bluuebunny/crossref_metadata_2025 huggingface dataset:
Constructing Elementary Teachers' Beliefs, Attitudes, and Practical Knowledge Through an Inquiry‐Based Elementary Science Course
Authors: Choi Sanghee , Ramsey John
Published: March 2010

This study examines inservice elementary school teachers' beliefs, attitudes, and practical knowledge toward inquiry‐based science instruction and the influence of an inquiry‐based elementary science course on teachers' beliefs, attitudes, and practical knowledge regarding inquiry. Both surveys and a case study were administered to the 14 elementary school teachers before and after completing a three‐credit elementary science methods course that was inquiry‐based. The findings showed that the teachers' beliefs, attitudes, and practical knowledge about inquiry were clearly influenced by the course. Through this course, the teachers developed fairly positive beliefs and attitudes that promoted inquiry instruction. The majority of participants also improved their knowledge and skills of conducting inquiry as they successfully practiced inquiry‐instruction in their science teachings.
Computational Estimation Performance on Whole‐Number Multiplication by Third‐ and Fifth‐Grade Chinese Students
Authors: Liu Fuchang
Published: March 2010

Four hundred and three 3rd‐ and 5th‐grade Chinese students took the Multiplication Estimation Test or participated in the interview on it, designed to assess their computational estimation performance on whole‐number multiplication. Students perform better when tasks are presented visually than orally. Third graders tend to use rounding based while fifth graders tend to use written algorithm based strategies, but boys' and girls ‘performances do not differ. It is concluded that students often will not estimate simply at the request to estimate if an exact answer is within their mental computation capability, and a two‐step process is suggested for helping students decide what route to take when given arithmetic problems.
Interesting Science and Mathematics Graduate Students in Secondary Teaching
Authors: Latterell Carmen M.
Published: March 2010

State and national initiatives attempt to increase the quantity and quality of secondary mathematics and science teachers. Research suggests that if one could appeal to something inside of people or about the process of teaching and learning itself, then one might draw current mathematics and science graduate students into secondary teaching. This study placed eight mathematics and science graduate students in secondary schools for ten hours a week. Pre‐ and post‐measures of their interest level in becoming secondary teachers were made. Overall, graduate students decreased in their desire to become secondary teachers. The main reasons were (1) fellows wanted to work with higher‐level mathematics and science; (2) fellows felt students were not behaved and unmotivated; (3) fellows did not view being a teacher as a career, but only as a job; and (4) fellows felt school systems had to do too many things that fellows did not want to do.
A Cross Discipline Study of Reformed Teaching by University Science and Mathematics Faculty
Authors: Flick Lawrence B. , Sadri Pejmon , Morrell Patricia D. , Wainwright Camille , Schepige Adele
Published: March 2010

Researchers observed 28 university faculty in graduate and undergraduate science and mathematics courses in a three‐year, longitudinal study of teaching. Subjects were selected from five higher education institutions in the state where faculty had a two‐ to five‐year association with a reform‐based NSF professional development program. Observer field notes and a researcher‐designed observation instrument were used to describe and compare teaching practices. The participants in this study taught science and mathematics content courses for majors, non‐majors, as well as education majors. Our observation method created a descriptive profile that enabled comparisons across a variety of teaching contexts. The main results showed that the faculty lacked practices that supported development of divergent thinking, conceptual thinking, and metacognitive thinking skills. Observed differences in the profiles of instruction included greater emphasis on real‐world connections in science and greater use of cooperative problem solving in mathematics. A case study of a large lecture course describes reformed teaching practices in even the most challenging selling.
Crossing the Barriers Between Preservice and Inservice Mathematics Teacher Education: An Evaluation of the Grant School Professional Development Program
Authors: Bahr Damon , Monroe Eula Ewing , Balzotti Mark , Eggett Dennis
Published: March 2010

A 2‐year school‐based mathematics professional development program is described and evaluated after its first year of implementation. Included in this program as its first course was a unique methods course in elementary education involving both preservice students and inservice teachers who cooperatively studied and applied reform pedagogy. The program resulted from the collaborative efforts of two institutions of higher education, a neighboring school district, the principal and teachers of one school within that district, and the state office of education. Evaluation of the first year of the program consisted of assessing the beliefs and perceptions of both preservice students and inservice teachers, along with an assessment of the mathematical achievement of the children within the classes of those teachers. Pre‐ and post‐assessments of the preservice students and inservice teachers' beliefs regarding reform pedagogy were administered using the IMAP [Integrating Mathematics and Pedagogy] Web‐Based Beliefs Survey (2006). Likert scale surveys were used to assess perceptions regarding course climate and participant relationships from both teacher groups. The mathematical achievement of children was assessed in three ways: The Wide Range Achievement Test‐3 (Stone, Jastak, & Wilkinson, 1995), the Utah state criterion‐referenced assessment, and performance assessments developed specifically for use at the school. Data obtained from all sources indicated positive effects upon teachers and children, thus providing substantial evidence in support of both the value of the methods course itself and the overall professional development program. An additional evaluation will be conducted following the second year of the program.
Therapeutic strategies for targeting excessive central sympathetic activation in human hypertension
Authors: Fisher James P. , Fadel Paul J.
Published: March 2010

The pathogenesis of hypertension and its mode of progression are complex, multifactoral and incompletely understood. However, there is accumulating evidence from humans and animal models of hypertension indicating that excessive central sympathetic nerve activity (SNA) plays a pathogenic role in triggering and sustaining the essential hypertensive state (the so‐called ‘neuroadrenergic hypothesis’). Importantly, augmented central sympathetic outflow has also been implicated in the initiation and progression of a plethora of pathophysiological processes independent of any increase in blood pressure, such as left ventricular hypertrophy and cardiac arrhythmias. Thus, the sympathetic nervous system constitutes an important putative drug target in hypertension. However, traditional pharmacological approaches for the management of essential hypertension appear ineffective in reducing central sympathetic outflow. Recently, several new and promising therapeutic strategies targeting neurogenic hypertension have been developed. The present report will provide a brief update of this topic with a particular emphasis on human studies examining the efficacy of novel pharmacological approaches (central sympatholytics and statins), lifestyle modification (aerobic exercise training, weight loss and stress reduction) and surgical intervention (renal denervation, chronic carotid baroreflex stimulation and deep brain stimulation) in reducing excessive central sympathetic activation in hypertension.
Sequences Essential for Transmission of
Spiroplasma citri
by Its Leafhopper Vector,
Circulifer haematoceps
, Revealed by Plasmid Curing and Replacement Based on Incompatibility
Authors: Breton Marc , Duret Sybille , Danet Jean-Luc , Dubrana Marie-Pierre , Renaudin Joël
Published: March 2010

Spiroplasma citri
GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as
S. citri
adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed
S. citri
mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these
S. citri
plasmid mutants through injection into the leafhopper vector
Circulifer haematoceps
revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though
S. citri
mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.
Application of Molecular Techniques To Elucidate the Influence of Cellulosic Waste on the Bacterial Community Structure at a Simulated Low-Level-Radioactive-Waste Site
Authors: Field Erin K. , D'Imperio Seth , Miller Amber R. , VanEngelen Michael R. , Gerlach Robin , Lee Brady D. , Apel William A. , Peyton Brent M.
Published: March 2010

Low-level-radioactive-waste (low-level-waste) sites, including those at various U.S. Department of Energy sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a nonradioactive model low-level-waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more operational taxonomic units, and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the fill and fill-waste interface (FW) layers and greater in the wood waste and waste-clay interface layers. Principal-coordinate analysis and lineage-specific analysis determined that the
Bacteroidetes
and
Actinobacteria
phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose-degrading microorganisms suggest that the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.
Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine
Authors: Page Martin A. , Shisler Joanna L. , Mariñas Benito J.
Published: March 2010

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins
in vitro
. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.
Abundance and Expression of Enantioselective
rdpA
and
sdpA
Dioxygenase Genes during Degradation of the Racemic Herbicide (
R
,
S
)-2-(2,4-Dichlorophenoxy)Propionate in Soil
Authors: Paulin Mélanie M. , Nicolaisen Mette H. , Sørensen Jan
Published: March 2010

The
rdpA
and
sdpA
genes encode two enantioselective α-ketoglutarate-dependent dioxygenases catalyzing the initial step of microbial degradation of the chiral herbicide (
R
,
S
)-2-(2,4-dichlorophenoxy)propionate (
R
,
S
-dichlorprop). Primers were designed to assess abundance and transcription dynamics of
rdpA
and
sdpA
genes in a natural agricultural soil. No indigenous
rdpA
genes were detected, but
sdpA
genes were present at levels of approximately 10
3
copies g of soil
−1
. Cloning and sequencing of partial
sdpA
genes revealed a high diversity within the natural
sdpA
gene pool that could be divided into four clusters by phylogenetic analysis. BLASTp analysis of deduced amino acids revealed that members of cluster I shared 68 to 69% identity, cluster II shared 78 to 85% identity, cluster III shared 58 to 64% identity, and cluster IV shared 55% identity to their closest SdpA relative in GenBank. Expression of
rdpA
and
sdpA
in
Delftia acidovorans
MC1 inoculated in soil was monitored by reverse transcription quantitative real-time PCR (qPCR) during
in situ
degradation of 2 and 50 mg kg
−1
of (
R
,
S
)-dichlorprop. (
R
,
S
)-Dichlorprop amendment created a clear upregulation of both
rdpA
and
sdpA
gene expression during the active phase of
14
C-labeled (
R
,
S
)-dichlorprop mineralization, particularly following the second dose of 50 mg kg
−1
herbicide. Expression of both genes was maintained at a low constitutive level in nonamended soil microcosms. This study is the first to report the presence of indigenous
sdpA
genes recovered directly from natural soil and also comprises the first investigation into the transcription dynamics of two enantioselective dioxygenase genes during the
in situ
degradation of the herbicide (
R
,
S
)-dichlorprop in soil.
Systematic Evaluation of the Efficacy of Chlorine Dioxide in Decontamination of Building Interior Surfaces Contaminated with Anthrax Spores
Authors: Rastogi Vipin K. , Ryan Shawn P. , Wallace Lalena , Smith Lisa S. , Shah Saumil S. , Martin G. Blair
Published: March 2010

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of
Bacillus anthracis
spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of
B
acillus atrophaeus
on stainless steel) was significantly less than that required for decontamination of most of the building materials tested.
Role of Antioxidant Enzymes in Bacterial Resistance to Organic Acids
Authors: Bruno-Bárcena Jose M. , Azcárate-Peril M. Andrea , Hassan Hosni M.
Published: March 2010

Growth in aerobic environments has been shown to generate reactive oxygen species (ROS) and to cause oxidative stress in most organisms. Antioxidant enzymes (i.e., superoxide dismutases and hydroperoxidases) and DNA repair mechanisms provide protection against ROS. Acid stress has been shown to be associated with the induction of Mn superoxide dismutase (MnSOD) inLactococcus lactisandStaphylococcus aureus. However, the relationship between acid stress and oxidative stress is not well understood. In the present study, we showed that mutations in the gene coding for MnSOD (sodA) increased the toxicity of lactic acid at pH 3.5 inStreptococcus thermophilus. The inclusion of the iron chelators 2,2′-dipyridyl (DIP), diethienetriamine-pentaacetic acid (DTPA), andO-phenanthroline (O-Phe) provided partial protection against 330 mM lactic acid at pH 3.5. The results suggested that acid stress triggers an iron-mediated oxidative stress that can be ameliorated by MnSOD and iron chelators. These findings were further validated inEscherichia colistrains lacking both MnSOD and iron SOD (FeSOD) but expressing a heterologous MnSOD fromS. thermophilus. We also found that, inE. coli, FeSOD did not provide the same protection afforded by MnSOD and that hydroperoxidases are equally important in protecting the cells against acid stress. These findings may explain the ability of some microorganisms to survive better in acidified environments, as in acid foods, during fermentation and accumulation of lactic acid or during passage through the low pH of the stomach.
Characterization of Triclosan-Resistant Mutants Reveals Multiple Antimicrobial Resistance Mechanisms in
Rhodospirillum rubrum
S1H
Authors: Pycke Benny F. G. , Crabbé Aurélie , Verstraete Willy , Leys Natalie
Published: March 2010

Antimicrobial resistance mechanisms were identified in 11 spontaneous high- and low-level triclosan resistance (Tcs
r
) mutants of
Rhodospirillum rubrum
S1H by genotyping complemented with transcriptional analyses, antibiotic resistance screening, and membrane permeability analyses. High-end Tcs
r
(MIC = 8 mg/liter) was the result of a FabI1(G98V) mutation. This point mutation led to an even higher level of Tcs
r
(MIC ≥ 16 mg/liter) in combination with constitutive upregulation of
mexB
and
mexF
efflux pump homologs. Hence, a mechanistic synergy of constitutive efflux pump expression and a FabI1 point mutation could prevent TCS-induced cell permeabilization, which was shown to occur between 4 and 8 mg/liter TCS in the
R. rubrum
S1H parent strain. Low-level Tcs
r
mutants constitutively upregulated the
emrAB
,
mexAB
, and/or
mexF
homolog. The mutants that overexpressed
emrAB
also derepressed the micropollutant-upregulated factors
mufA1
and
mufM
. In some cases, low-level Tcs
r
decreased innate resistance to ampicillin and tetracycline, while in others, a triclosan-induced antibiotic cross-resistance was shown for chloramphenicol and carbenicillin. This study showed that the TCS resistance degree is dependent of the initial exposure concentration in
Rhodospirillum rubrum
S1H and that similar resistance degrees can be the result of different defense mechanisms, which all have distinct antibiotic cross-resistance profiles.
Denitrifying Bacteria Isolated from Terrestrial Subsurface Sediments Exposed to Mixed-Waste Contamination
Authors: Green Stefan J. , Prakash Om , Gihring Thomas M. , Akob Denise M. , Jasrotia Puja , Jardine Philip M. , Watson David B. , Brown Steven D. , Palumbo Anthony V. , Kostka Joel E.
Published: March 2010

In terrestrial subsurface environments where nitrate is a critical groundwater contaminant, few cultivated representatives are available to verify the metabolism of organisms that catalyze denitrification. In this study, five species of denitrifying bacteria from three phyla were isolated from subsurface sediments exposed to metal radionuclide and nitrate contamination as part of the U.S. Department of Energy's Oak Ridge Integrated Field Research Challenge (OR-IFRC). Isolates belonged to the genera
Afipia
and
Hyphomicrobium
(
Alphaproteobacteria
),
Rhodanobacter
(
Gammaproteobacteria
),
Intrasporangium
(
Actinobacteria
), and
Bacillus
(
Firmicutes
). Isolates from the phylum
Proteobacteria
were complete denitrifiers, whereas the Gram-positive isolates reduced nitrate to nitrous oxide. rRNA gene analyses coupled with physiological and genomic analyses suggest that bacteria from the genus
Rhodanobacter
are a diverse population of denitrifiers that are circumneutral to moderately acidophilic, with a high relative abundance in areas of the acidic source zone at the OR-IFRC site. Based on genome analysis,
Rhodanobacter
species contain two nitrite reductase genes and have not been detected in functional-gene surveys of denitrifying bacteria at the OR-IFRC site. Nitrite and nitrous oxide reductase gene sequences were recovered from the isolates and from the terrestrial subsurface by designing primer sets mined from genomic and metagenomic data and from draft genomes of two of the isolates. We demonstrate that a combination of cultivation and genomic and metagenomic data is essential to the
in situ
characterization of denitrifiers and that current PCR-based approaches are not suitable for deep coverage of denitrifiers. Our results indicate that the diversity of denitrifiers is significantly underestimated in the terrestrial subsurface.
A Plasmodium falciparum Transcriptional Cyclin-Dependent Kinase-Related Kinase with a Crucial Role in Parasite Proliferation Associates with Histone Deacetylase Activity
Authors: Halbert Jean , Ayong Lawrence , Equinet Leila , Le Roch Karine , Hardy Mary , Goldring Dean , Reininger Luc , Waters Norman , Chakrabarti Debopam , Doerig Christian
Published: March 2010

Cyclin-dependent protein kinases (CDKs) are key regulators of the eukaryotic cell cycle and of the eukaryotic transcription machinery. Here we report the characterization of Pfcrk-3 (
Plasmodium falciparum
CDK-related kinase 3; PlasmoDB identifier PFD0740w), an unusually large CDK-related protein whose kinase domain displays maximal homology to those CDKs which, in other eukaryotes, are involved in the control of transcription. The closest enzyme in
Saccharomyces cerevisiae
is BUR1 (
b
ypass
u
pstream activating sequence
r
equirement 1), known to control gene expression through interaction with chromatin modification enzymes. Consistent with this, immunofluorescence data show that Pfcrk-3 colocalizes with histones. We show that recombinant Pfcrk-3 associates with histone H1 kinase activity in parasite extracts and that this association is detectable even if the catalytic domain of Pfcrk-3 is rendered inactive by site-directed mutagenesis, indicating that Pfcrk-3 is part of a complex that includes other protein kinases. Immunoprecipitates obtained from extracts of transgenic parasites expressing hemagglutinin (HA)-tagged Pfcrk-3 by using an anti-HA antibody displayed both protein kinase and histone deacetylase activities. Reverse genetics data show that the
pfcrk-3
locus can be targeted only if the genetic modification does not cause a loss of function. Taken together, our data strongly suggest that Pfcrk-3 fulfils a crucial role in the intraerythrocytic development of
P. falciparum
, presumably through chromatin modification-dependent regulation of gene expression.
Abundant 5S rRNA-Like Transcripts Encoded by the Mitochondrial Genome in Amoebozoa
Authors: Bullerwell Charles E. , Burger Gertraud , Gott Jonatha M. , Kourennaia Olga , Schnare Murray N. , Gray Michael W.
Published: March 2010

5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in
Acanthamoeba castellanii
, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba
Hartmannella vermiformis
and in the myxomycete slime mold
Physarum polycephalum
. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold,
Didymium nigripes
, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the
Physarum
small RNA. A mtDNA-encoded small RNA previously identified in
Dictyostelium discoideum
is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus
Dictyostelium
as well as in a related genus,
Polysphondylium
. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.
Fungal Homoserine Kinase (
thr1
Δ) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation
Authors: Kingsbury Joanne M. , McCusker John H.
Published: March 2010

The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in
Cryptococcus neoformans
, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of
Saccharomyces cerevisiae
homoserine kinase (
thr1
Δ) and threonine synthase (
thr4
Δ) mutants. In striking contrast to aspartate kinase (
hom3
Δ) mutants,
S. cerevisiae
thr1
Δ and
thr4
Δ mutants were severely depleted after only 4 h
in vivo
. Similarly,
Candida albicans
thr1
Δ mutants, but not
hom3
Δ mutants, were significantly attenuated in virulence. Consistent with the
in vivo
phenotypes,
S. cerevisiae
thr1
Δ and
thr4
Δ mutants as well as
C. albicans
thr1
Δ mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the
HOM3
and
HOM6
genes, required for the production of the toxic pathway intermediate homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of
C. neoformans
threonine synthesis, the cross-species serum sensitivity of
thr1
Δ mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger
Authors: Aguilar-Osorio Guillermo , vanKuyk Patricia A. , Seiboth Bernhard , Blom Dirk , Solomon Peter S. , Vinck Arman , Kindt Frits , Wösten Han A. B. , de Vries Ronald P.
Published: March 2010

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of
Aspergillus niger
. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of
A. niger
. Northern analysis demonstrated that
mpdA
was expressed in both sporulating and nonsporulating mycelia, while expression of
mtdA
was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of
mtdA
and
mpdA
, respectively, demonstrated that expression of
mpdA
occurs in vegetative hyphae while
mtdA
expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in
A. niger
as two of the enzymes of the cycle are not present in the same part of
A. niger
colonies. The results also demonstrate the existence of spore-specific genes and enzymes in
A. niger
.
Genetic and Molecular Characterization of a Cryptochrome from the Filamentous Fungus Neurospora crassa
Authors: Froehlich Allan C. , Chen Chen-Hui , Belden William J. , Madeti Cornelia , Roenneberg Till , Merrow Martha , Loros Jennifer J. , Dunlap Jay C.
Published: March 2010

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus
Neurospora crassa
and demonstrate that
Neurospora cry
encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The
cry
transcript and CRY protein levels are strongly induced by blue light in a
wc-1
-dependent manner, and
cry
transcript is circadianly regulated, with a peak abundance opposite in phase to
frq
. Neither deletion nor overexpression of
cry
appears to perturb the free-running circadian clock. However,
cry
disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of
cry
under our laboratory conditions.
RecA4142 Causes SOS Constitutive Expression by Loading onto Reversed Replication Forks inEscherichia coliK-12
Authors: Long Jarukit Edward , Massoni Shawn C. , Sandler Steven J.
Published: March 2010

Escherichia coliinitiates the SOS response when single-stranded DNA (ssDNA) produced by DNA damage is bound by RecA and forms a RecA-DNA filament.recASOS constitutive [recA(Con)] mutants induce the SOS response in the absence of DNA damage. It has been proposed thatrecA(Con) mutants bind to ssDNA at replication forks, although the specific mechanism is unknown. Previously, it had been shown thatrecA4142(F217Y), a novelrecA(Con) mutant, was dependent on RecBCD for its high SOS constitutive [SOS(Con)] expression. This was presumably because RecA4142 was loaded at a double-strand end (DSE) of DNA. Herein, it is shown thatrecA4142SOS(Con) expression is additionally dependent onruvAB(replication fork reversal [RFR] activity only) andrecJ(5′→3′ exonuclease),xonA(3′→5′ exonuclease) and partially dependent onrecQ(helicase). Lastly,sbcCDmutations (Mre11/Rad50 homolog) inrecA4142strains caused full SOS(Con) expression in anruvAB-,recBCD-,recJ-, andxonA-independent manner. It is hypothesized that RuvAB catalyzes RFR, RecJ and XonA blunt the DSE (created by the RFR), and then RecBCD loads RecA4142 onto this end to produce SOS(Con) expression. InsbcCDmutants, RecA4142 can bind other DNA substrates by itself that are normally degraded by the SbcCD nuclease.
Thiol Peroxidase Protects
Salmonella enterica
from Hydrogen Peroxide Stress
In Vitro
and Facilitates Intracellular Growth
Authors: Horst Sarah A. , Jaeger Timo , Denkel Luisa A. , Rouf Syed Fazle , Rhen Mikael , Bange Franz-Christoph
Published: March 2010

At present,
Salmonella
is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in
Salmonella enterica
and show that a single
tpx
mutant is susceptible to exogenous hydrogen peroxide (H
2
O
2
), that it has a reduced capacity to degrade H
2
O
2
compared to the
ahpCF
and
tsaA
mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.
GlnB/GlnK PII Proteins and Regulation of the
Sinorhizobium meliloti
Rm1021 Nitrogen Stress Response and Symbiotic Function
Authors: Yurgel Svetlana N. , Rice Jennifer , Mulder Monika , Kahn Michael L.
Published: March 2010

The
Sinorhizobium meliloti
Rm1021Δ
glnD
-sm2 mutant, which is predicted to make a GlnD nitrogen sensor protein truncated at its amino terminus, fixes nitrogen in symbiosis with alfalfa, but the plants cannot use this nitrogen for growth (S. N. Yurgel and M. L. Kahn, Proc. Natl. Acad. Sci. U. S. A. 105:18958-18963, 2008). The mutant also has a generalized nitrogen stress response (NSR) defect. These results suggest a connection between GlnD, symbiotic metabolism, and the NSR, but the nature of this connection is unknown. In many bacteria, GlnD modifies the PII proteins, GlnB and GlnK, as it transduces a measurement of bacterial nitrogen status to a cellular response. We have now constructed and analyzed Rm1021 mutants missing GlnB, GlnK, or both proteins. Rm1021Δ
glnK
Δ
glnB
was much more defective in its NSR than either single mutant, suggesting that GlnB and GlnK overlap in regulating the NSR in free-living Rm1021. The single mutants and the double mutant all formed an effective symbiosis, indicating that symbiotic nitrogen exchange could occur without the need for either GlnB or GlnK. N-terminal truncation of the GlnD protein interfered with PII protein modification
in vitro
, suggesting either that unmodified PII proteins were responsible for the
glnD
mutant's ineffective phenotype or that connecting GlnD and appropriate symbiotic behavior does not require the PII proteins.
Quantitative PCR Assessments of Bacterial Species in Women with and without Bacterial Vaginosis
Authors: Zozaya-Hinchliffe Marcela , Lillis Rebecca , Martin David H. , Ferris Michael J.
Published: March 2010

Knowledge of the abundance of bacterial species in vaginal communities will help us to better understand their role in health and disease. However, progress in this field has been limited because quantifying bacteria in natural specimens is an arduous process. We developed quantitative real-time PCR (qPCR) assays to facilitate assessments of bacterial abundance in vaginal specimens and evaluated the utility of these assays by measuring species abundance in patients whose vaginal floras were clinically described as normal, intermediate, or bacterial vaginosis (BV) as defined by Nugent's criteria. The qPCR measurements showed that
Lactobacillus
species were predominant in normal vaginal specimens and that high
Lactobacillus crispatus
and
Lactobacillus jensenii
abundance was specific to normal specimens, while
Lactobacillus iners
abundance was high in all categories including BV. The abundances of all non-
Lactobacillus
species were higher in BV specimens than in normal specimens.
Prevotella
species were prevalent in all specimens and represented a high percentage of total species in BV specimens. qPCR assays can be a useful tool for describing the structure of vaginal communities and elucidating their role in health and disease.
Isolation and Identification of ATP-Secreting Bacteria from Mice and Humans
Authors: Iwase Tadayuki , Shinji Hitomi , Tajima Akiko , Sato Fumiya , Tamura Taku , Iwamoto Takeo , Yoneda Minoru , Mizunoe Yoshimitsu
Published: March 2010

In a recent report, ATP, which was possibly secreted by some intestinal bacteria, was shown to cause colitis in mice via Th17 cell differentiation. However, the ATP-secreting bacteria have not been isolated and identified. In the present study, we report that
Enterococcus gallinarum
, which is a vancomycin-resistant Gram-positive coccus isolated from mice and humans, secretes ATP.
Prevalence of Rotavirus, Adenovirus, Norovirus, and Astrovirus Infections and Coinfections among Hospitalized Children in Northern France
Authors: Tran Adissa , Talmud Déborah , Lejeune Benoît , Jovenin Nicolas , Renois Fanny , Payan Christopher , Leveque Nicolas , Andreoletti Laurent
Published: March 2010

From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (
P
= 0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments.
Measurements of changes in left ventricular volume, strain, and twist during isovolumic relaxation using MRI
Authors: Cheng-Baron June , Chow Kelvin , Khoo Nee Scze , Esch Ben T. , Scott Jessica M. , Haykowsky Mark J. , Tyberg John V. , Thompson Richard B.
Published: March 2010

Left ventricular (LV) active relaxation begins before aortic valve closure and is largely completed during isovolumic relaxation (IVR), before mitral valve opening. During IVR, despite closed mitral and aortic valves, indirect assessments of LV volume have suggested volume increases during this period. The aim of this study is to measure LV volume throughout IVR and to determine the sources of any volume changes. For 10 healthy individuals (26.0 ± 3.8 yr), magnetic resonance imaging was used to measure time courses of LV volume, principal myocardial strains (circumferential, longitudinal, radial), and LV twist. Mitral leaflet motion was observed using echocardiography. During IVR, LV volume measurements showed an apparent increase of 4.6 ± 1.5 ml (5.0 ± 2.0% of the early filling volume change), the LV untwisted by 4.5 ± 1.9° (36.6 ± 18.0% of peak systolic twist), and changes in circumferential, longitudinal, and radial strains were +0.87 ± 0.64%, +0.93 ± 0.57%, and −1.46 ± 1.66% (4.2 ± 3.3%, 5.9 ± 3.3%, and 5.3 ± 7.5% of peak systolic strains), respectively. The apparent changes in volume correlated ( P < 0.01) with changes in circumferential, longitudinal, and radial strains ( r = 0.86, 0.69, and −0.37, respectively) and untwisting ( r = 0.83). The closed mitral valve leaflets were observed to descend into the LV throughout IVR in all subjects in apical four- and three-chamber and parasternal long-axis views by 6.0 ± 3.3, 5.1 ± 2.4, and 2.1 ± 5.0 mm, respectively. In conclusion, LV relaxation during IVR is associated with changes in principal strains and untwisting, which are all correlated with an apparent increase in LV volume. Since closed mitral and aortic valves ensure true isovolumic conditions, the apparent volume change likely reflects expansion of the LV myocardium and the inward bowing of the closed mitral leaflets toward the LV interior.
Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure
Authors: Rengo Giuseppe , Leosco Dario , Zincarelli Carmela , Marchese Massimo , Corbi Graziamaria , Liccardo Daniela , Filippelli Amelia , Ferrara Nicola , Lisanti Michael P. , Koch Walter J. , Lymperopoulos Anastasios
Published: March 2010

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory α2-adrenoceptors (α2-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-α2-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of β-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 ± 5% reduction vs. post-MI untrained) and α2-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-α2-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.
Cardiac oxidative stress is involved in heart failure induced by thiamine deprivation in rats
Authors: Gioda Carolina Rosa , de Oliveira Barreto Tatiane , Prímola-Gomes Thales Nicolau , de Lima Daniel Carvalho , Campos Paula Peixoto , Capettini Luciano dos Santos Aggunn , Lauton-Santos Sandra , Vasconcelos Anilton César , Coimbra Cândido C. , Lemos Virginia Soares , Pesquero Jorge L. , Cruz Jader S.
Published: March 2010

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (∼40%) and H2O2(2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 ± 28.6 vs. TD 199.4 ± 30.2 nmol NADPH·min−1·ml−1) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis.
Weight loss in obese C57BL/6 mice limits adventitial expansion of established angiotensin II-induced abdominal aortic aneurysms
Authors: Police Sara B. , Putnam Kelly , Thatcher Sean , Batifoulier-Yiannikouris Frederique , Daugherty Alan , Cassis Lisa A.
Published: March 2010

Previous studies demonstrated that obesity increases inflammation in periaortic adipose tissue and promotes angiotensin II (ANG II)-induced abdominal aortic aneurysms (AAAs). We sought to determine whether weight loss of obese C57BL/6 mice would influence the progression of established AAAs. Male C57BL/6 mice were fed a high-fat diet (HF) for 4 mo and then infused with either saline or ANG II (1,000 ng·kg−1·min−1) for 3 mo. Mice with dilated suprarenal aortas at 28 days of ANG II infusion were designated to groups fed the HF (HF/HF) or a low-fat diet (LF; 10% kcal as fat; HF/LF) to induce weight loss for the last 2 mo of infusions. Suprarenal aortic lumen diameters of obese mice were increased by ANG II infusion at day 28 ( day 0: 1.03 ± 0.02; day 28: 1.86 ± 0.14 mm; P < 0.05), but did not progress with continued infusion in HF/HF mice. Moreover, aortic lumen diameters were not different between groups (HF/HF: 1.89 ± 0.15; HF/LF: 1.79 ± 0.18 mm). However, maximal diameters of excised AAAs were decreased with weight loss (HF/HF: 2.00 ± 0.11; HF/LF: 1.55 ± 0.13 mm; P < 0.05) and had reduced adventitial areas (HF/HF: 1.18 ± 0.10; HF/LF: 0.54 ± 0.02 mm2; P < 0.05). Neovascularization of aortic adventitias was strikingly decreased in HF/LF mice (HF/HF: 43 ± 5; HF/LF: 12 ± 2 endothelial cells/adventitial area; P < 0.05). ANG II-induced elevations in adipose mRNA abundance of CD105, an adipose-derived stem cell marker, were abolished with weight loss. These results demonstrate that weight loss limits adventitial expansion of ANG II-induced AAAs. Reduced neovascularization from weight loss may limit progression of AAAs.