assumed to follow some specific parametric distribution function. The present work performs a better selection of marginal distribution functions for flood characteristics as both parametric and nonparametric estimation procedures are extensively followed. The methodology is demonstrated with 70‐year stream flow data of Red River at Grand Forks of North Dakota, USA.
Effects of deep and superficial experimentally induced acute pain on muscle sympathetic nerve activity in human subjects
Authors: Burton A. R. , Birznieks I. , Bolton P. S. , Henderson L. A. , Macefield V. G.
Published: November 2008

Human studies conducted more than half a century ago have suggested that superficial pain induces excitatory effects on the sympathetic nervous system, resulting in increases in blood pressure (BP) and heart rate (HR), whereas deep pain is believed to cause vasodepression. To date, no studies have addressed whether deep or superficial pain produces such differential effects on muscle sympathetic nerve activity (MSNA). Using microneurography we recorded spontaneous MSNA from the common peroneal nerve in 13 awake subjects. Continuous blood pressure was recorded by radial arterial tonometry. Deep pain was induced by intramuscular injection of 0.5 ml hypertonic saline (5%) into the tibialis anterior muscle, superficial pain by subcutaneous injection of 0.2 ml hypertonic saline into the overlying skin. Muscle pain, with a mean rating of 4.9 ± 0.8 (s.e.m.) on a 0–10 visual analog scale (VAS) and lasting on average 358 ± 32 s, caused significant increases in MSNA (43.9 ± 10.0%), BP (5.4 ± 1.1%) and HR (7.0 ± 2.0%) – not the expected decreases. Skin pain, rated at 4.9 ± 0.6 and lasting 464 ± 54 s, also caused significant increases in MSNA (38.2 ± 12.8%), BP (5.1 ± 2.1%) and HR (5.6 ± 2.0%). The high‐frequency (HF) to low‐frequency (LF) ratio of heart rate variability (HRV) increased from 1.54 ± 0.25 to 2.90 ± 0.45 for muscle pain and 2.80 ± 0.52 for skin pain. Despite the different qualities of deep (dull and diffuse) and superficial (burning and well‐localized) pain, we conclude that pain originating in muscle and skin does not exert a differential effect on muscle sympathetic nerve activity, both causing an increase in MSNA and an increase in the LF : HF ratio of HRV. Whether this holds true for longer lasting experimental pain remains to be seen.
Opioid‐mediated muscle afferents inhibit central motor drive and limit peripheral muscle fatigue development in humans
Authors: Amann Markus , Proctor Lester T. , Sebranek Joshua J. , Pegelow David F. , Dempsey Jerome A.
Published: November 2008

We investigated the role of somatosensory feedback from locomotor muscles on central motor drive (CMD) and the development of peripheral fatigue during high‐intensity endurance exercise. In a double‐blind, placebo‐controlled design, eight cyclists randomly performed three 5 km time trials: control, interspinous ligament injection of saline (5KPlac, L3–L4) or intrathecal fentanyl (5KFent, L3–L4) to impair cortical projection of opioid‐mediated muscle afferents. Peripheral quadriceps fatigue was assessed via changes in force output pre‐ versus postexercise in response to supramaximal magnetic femoral nerve stimulation (ΔQtw). The CMD during the time trials was estimated via quadriceps electromyogram (iEMG). Fentanyl had no effect on quadriceps strength. Impairment of neural feedback from the locomotor muscles increased iEMG during the first 2.5 km of 5KFentversus 5KPlac by 12 ± 3% (P < 0.05); during the second 2.5 km, iEMG was similar between trials. Power output was also 6 ± 2% higher during the first and 11 ± 2% lower during the second 2.5 km of 5KFentversus 5KPlac (both P < 0.05). Capillary blood lactate was higher (16.3 ± 0.5 versus 12.6 ± 1.0%) and arterial haemoglobin O2 saturation was lower (89 ± 1 versus 94 ± 1%) during 5KFentversus 5KPlac. Exercise‐induced ΔQtw was greater following 5KFentversus 5KPlac (−46 ± 2 versus−33 ± 2%, P < 0.001). Our results emphasize the critical role of somatosensory feedback from working muscles on the centrally mediated determination of CMD. Attenuated afferent feedback from exercising locomotor muscles results in an overshoot in CMD and power output normally chosen by the athlete, thereby causing a greater rate of accumulation of muscle metabolites and excessive development of peripheral muscle fatigue.
Voriconazole Increases while Itraconazole Decreases Plasma Meloxicam Concentrations
Authors: Hynninen V. V. , Olkkola K. T. , Bertilsson L. , Kurkinen K. J. , Korhonen T. , Neuvonen P. J. , Laine K.
Published: November 2008

This study investigated the effect of voriconazole, an inhibitor of cytochrome P450 2C9 (CYP2C9) and CYP3A4, and itraconazole, an inhibitor of CYP3A4, on the pharmacokinetics and pharmacodynamics of meloxicam. Twelve healthy volunteers in a crossover study ingested 15 mg of meloxicam without pretreatment (control), after voriconazole pretreatment, and after itraconazole pretreatment. The plasma concentrations of meloxicam, voriconazole, itraconazole, and thromboxane B2(TxB2) generation were monitored. Compared to the control phase, voriconazole increased the mean area under the plasma concentration-time curve from 0 to 72 h (AUC0-72) of meloxicam by 47% (P< 0.001) and prolonged its mean half-life (t1/2) by 51% (P< 0.01), without affecting its mean peak concentration (Cmax). In contrast, itraconazole decreased the mean AUC0-72andCmaxof meloxicam by 37% (P< 0.001) and by 64% (P< 0.001), respectively, and prolonged itst1/2and time toCmax. The plasma protein unbound fraction of meloxicam was unchanged by voriconazole and itraconazole. Lowered plasma meloxicam concentrations during the itraconazole phase were associated with decreased pharmacodymic effects of meloxicam, as observed by weaker inhibition of TxB2synthesis compared to the control and voriconazole phases. Voriconazole increases plasma concentrations of meloxicam, whereas itraconazole, unexpectedly, decreases plasma meloxicam concentrations, possibly by impairing its absorption.
Nalidixic Acid Disk for Laboratory Detection of Ciprofloxacin Resistance in
Neisseria meningitidis
Authors: Enríquez Rocío , Abad Raquel , Salcedo Celia , Vázquez Julio A.
Published: November 2008

Recently the CLSI recommended a disk diffusion method and breakpoints for meningococci which include breakpoints derived for nalidixic acid which serve as surrogate markers for gyrase A mutations associated with diminished fluoroquinolone susceptibility. This study presents the application of this methodology to a panel of 57 meningococcal strains isolated in Spain that include all levels of susceptibility to ciprofloxacin. In conclusion, the most useful method to predict isolates with
gyrA
mutations that decrease the activity of fluoroquinolones is the use of 30-μg nalidixic acid disks.
Chitosan Acetate Bandage as a Topical Antimicrobial Dressing for Infected Burns
Authors: Dai Tianhong , Tegos George P. , Burkatovskaya Marina , Castano Ana P. , Hamblin Michael R.
Published: November 2008

An engineered chitosan acetate bandage preparation (HemCon) is used as a hemostatic dressing, and its chemical structure suggests that it should also be antimicrobial. We previously showed that when a chitosan acetate bandage was applied to full-thickness excisional wounds in mice that had been infected with pathogenic bioluminescent bacteria (Pseudomonas aeruginosa, Proteus mirabilis, andStaphylococcus aureus), it was able to rapidly kill the bacteria and save the mice from developing fatal infections. Wound healing was also stimulated. In the present study, we asked whether a chitosan acetate bandage could act as a topical antimicrobial dressing when it was applied to third-degree burns in mice contaminated with two of these bacterial species (P. aeruginosaandP. mirabilis). Preliminary experiments established the length of burn time and the number of bacteria needed to produce fatal infections in untreated mice and established that the chitosan acetate bandage could adhere to the infected burn for up to 21 days. In the case ofP. aeruginosainfections, the survival rate of mice treated with the chitosan acetate bandage was 73.3% (whereas the survival rate of mice treated with a nanocrystalline silver dressing was 27.3% [P= 0.0055] and that of untreated mice was 13.3% [P< 0.0002]). ForP. mirabilisinfections, the comparable survival rates were 66.7%, 62.5%, and 23.1% respectively. Quantitative bioluminescent signals showed that the chitosan acetate bandage effectively controlled the growth of bacteria in the burn and prevented the development of systemic sepsis, as shown by blood culture. These data suggest that chitosan acetate bandage is efficacious in preventing fatal burn infections.
Drug-Drug Interaction Study of Ketoconazole and Ritonavir-Boosted Saquinavir
Authors: Kaeser Benoite , Zandt Hagen , Bour Fabrice , Zwanziger Elke , Schmitt Christophe , Zhang Xiaoping
Published: November 2008

Saquinavir, a potent human immunodeficiency virus protease inhibitor, is extensively metabolized by CYP3A4. Saquinavir is coadministered with ritonavir, a strong CYP3A4 inhibitor, to boost its exposure. Ketoconazole is a potent CYP3A inhibitor. The objectives of this study were to investigate the effect of ketoconazole on the pharmacokinetics of saquinavir/ritonavir and vice versa using the approved dosage regimens of saquinavir/ritonavir at 1,000/100 mg twice daily and ketoconazole at 200 mg once daily. This was an open-label, randomized two-arm, one-sequence, two-period crossover study in healthy subjects. In study arm 1, 20 subjects received saquinavir/ritonavir treatment alone for 14 days, followed in combination with ketoconazole treatment for 14 days. In arm 2, 12 subjects received ketoconazole treatment for 6 days, followed in combination with saquinavir/ritonavir treatment for 14 days. The pharmacokinetics were assessed on the last day of each treatment (days 14 and 28 in arm 1 and days 6 and 20 in arm 2). The exposures
C
max
and the area under the concentration-time curve from 0 to 12 h (AUC
0-12
) of saquinavir and ritonavir with or without ketoconazole were not substantially altered after 2 weeks of concomitant dosing with ketoconazole. The
C
max
and AUC
0-12
of ketoconazole, dosed at 200 mg once daily, were increased by 45% (90% confidence interval = 32 to 59%) and 168% (90% confidence interval = 146 to 193%), respectively, after 2 weeks of concomitant dosing with ritonavir-boosted saquinavir (1,000 mg of saquinavir/100 mg of ritonavir given twice daily). The greater exposure to ketoconazole when given in combination with saquinavir/ritonavir was not associated with unacceptable safety or tolerability. No dose adjustment for saquinavir/ritonavir (1,000/100 mg twice daily) is required when coadministered with 200 mg of ketoconazole once daily, and high doses of ketoconazole (>200 mg/day) are not recommended.
Giardia
,
Entamoeba
, and
Trichomonas
Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases)
Authors: Pal Dibyarupa , Banerjee Sulagna , Cui Jike , Schwartz Aaron , Ghosh Sudip K. , Samuelson John
Published: November 2008

Infections with
Giardia lamblia
,
Entamoeba histolytica
, and
Trichomonas vaginalis
, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that
Giardia
,
Entamoeba
, and
Trichomonas
have oxygen-insensitive nitroreductase (
ntr
) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant
Helicobacter pylori
isolates.
Entamoeba
and
Trichomonas
also have
nim
genes which are homologous to those genes expressed in metronidazole-resistant
Bacteroides fragilis
isolates. Recombinant
Giardia
,
Entamoeba
, and
Trichomonas
nitroreductases used NADH rather than the NADPH used by
Helicobacter
, and two recombinant
Entamoeba
nitroreductases increased the metronidazole sensitivity of transformed
Escherichia coli
strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of
Entamoeba
and
Trichmonas
conferred very strong metronidazole resistance to transformed bacteria. The Eh
ntr1
gene of the genome project HM-1:IMSS strain of
Entamoeba histolytica
had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of
Entamoeba
. While
ntr
and
nim
mRNAs were variably expressed by cultured
Entamoeba
and
Trichomonas
isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While
Entamoeba
and
Trichomonas
displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.
A 6-Aminoquinolone Compound, WC5, with Potent and Selective Anti-Human Cytomegalovirus Activity
Authors: Mercorelli Beatrice , Muratore Giulia , Sinigalia Elisa , Tabarrini Oriana , Biasolo Maria Angela , Cecchetti Violetta , Palù Giorgio , Loregian Arianna
Published: November 2008

We identified a 6-aminoquinolone compound, WC5, that inhibits human cytomegalovirus (HCMV) replication with a selectivity index of ∼500. WC5 also showed activity against drug-resistant HCMV strains. In contrast, it did not significantly affect the replication of human herpesvirus 6 and 8 and was ∼10-fold less active against murine cytomegalovirus. Thus, WC5 may represent a lead for the development of new, potent, and selective anti-HCMV compounds.
Abc1p Is a Multidrug Efflux Transporter That Tips the Balance in Favor of Innate Azole Resistance in
Candida krusei
Authors: Lamping Erwin , Ranchod Amrita , Nakamura Kenjirou , Tyndall Joel D. A. , Niimi Kyoko , Holmes Ann R. , Niimi Masakazu , Cannon Richard D.
Published: November 2008

Most
Candida krusei
strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of
C. krusei
has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate
ERG11
and
ABC1
expression in
C. krusei
. Examination of the
ERG11
promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct
ERG11
alleles in one of two
C. krusei
strains. Functional overexpression of
ERG11
and
ABC1
in
Saccharomyces cerevisiae
conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized
C. krusei
to azole antifungals. Our data show that despite the presence of multiple alleles of
ERG11
in some, likely aneuploid,
C. krusei
strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of
C. krusei
.
Differential Effects of Inhibiting Chitin and 1,3-β-
d
-Glucan Synthesis in Ras and Calcineurin Mutants of
Aspergillus fumigatus
Authors: Fortwendel Jarrod R. , Juvvadi Praveen Rao , Pinchai Nadthanan , Perfect B. Zachary , Alspaugh J. Andrew , Perfect John R. , Steinbach William J.
Published: November 2008

Aspergillus fumigatus
must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools. Strains deleted for three genes encoding proteins of these pathways,
rasA
(the Ras protein),
cnaA
(calcineurin), or
crzA
(the zinc finger transcription factor downstream of calcineurin), all displayed decreased cell wall 1,3-β-
d
-glucan content. Echinocandin treatment further decreased the levels of 1,3-β-
d
-glucan for all strains tested yet also partially corrected the hyphal growth defect of the Δ
rasA
strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active
rasA
, and Δ
rasA
strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (Δ
cnaA
and Δ
crzA
). Taken together, our data suggest that the Ras and calcineurin pathways act in parallel to regulate cell wall formation and hyphal growth. Additionally, the calcineurin pathway elements
cnaA
and
crzA
play a major role in proper chitin and glucan incorporation into the
A. fumigatus
cell wall.
Modeling the Growth/No-Growth Boundaries of Postprocessing
Listeria monocytogenes
Contamination on Frankfurters and Bologna Treated with Lactic Acid
Authors: Yoon Yohan , Kendall Patricia A. , Belk Keith E. , Scanga John A. , Smith Gary C. , Sofos John N.
Published: November 2008

This study developed models to predict lactic acid concentration, dipping time, and storage temperature combinations determining growth/no-growth interfaces of
Listeria monocytogenes
at desired probabilities on bologna and frankfurters.
L
.
monocytogenes
was inoculated on bologna and frankfurters, and 75 combinations of lactic acid concentrations, dipping times, and storage temperatures were tested. Samples were stored in vacuum packages for up to 60 days, and bacterial populations were enumerated on tryptic soy agar plus 0.6% yeast extract and Palcam agar on day zero and at the end point of storage. The combinations that allowed
L
.
monocytogenes
increases of ≥1 log CFU/cm
2
were assigned the value of 1 (growth), and the combinations that had increases of <l log CFU/cm
2
were given the value of 0 (no growth). These binary growth response data were fitted to logistic regression to develop a model predicting probabilities of growth. Validation with existing data and various indices showed acceptable model performance. Thus, the models developed in this study may be useful in determining probabilities of growth and in selecting lactic acid concentrations and dipping times to control
L
.
monocytogenes
growth on bologna and frankfurters, while the procedures followed may also be used to develop models for other products, conditions, or pathogens.
Coxiella burnetii
Shedding Routes and Antibody Response after Outbreaks of Q Fever-Induced Abortion in Dairy Goat Herds
Authors: Rousset Elodie , Berri Mustapha , Durand Benoit , Dufour Philippe , Prigent Myriam , Delcroix Thibault , Touratier Anne , Rodolakis Annie
Published: November 2008

Q fever is a zoonosis caused by
Coxiella burnetii
, a bacterium largely carried by ruminants and shed into milk, vaginal mucus, and feces. The main potential hazard to humans and animals is due to shedding of bacteria that can then persist in the environment and be aerosolized. The purpose of this study was to evaluate shedding after an outbreak of Q fever abortion in goat herds and to assess the relationship with the occurrence of abortions and antibody responses. Aborting and nonaborting goats were monitored by PCR for
C. burnetii
shedding 15 and 30 days after the abortion episodes. PCR analysis of all samples showed that 70% (
n
= 50) of the aborting and 53% (
n
= 70) of the nonaborting goats were positive.
C. burnetii
was shed into vaginal mucus, feces, and milk of 44%, 21%, and 38%, respectively, of goats that aborted and 27%, 20%, and 31%, respectively, of goats that delivered normally. Statistical comparison of these shedding results did not reveal any difference between these two groups. PCR results obtained for the vaginal and fecal routes were concordant in 81% of cases, whereas those for milk correlated with only 49% of cases with either vaginal or fecal shedding status. Serological analysis, using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and complement fixation tests, showed that at least 24% of the seronegative goats shed bacteria. Positive vaginal and fecal shedding, unlike positive milk shedding, was observed more often in animals that were weakly positive or negative by ELISA or IFA. Two opposite shedding trends were thus apparent for the milk and vaginal-fecal routes. Moreover, this study showed that a nonnegligible proportion of seronegative animals that delivered normally could excrete
C. burnetii
.
Potential Application ofN-Carbamoyl-β-Alanine Amidohydrolase fromAgrobacterium tumefaciensC58 for β-Amino Acid Production
Authors: Martínez-Gómez Ana Isabel , Martínez-Rodríguez Sergio , Pozo-Dengra Joaquín , Tessaro Davide , Servi Stefano , Clemente-Jiménez Josefa María , Rodríguez-Vico Felipe , Las Heras-Vázquez Francisco Javier
Published: November 2008

AnN-carbamoyl-β-alanine amidohydrolase of industrial interest fromAgrobacterium tumefaciensC58 (βcarAt) has been characterized. βcarAtis most active at 30°C and pH 8.0 withN-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn2+, Ni2+, and Co2+. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstitutedN-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency forN-carbamoyl-β-alanine. βcarAtalso recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcarAtis able to produce monosubstituted β2- and β3-amino acids, showing better catalytic efficiency (kcat/Km) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzesN-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcarAtan outstanding candidate for application in the biotechnology industry.
Genotypic and Phenotypic Diversity among Induced,
stx
2
-Carrying Bacteriophages from Environmental
Escherichia coli
Strains
Authors: García-Aljaro Cristina , Muniesa Maite , Jofre Juan , Blanch Anicet R.
Published: November 2008

Shiga toxin 2 (
stx
2
) gene-carrying bacteriophages have been shown to convert
Escherichia coli
strains to Shiga toxin-producing
Escherichia coli
(STEC). In this study, 79
E. coli
strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of

stx
2

-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected
Shigella sonnei
host strain 866. In summary, in spite of carrying either the same or different
stx
2
variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the
stx
2
gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.
Evidence from Internally Transcribed Spacer Sequence Analysis of Soybean Strains that ExtantBradyrhizobiumspp. Are Likely the Products of Reticulate Evolutionary Events
Authors: van Berkum Peter , Fuhrmann Jeffry J.
Published: November 2008

The internally transcribed spacer (ITS) sequences of several members within each of 17 soybean bradyrhizobial serogroups were determined to establish whether the regions within all members of each serogroup were identical. The rationale was to provide a sequence-based alternative to serology. The objective also was to link the extensive older literature on soybean symbiosis based on serology with ITS sequence data for more recent isolates from both soybean and other legumes nodulated by rhizobia within the genusBradyrhizobium. With the exception of serogroup 31 and 110 strains, sequence identity was established within each serogroup. Variation ranged from 0 to 23 nucleotides among serogroup 31 strains, and the regions in the type strains USDA 31 (serogroup 31) and USDA 130 (serogroup 130) were identical. Sequence identity was established among most strains within serogroup 110. The exceptions were USDA 452 and USDA 456, which had ITS sequences that were identical with those of the serotype 124 strain, USDA 124. Perhaps this would imply that USDA 452, USDA 456, and serogroup 31 strains are members of rhizobial lineages resulting from genetic exchange and homologous recombination events. This conclusion would be supported by the construction of a phylogenetic network from the ITS sequence alignment implying that the genomes of extant members of the genusBradyrhizobiumare likely the products of reticulate evolutionary events. A pairwise homoplasy index (phi or Φw) test was used to obtain further evidence for recombination. The ITS sequences of USDA 110 and USDA 124 were more divergent (53 nucleotides) than this region between the type strainBradyrhizobium japonicumUSDA 6Tand the proposed speciesBradyrhizobium yuanmingense(28 nucleotides) andBradyrhizobium liaoningense(48 nucleotides). Therefore, support for assigning discrete species boundaries among these three proposed species appears limited, considering the evidence for recombination, the narrow divergence of the ITS sequence, and their relative placement on the phylogenetic network.
In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by
Bifidobacterium
Species Reveals Four Different Phenotypes
Authors: Falony Gwen , Lazidou Katerina , Verschaeren An , Weckx Stefan , Maes Dominique , De Vuyst Luc
Published: November 2008

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18
Bifidobacterium
strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a β-fructofuranosidase gene previously encountered in
Bifidobacterium animalis
subsp.
lactis
DSM 10140
T
, revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the β-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.
Efficient Production of Optically Pure
d
-Lactic Acid from Raw Corn Starch by Using a Genetically Modified
l
-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting
Lactobacillus plantarum
Strain
Authors: Okano Kenji , Zhang Qiao , Shinkawa Satoru , Yoshida Shogo , Tanaka Tsutomu , Fukuda Hideki , Kondo Akihiko
Published: November 2008

In order to achieve direct and efficient fermentation of optically pure
d
-lactic acid from raw corn starch, we constructed
l
-lactate dehydrogenase gene (
ldhL1
)-deficient
Lactobacillus plantarum
and introduced a plasmid encoding
Streptococcus bovis
148 α-amylase (AmyA). The resulting strain produced only
d
-lactic acid from glucose and successfully expressed
amyA
. With the aid of secreting AmyA, direct
d
-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the
ldhL1
gene with an
amyA
-secreting expression cassette was constructed. Using this strain, direct
d
-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct
d
-lactic acid fermentation from raw starch.
Virulent Bacteriophage for Efficient Biocontrol of
Listeria monocytogenes
in Ready-To-Eat Foods
Authors: Guenther Susanne , Huwyler Dominique , Richard Simon , Loessner Martin J.
Published: November 2008

Food-borne
Listeria monocytogenes
is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of
L. monocytogenes
strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 10
3
CFU/g), phage added thereafter (3 × 10
6
to 3 × 10
8
PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 10
8
PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log
10
. In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of
L. monocytogenes
in contamination-sensitive RTE foods.
Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length, Degree of Coverage, and DNA Fractionation on Classification and Ecological Parameters
Authors: Morales Sergio E. , Cosart Theodore F. , Johnson Jesse V. , Holben William E.
Published: November 2008

To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.
In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6
Authors: Koch Daniel J. , Chen Mike M. , van Beilen Jan B. , Arnold Frances H.
Published: November 2008

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from
Pseudomonas putida
GPo1 and the class II-type soluble CYP153A6 from
Mycobacterium
sp. strain HXN-1500, to enhance their activity on small alkanes. We established a
P. putida
evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted
P. putida
GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.
In Silico
Geobacter sulfurreducens
Metabolism and Its Representation in Reactive Transport Models
Authors: King E. L. , Tuncay K. , Ortoleva P. , Meile C.
Published: November 2008

Microbial activity governs elemental cycling and the transformation of many anthropogenic substances in aqueous environments. Through the development of a dynamic cell model of the well-characterized, versatile, and abundant
Geobacter sulfurreducens
, we showed that a kinetic representation of key components of cell metabolism matched microbial growth dynamics observed in chemostat experiments under various environmental conditions and led to results similar to those from a comprehensive flux balance model. Coupling the kinetic cell model to its environment by expressing substrate uptake rates depending on intra- and extracellular substrate concentrations, two-dimensional reactive transport simulations of an aquifer were performed. They illustrated that a proper representation of growth efficiency as a function of substrate availability is a determining factor for the spatial distribution of microbial populations in a porous medium. It was shown that simplified model representations of microbial dynamics in the subsurface that only depended on extracellular conditions could be derived by properly parameterizing emerging properties of the kinetic cell model.
Autoinducer-2 Production in
Campylobacter jejuni
Contributes to Chicken Colonization
Authors: Quiñones Beatriz , Miller William G. , Bates Anna H. , Mandrell Robert E.
Published: November 2008

Inactivation of
luxS
, encoding an AI-2 biosynthesis enzyme, in
Campylobacter jejuni
strain 81-176 significantly reduced colonization of the chick lower gastrointestinal tract, chemotaxis toward organic acids, and in vitro adherence to LMH chicken hepatoma cells. Thus, AI-2 production in
C. jejuni
contributes to host colonization and interactions with epithelial cells.
Detection of Differential Host Susceptibility to the Marine Oomycete Pathogen
Eurychasma dicksonii
by Real-Time PCR: Not All Algae Are Equal
Authors: Gachon Claire M. M. , Strittmatter Martina , Müller Dieter G. , Kleinteich Julia , Küpper Frithjof C.
Published: November 2008

In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen
Eurychasma dicksonii
in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against
Eurychasma
, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of
Eurychasma
in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed
Ectocarpus siliculosus
to improve our understanding of brown algal diseases.
Helix α4 of the
Bacillus thuringiensis
Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation
Authors: Girard Frédéric , Vachon Vincent , Préfontaine Gabrielle , Marceau Lucie , Schwartz Jean-Louis , Masson Luke , Laprade Raynald
Published: November 2008

Helix α4 of
Bacillus thuringiensis
Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from
Manduca sexta
was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.
In Vitro Activity of
Acanthamoeba castellanii
on Human Platelets and Erythrocytes
Authors: Mattana A. , Alberti L. , Delogu G. , Fiori P. L. , Cappuccinelli P.
Published: November 2008

The effect of
Acanthamoeba
on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from
A. castellanii
can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that
A. castellanii
can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by
A. castellanii
trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during
Acanthamoeba
infection.
Klebsiella pneumoniaeIncreases the Levels of Toll-Like Receptors 2 and 4 in Human Airway Epithelial Cells
Authors: Regueiro Verónica , Moranta David , Campos Miguel A. , Margareto Javier , Garmendia Junkal , Bengoechea José A.
Published: November 2008

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whetherKlebsiella pneumoniaeupregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection withK. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively.Klebsiella-dependent upregulation of TLR expression occurred via a positive IκBα-dependent NF-κΒ pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed thatKlebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.
Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-StagePlasmodium chabaudiAS Infection in Mice
Authors: Ing Rebecca , Stevenson Mary M.
Published: November 2008

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response toPlasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity toP. chabaudiAS infection. During the first week of infection, the uptake of iRBC by splenic CD11c+DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15−/−(IL-15−/−) and IL-12p40−/−mice, which differ in the severity ofP. chabaudiAS infection. DCs from infected IL-15−/−mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40−/−mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4+T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4+Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution ofP. chabaudiAS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.