bluuebunny/crossref_metadata_2025 huggingface dataset:
ZOOSPOROGENESIS, MORPHOLOGY, ULTRASTRUCTURE, PIGMENT COMPOSITION, AND PHYLOGENETIC POSITION OF TRACHYDISCUS MINUTUS (EUSTIGMATOPHYCEAE, HETEROKONTOPHYTA)1
Authors: Přibyl Pavel , Eliáš Marek , Cepák Vladislav , Lukavský Jaromír , Kaštánek Petr
Published: January 2012

The traditional order Mischococcales (Xanthophyceae) is polyphyletic with some original members now classified in a separate class, Eustigmatophyceae. However, most mischococcalean species have not yet been studied in detail, raising the possibility that many of them still remain misplaced. We established an algal culture (strain CCALA 838) determined as one such species, Trachydiscus minutus (Bourr.) H. Ettl, and studied the morphology, ultrastructure, life cycle, pigment composition, and phylogeny using the 18S rRNA gene. We discovered a zoosporic part of the life cycle of this alga. Zoospore production was induced by darkness, suppressed by light, and was temperature dependent. The zoospores possessed one flagellum covered with mastigonemes and exhibited a basal swelling, but a stigma was missing. Ultrastructural investigations of vegetative cells revealed plastids lacking both a connection to the nuclear envelope and a girdle lamella. Moreover, we described biogenesis of oil bodies on the ultrastructural level. Photosynthetic pigments of T. minutus included as the major carotenoids violaxanthin and vaucheriaxanthin (ester); we detected no chl c. An 18S rRNA gene‐based phylogenetic analysis placed T. minutus in a clade with species of the genus Pseudostaurastrum and with Goniochloris sculpta Geitler, which form a sister branch to initially studied Eustigmatophyceae. In summary, our results are inconsistent with classifying T. minutus as a xanthophycean and indicate that it is a member of a novel deep lineage of the class Eustigmatophyceae.
Ethnic Minorities and Integration Process in France and the Netherlands: An Institutionalist Perspective
Authors: EL KAROUNI ILYESS
Published: January 2012

Abstract

This article examines the ethnic minorities integration policies as implemented in France and the Netherlands. This study will be based on an institutionalist approach relying on North's analytical framework in order to explain the recent changes in these policies. I emphasize the existence of four patterns of social integration: reciprocity, redistribution, exchange, and coercion. I will also be insisting on the importance of ideology and the way it has evolved over time. This theoretical framework is then used to “tell the story” of the two countries. While France's historical and ideological backgrounds have forced it to adopt a universalist view and fight against communautarisme, the Dutch position was quite different as it does not deny the ethnic minorities cultural and religious freedom. Nevertheless, there is a hardening of the dominant ideology taking place in France. As to the Netherlands, there is a noticeable shift in the government's attitude toward ethnic minorities.
Homocysteine Impairs Endothelial Wound Healing by Activating Metabotropic Glutamate Receptor 5
Authors: CHEN CHENG‐HUNG , BEARD RICHARD S. , BEARDEN SHAWN E.
Published: January 2012

Please cite this paper as:Chen C‐H, Beard RS, Bearden SE. Homocysteine impairs endothelial wound healing by activating metabotropic glutamate receptor 5.Microcirculation19: 285–295, 2012.

Objective: Hcy is an independent risk factor for cerebrovascular disease and cognitive impairment. The purpose of this study was to elucidate the role of mGluR5 in Hcy‐mediated impairment of cerebral endothelial wound repair.

Methods: Mouse CMVECs (bEnd.3) were used in conjunction with directed pharmacology and shRNA. AutoDock was used to simulate the docking of ligand–receptor interactions.

Results: Hcy (20 μM) significantly increased Cx43‐pS368 by mGluR5‐ and PKC‐dependent mechanisms. Hcy attenuated wound repair by an mGluR5‐dependent mechanism over the six‐day study period but did not alter cell proliferation in a proliferation assay, suggesting that the attenuation of wound repair may be due to dysfunctional migration in HHcy. Hcy increased the expression of Cx43 and Cx43‐pS368 at the wound edge by activating mGluR5. Direct activation of mGluR5, using the specific agonist CHPG, was sufficient to reproduce the results whereas KO of mGluR5 with shRNA, or inhibition with MPEP, blocked the response to Hcy.

Conclusions: Inhibition of mGluR5 activation could be a novel strategy for promoting endothelial wound repair in patients with HHcy. Activation of mGluR5 may be a viable strategy for disrupting angiogenesis.
Is a Standardized Questionnaire Useful for Tubal Rupture Screening in Patients With Ectopic Pregnancy?
Authors: Huchon Cyrille , Panel Pierre , Kayem Gilles , Bassot Agnès , Nguyen Thuy , Falissard Bruno , Fauconnier Arnaud
Published: January 2012

ACADEMIC EMERGENCY MEDICINE 2012; 19:24–30 © 2012 by the Society for Academic Emergency Medicine

Objectives:  Physical examination, ultrasonography, and laboratory tests fail to reliably establish the preoperative diagnosis of tubal rupture in patients with ectopic pregnancy (EP), leading to a high rate of diagnostic laparoscopy. The aim of this study was to construct and to evaluate a clinical prediction rule for tubal rupture screening based on a self‐assessment questionnaire, among patients with EP.

Methods:  A standardized questionnaire was constructed via semistructured interviews of patients with acute pelvic pain. Features associated with tubal rupture were then identified in 141 prospectively included patients with tubal pregnancy, including 30 with tubal rupture, in five hospitals. Multiple logistic regression was used to select the best combination of independent features for predicting tubal rupture. Cross‐validation was with the jackknife method. The main outcome measure was diagnostic accuracy of the questionnaire for ruling out tubal rupture.

Results:  Eighty‐nine items characterizing acute pelvic pain were identified. Among them, four contributed independently to the diagnosis of tubal rupture: vomiting during pain, diffuse abdominal pain, acute pain for longer than 30 minutes, and flashing pain. The presence of one or more of these features had 93% sensitivity (95% confidence interval [CI] = 84% to 100%) and 44% specificity (95% CI = 35% to 53%) for tubal rupture, with a negative likelihood ratio for ruling out tubal rupture of 0.16.

Conclusions:  These results suggest that a standardized questionnaire may contribute to ruling out tubal rupture in patients with EP.
The histological evaluation of osseointegration of surface enhanced microimplants immediately loaded in conjunction with sinuslifting in humans
Authors: Browaeys Hilde , Vandeweghe Stefan , Johansson Carina B. , Jimbo Ryo , Deschepper Ellen , De Bruyn Hugo
Published: January 2012

Objective

The aim was to investigate histomorphometrically whether or not implant surface nanotopography improves the bone response under immediate loading simultaneous to sinus grafting.

Materials and methods

Dual acid‐etched titanium microimplants with/without crystalline surface deposition of calcium phosphate particles were placed in bilateral sinuslift areas grafted with a mixture of iliac crest bone and BioOss. Surface topography of microimplants was characterized using interferometry. Loaded microimplants (MsL) were immobilized in a provisional bridge supported by four normal size implants. Some patients had unloaded microimplants as controls (MsU). Biopsies were trephined after 2 or 4 months and histomorphometric analysis of bone area (BA) and bone‐to‐implant contact (BC) was performed. Nonparametric methods for dependent data were used to compare effect of surface modification, and healing time (2 vs. 4 months).

Results

A total of 53 biopsies were available from 13 patients. A total of 4/28 and 1/11 MsL failed after resp. 2 and 4 months vs. 0/6 and 1/5 MsU. Many loaded biopsies were damaged at the apical portion and showed no bone adhesion. MsL decreased in BA from coronal to apical from 2 to 4 months; Coronal > Middle (P = 0.047), Coronal > Apical (P < 0.001) and Middle > Apical (P < 0.001). This gradual decrease was not observed for BC; Coronal < Middle and Middle > Apical (P < 0.001). Only the middle part showed significant bone contact after 2 months. For MsL there was no statistically significant difference between surface or time indicating that improvement of osseointegration over time due to maturation of the graft was poor. The MsU did not show any difference between Osseotite and Nanotite for BIC and BA (P > 0.05) but doubled both their BA and BIC (P < 0.05) between 2 and 4 months.

Conclusions

Osseointegration in sinus‐grafted bone mixed with BioOss was poor irrespective of healing time or nanotopographical surface modification. The apex of MsL showed minimal bone contact suggesting that the graft does not add to the loading capacity. Surface enhancement was not beneficial despite the enlarged surface area. Overloading, most critical coronally of an implant, increases risks for implant failure and jeopardizes bone healing especially under immediate loading conditions with high load.
Adhesion of Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum on bioactive TiO2 surfaces
Authors: Närhi Timo O. , Leminen Heidi , Haukioja Anna , Söderling Eva
Published: January 2012

Background

Bioactive TiO2 coatings have been found to enhance fibroblast adhesion and gingival attachment on the titanium surfaces, but no information is available whether the coatings also promote the adhesion of periodontal pathogens.

Aim

The purpose of this study was to investigate protein adsorption and the adhesion of Aggregatibacter actinomycetemcomitans (Aa) and Fusobacterium nucleatum (Fn) on bioactive TiO2 surfaces.

Materials and methods

Commercially pure titanium discs (diameter 11.0 mm, grade 2) were coated with sol‐gel derived bioactive TiO2 coatings (MetAlive, Vivoxid, Turku, Finland) and preincubated in 1.5 ml PBS/diluted serum/diluted saliva at room temperature to mimic the clinical situation after implantation and to allow serum/saliva proteins to adhere on the substrates. Uncoated titanium discs were used as controls.

Results

SDS‐PAGE revealed similar protein profiles on bioactive and control titanium substrates. No differences were noticed in Aa or Fn adhesion between bioactive and control titanium. However, serum and saliva conditioning diminished Aa adhesion on both surfaces (p<0.001).

Conclusion

It can be concluded that bioactive TiO2 coating does not promote adhesion of Aa and Fn.
The Fe(II)/α‐ketoglutarate‐dependent taurine dioxygenases from Pseudomonas putida and Escherichia coli are tetramers
Authors: Knauer Stefan H. , Hartl‐Spiegelhauer Olivia , Schwarzinger Stephan , Hänzelmann Petra , Dobbek Holger
Published: January 2012

Fe(II)/α‐ketoglutarate‐dependent oxygenases are versatile catalysts associated with a number of different biological functions in which they use the oxidizing power of activated dioxygen to convert a variety of substrates. A mononuclear nonheme iron center is used to couple the decarboxylation of the cosubstrate α‐ketoglutarate with a two‐electron oxidation of the substrate, which is a hydroxylation in most cases. Although Fe(II)/α‐ketoglutarate‐dependent oxygenases have diverse amino acid sequences and substrate specifity, it is assumed that they share a common mechanism. One representative of this enzyme family is the Fe(II)/α‐ketoglutarate‐dependent taurine dioxygenase that catalyzes the hydroxylation of taurine yielding sulfite and aminoacetaldehyde. Its mechanism has been studied in detail becoming a model system for the whole enzyme family. However, its oligomeric state and architecture have been disputed. Here, we report the biochemical and kinetic characterization of the Fe(II)/α‐ketoglutarate‐dependent taurine dioxygenase from Pseudomonas putida KT2440 (TauDPp). We also present three crystal structures of the apo form of this enzyme. Comparisons with taurine dioxygenase from Escherichia coli (TauDEc) demonstrate that both enzymes are quite similar regarding their spectra, structure and kinetics, and only minor differences for the accumulation of intermediates during the reaction have been observed. Structural data and analytical gel filtration, as well as sedimentation velocity analytical ultracentrifugation, show that both TauDPp and TauDEc are tetramers in solution and in the crystals, which is in contrast to the earlier description of taurine dioxygenase from E. coli as a dimer.

Database

The atomic coordinates and structure factors have been deposited with the Brookhaven Protein Data Bank (entry 3PVJ, 3V15, 3V17)

Structured digital abstract

tauDpp
 and tauDpp bind by molecular sieving (View interaction)

tauDpp
 and tauDpp bind by x‐ray crystallography (View interaction)

tauDEc
 and tauDEc bind by molecular sieving (View interaction)
Evaluation of Toll‐like receptor and adaptor molecule polymorphisms for susceptibility to tuberculosis in a Colombian population
Authors: Sánchez D. , Lefebvre C. , Rioux J. , García L. F. , Barrera L. F.
Published: January 2012

Immunological studies have supported the idea that innate immunity is critical for the control of Mycobacterium tuberculosis (Mtb) infection in humans. Despite the overwhelming evidence showing the critical role of Toll‐like receptors (TLRs) in the in vitro recognition of Mtb, the in vivo significance of individual TLRs has been more difficult to demonstrate consistently. We were interested in examining the role of genes of TLRs and molecules involved in their signalling cascades, and a case–control study was designed to test the association of polymorphisms of these innate immune genes with pulmonary tuberculosis (TB) in a Colombian population. In this study, we did not find an association with TLR2, TLR4, TLR9, MyD88 or MAL/TIRAP polymorphic variants. These findings suggest that those genes are not involved as risk factors for pulmonary TB in our population.
Studying Large Mammals With Imperfect Detection: Status and Habitat Preferences of Wild Cattle and Large Carnivores in Eastern Cambodia
Authors: Gray Thomas N. E.
Published: January 2012

Studying large mammal species in tropical forests is a conservation challenge with species’ behavior and ecology often increasing the probability of non‐detection during surveys. Consequently, knowledge of the distribution, status, and natural history of many large mammal species in Southeast Asia is limited. I developed occupancy models from camera‐trapping data, thereby accounting for imperfect detection at sampling sites, to clarify the status and habitat requirements of four globally threatened or near threatened large mammals (banteng Bos javanicus, gaur Bos gaurus, dhole Cuon alpinus, and leopard Panthera pardus) in Mondulkiri Protected Forest, eastern Cambodia. Camera traps were operational for >3500 trap nights with 202 photographic encounters of the four study species. Model averaged occupancy estimates were between 5 percent (leopard) and 140 percent (gaur) higher than naive estimates (i.e., proportion of camera‐trap sites species recorded from) thus highlighting the importance of accounting for detectability during conservation surveys. I recommend the use of an occupancy framework when using camera‐trap data to study the status, ecology, and habitat preferences of poorly known and elusive species. The results highlight the importance of mixed deciduous and semi‐evergreen forest for wild cattle in eastern Cambodia and I emphasize that these habitats must be considered in conservation planning across the Lower Mekong Dry Forest Ecoregion.
Lysosomal Signaling Enhances Mitochondria‐Mediated Photodynamic Therapy in A431 Cancer Cells: Role of Iron
Authors: Saggu Shalini , Hung Hsin‐I , Quiogue Geraldine , Lemasters John J. , Nieminen Anna‐Liisa
Published: January 2012

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria‐mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT‐induced cell killing by mitochondria‐targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4‐PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4‐PDT‐induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin‐induced killing, cells were incubated with ammonium ferric citrate (30 μm) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin‐induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch‐desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin‐mediated PDT and that lysosomal disruption augments PDT with Pc 4.
Catalytic dechlorination of diclofenac by biogenic palladium in a microbial electrolysis cell
Authors: Gusseme Bart De , Soetaert Maarten , Hennebel Tom , Vanhaecke Lynn , Boon Nico , Verstraete Willy
Published: January 2012

Diclofenac is one of the most commonly detected pharmaceuticals in wastewater treatment plant (WWTP) effluents and the receiving water bodies. In this study, biogenic Pd nanoparticles (‘bio‐Pd’) were successfully applied in a microbial electrolysis cell (MEC) for the catalytic reduction of diclofenac. Hydrogen gas was produced in the cathodic compartment, and consumed as a hydrogen donor by the bio‐Pd on the graphite electrodes. In this way, complete dechlorination of 1 mg diclofenac l−1 was achieved during batch recirculation experiments, whereas no significant removal was observed in the absence of the biocatalyst. The complete dechlorination of diclofenac was demonstrated by the concomitant production of 2‐anilinophenylacetate (APA). Through the addition of −0.8 V to the circuit, continuous and complete removal of diclofenac was achieved in synthetic medium at a minimal HRT of 2 h. Continuous treatment of hospital WWTP effluent containing 1.28 µg diclofenac l−1 resulted in a lower removal efficiency of 57%, which can probably be attributed to the affinity of other environmental constituents for the bio‐Pd catalyst. Nevertheless, reductive catalysis coupled to sustainable hydrogen production in a MEC offers potential to lower the release of micropollutants from point‐sources such as hospital WWTPs.
Ohm's Law in a Quantum World
Authors: Ferry David K.
Published: January 2012

Conductivity measurements reveal unexpected classical electronic behavior at the quantum level.
Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation
Authors: Lee Joungmin , Jang Yu-Sin , Choi Sung Jun , Im Jung Ae , Song Hyohak , Cho Jung Hee , Seung Do Young , Papoutsakis E. Terry , Bennett George N. , Lee Sang Yup
Published: December 2011

Clostridium acetobutylicumnaturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineeredC. acetobutylicumcan be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene fromClostridium beijerinckiiNRRL B-593 (i.e.,adhB-593) inC. acetobutylicumATCC 824. To increase the total alcohol titer, a synthetic acetone operon (actoperon;adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in thebukgene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged byin situremoval of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.
Time-Dependent Profiles of Transcripts Encoding Lignocellulose-Modifying Enzymes of the White Rot Fungus Phanerochaete carnosa Grown on Multiple Wood Substrates
Authors: MacDonald Jacqueline , Master Emma R.
Published: December 2011

The abundances of nine transcripts predicted to encode lignocellulose-modifying enzymes were measured over the course of
Phanerochaete carnosa
cultivation on four wood species. Profiles were consistent with sequential decay; transcripts encoding lignin-degrading peroxidases featured a significant substrate-dependent response. The chitin synthase gene was identified as the optimal internal reference gene for transcript quantification.
Wolbachia Strain wAlbB Enhances Infection by the Rodent Malaria Parasite Plasmodium berghei in Anopheles gambiae Mosquitoes
Authors: Hughes Grant L. , Vega-Rodriguez Joel , Xue Ping , Rasgon Jason L.
Published: December 2011

Wolbachia
, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent
Wolbachia
strains, wAlbB from
Aedes albopictus
and wMelPop from
Drosophila melanogaster
, on the vector competence of
Anopheles gambiae
challenged with
Plasmodium berghei
. We show that the wAlbB strain significantly increases
P. berghei
oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on
P. berghei
levels suggest that
Wolbachia
strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.
Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-Limited Continuous Culture
Authors: Dang T. C. , Fujii M. , Rose A. L. , Bligh M. , Waite T. D.
Published: December 2011

A continuous culturing system (chemostat) made of metal-free materials was successfully developed and used to maintain Fe-limited cultures of
Microcystis aeruginosa
PCC7806 at nanomolar iron (Fe) concentrations (20 to 50 nM total Fe). EDTA was used to maintain Fe in solution, with bioavailable Fe controlled by absorption of light by the ferric EDTA complex and resultant reduction of Fe(III) to Fe(II). A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe (i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of the ferric EDTA complex. Prediction by chemostat theory modified to account for the light-mediated formation of bioavailable Fe rather than total Fe was in good agreement with growth characteristics of
M. aeruginosa
under Fe limitation. The cellular Fe quota increased with increasing dilution rates in a manner consistent with the Droop theory. Short-term Fe uptake assays using cells maintained at steady state indicated that
M. aeruginosa
cells vary their maximum Fe uptake rate (ρ
max
) depending on the degree of Fe stress. The rate of Fe uptake was lower for cells grown under conditions of lower Fe availability (i.e., lower dilution rate), suggesting that cells in the continuous cultures adjusted to Fe limitation by decreasing ρ
max
while maintaining a constant affinity for Fe.
Role for Rhizobium rhizogenes K84 Cell Envelope Polysaccharides in Surface Interactions
Authors: Abarca-Grau Ana M. , Burbank Lindsey P. , de Paz Héctor D. , Crespo-Rivas Juan C. , Marco-Noales Ester , López María M. , Vinardell Jose M. , von Bodman Susanne B. , Penyalver Ramón
Published: December 2011

Rhizobium rhizogenes
strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a
wcbD
ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of
wcbD
in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the
rkpK
gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The
rkpK
mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.
Enhanced Microbial Utilization of Recalcitrant Cellulose by an
Ex Vivo
Cellulosome-Microbe Complex
Authors: You Chun , Zhang Xiao-Zhou , Sathitsuksanoh Noppadon , Lynd Lee R. , Zhang Y.-H. Percival
Published: December 2011

A cellulosome-microbe complex was assembled
ex vivo
on the surface of
Bacillus subtilis
displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g.,
B. subtilis
) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.
Improvement of Modified Charcoal-Cefoperazone-Deoxycholate Agar by Supplementation with a High Concentration of Polymyxin B for Detection of Campylobacter jejuni and C. coli in Chicken Carcass Rinses
Authors: Chon Jung-Whan , Hyeon Ji-Yeon , Yim Jin-Hyuk , Kim Jong-Hyun , Song Kwang-Young , Seo Kun-Ho
Published: December 2011

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate
Campylobacter jejuni
and
C. coli
from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (
P
< 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.
Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of a
pfkB
Mutant
Authors: Wang Huiyan , Liu Xiangmei , Liu Shuangshuang , Yu Yangyang , Lin Jianqun , Lin Jianqiang , Pang Xin , Zhao Jian
Published: December 2011

The extremely acidophilic, chemolithoautotrophic
Acidithiobacillus ferrooxidans
is an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strain
A. ferrooxidans
ATCC 23270. A targeted phosphofructokinase (PFK) gene (
pfkB
) mutant of
A. ferrooxidans
ATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potential
pfkB
gene (AFE_1807) was also characterized by expression in PFK-deficient
Escherichia coli
cells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by the
pfkB
gene from
E. coli
K-12. The function of the potential PFKB protein in
A. ferrooxidans
was demonstrated by comparing the properties of the
pfkB
mutant with those of the wild type. The
pfkB
mutant strain displayed a relatively reduced growth capacity in S
0
medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H
2
SO
4
), but the mutation did not completely prevent
A. ferrooxidans
from assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest from
A. ferrooxidans
and multiple mutations could be made in a single
A. ferrooxidans
strain.
Production of Indole-3-Acetic Acid via the Indole-3-Acetamide Pathway in the Plant-Beneficial Bacterium Pseudomonas chlororaphis O6 Is Inhibited by ZnO Nanoparticles but Enhanced by CuO Nanoparticles
Authors: Dimkpa Christian O. , Zeng Jia , McLean Joan E. , Britt David W. , Zhan Jixun , Anderson Anne J.
Published: December 2011

The beneficial bacterium
Pseudomonas chlororaphis
O6 produces indole-3-acetic acid (IAA), a plant growth regulator. However, the pathway involved in IAA production in this bacterium has not been reported. In this paper we describe the involvement of the indole-3-acetamide (IAM) pathway in IAA production in
P. chlororaphis
O6 and the effects of CuO and ZnO nanoparticles (NPs). Sublethal levels of CuO and ZnO NPs differentially affected the levels of IAA secreted in medium containing tryptophan as the precursor. After 15 h of growth, CuO NP-exposed cells had metabolized more tryptophan than the control and ZnO NP-challenged cells. The CuO NP-treated cells produced higher IAA levels than control cultures lacking NPs. In contrast, ZnO NPs inhibited IAA production. Mixing of CuO and ZnO NPs resulted in an intermediate level of IAA production relative to the levels in the separate CuO and ZnO NP treatments. The effect of CuO NPs on IAA levels could be duplicated by ions at the concentrations released from the NPs. However, ion release did not account for the inhibition caused by the ZnO NPs. The mechanism underlying changes in IAA levels cannot be accounted for by effects on transcript accumulation from genes encoding a tryptophan permease or the IAM hydrolase in 15-h cultures. These findings raise the issue of whether sublethal doses of NPs would modify the beneficial effects of association between plants and bacteria.
Evaluation of Three Commercially Available Influenza A Type-Specific Blocking Enzyme-Linked Immunosorbent Assays for Seroepidemiological Studies of Influenza A Virus Infection in Pigs
Authors: Tse Maying , Kim Mia , Chan Chung-Hei , Ho Po-Lai , Ma Siu-Kit , Guan Yi , Peiris J. S. M.
Published: January 2012

The reverse zoonotic transmission of the pandemic H1N1 2009 influenza virus to swine necessitates enhanced surveillance of swine for influenza virus infection. Using a well-characterized panel of naturally infected swine sera, we evaluated and optimized the performances of three commercially available competitive enzyme-linked immunosorbent assays (ELISAs), namely, the IDEXX Influenza A Ab test, IDEXX AI MultiS-Screen Ab test, and IDVet ID Screen influenza A antibody competition ELISA, for detecting influenza A virus-reactive antibodies in swine. Receiver operating characteristic (ROC) analysis suggests that adjustment of the manufacturer-recommended cutoff values optimizes the sensitivity and specificity of these assays, making them applicable for seroepidemiology studies of swine influenza. Using such optimized cutoff levels, the sensitivity and specificity of the IDEXX Influenza A Ab test were 86% and 89%, respectively; those for the IDEXX AI MultiS-Screen Ab test were 91% and 87%, respectively; and those for the IDVet ID Screen influenza A test were 95% and 79%, respectively.
Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility
Authors: Daher Wassim , Klages Natacha , Carlier Marie-France , Soldati-Favre Dominique
Published: December 2011

Toxoplasma gondii
belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that
T. gondii
formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to
Toxoplasma
actin and nucleates rabbit actin assembly
in vitro
. TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the
TgFRM3
gene by double homologous recombination in a
ku80-ko
strain reveals no vital function for tachyzoite propagation
in vitro
, which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity.
Rapamycin Exerts Antifungal Activity
In Vitro
and
In Vivo
against Mucor circinelloides via FKBP12-Dependent Inhibition of Tor
Authors: Bastidas Robert J. , Shertz Cecelia A. , Lee Soo Chan , Heitman Joseph , Cardenas Maria E.
Published: December 2011

The zygomycete
Mucor circinelloides
is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in
M. circinelloides
are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene,
fkbA
, also conferred rapamycin and FK506 resistance. Expression of
M. circinelloides
FKBP12 (
Mc
FKBP12) complemented a
Saccharomyces cerevisiae
mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the
Mc
Tor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in
S. cerevisiae
, and
Mc
FKBP12 interacted in a rapamycin-dependent fashion with the
Mc
Tor FRB domain in a yeast two-hybrid assay, validating
Mc
FKBP12 and
Mc
Tor as conserved targets of rapamycin. We showed that
in vitro
, rapamycin exhibited potent growth inhibitory activity against
M. circinelloides
. In a
Galleria mellonella
model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.
A 54-Kilodalton Protein Encoded by pBtoxis Is Required for Parasporal Body Structural Integrity in Bacillus thuringiensis subsp. israelensis
Authors: Diaz-Mendoza Mercedes , Bideshi Dennis K. , Federici Brian A.
Published: December 2011

Strains of
Bacillus thuringiensis
such as
B. thuringiensis
subsp.
israelensis
(ONR-60A) and
B. thuringiensis
subsp.
morrisoni
(PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of
B. thuringiensis
subsp.
israelensis
, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in
B. thuringiensis
subsp.
israelensis
, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.
Analysis of the Pseudomonas aeruginosa Regulon Controlled by the Sensor Kinase KinB and Sigma Factor RpoN
Authors: Damron F. Heath , Owings Joshua P. , Okkotsu Yuta , Varga John J. , Schurr Jill R. , Goldberg Joanna B. , Schurr Michael J. , Yu Hongwei D.
Published: December 2011

Alginate overproduction by
Pseudomonas aeruginosa
, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ
22
; AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of
mucA
. However, the mucoid conversion can occur in wt
mucA
strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ
54
). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid
kinB
mutant and an isogenic nonmucoid
kinB rpoN
double mutant. In the
kinB
mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the
kinB
mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.
Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor
Authors: Asención Diez Matías D. , Peirú Salvador , Demonte Ana M. , Gramajo Hugo , Iglesias Alberto A.
Published: December 2011

Streptomyces coelicolor
exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of
S. coelicolor
A3(2). Each gene was heterologously expressed in
Escherichia coli
cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high
V
max
in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (
V
max
of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in
S. coelicolor
, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.
Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls
Authors: Sabui Subrata , Dutta Sanjucta , Debnath Anusuya , Ghosh Avishek , Hamabata T. , Rajendran K. , Ramamurthy T. , Nataro James P. , Sur Dipika , Levine Myron M. , Chatterjee Nabendu Sekhar
Published: January 2012

Enterotoxigenic
Escherichia coli
(ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes,
cssA
and
cssB
, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific
m
ismatch
a
mplification
m
utation
a
ssay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3′ ends of the reverse primers specific for
cssA
and
cssB
and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (
est
) alone or with the heat-labile toxin gene (
elt
), whereas the AIIBII allele was predominant in ETEC isolates harboring the
elt
gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.
An Alternative Method for Cultivation of Lawsonia intracellularis
Authors: Vannucci Fabio A. , Wattanaphansak Suphot , Gebhart Connie J.
Published: January 2012

An alternative method for the cultivation of
Lawsonia intracellularis
, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, was developed using an Original Space Bag inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. The flexibility of this protocol allows the testing of various environmental conditions for static cultivation of this bacterium and the development of diagnostic techniques.
Cationic β-vinyl substitutedmeso-tetraphenylporphyrins: synthesis and non-covalent interactions with a short poly(dGdC) duplex
Authors: Silva Eduarda M.P. , Ramos Catarina I.V. , Pereira Patrícia M.R. , Giuntini Francesca , Faustino Maria A.F. , Tomé João P.C. , Tomé Augusto C. , Silva Artur M.S. , Santana-Marques M. Graça , Neves M. Graça P.M.S. , Cavaleiro José A.S.
Published: January 2012

Several cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenylporphyrin derivatives were synthesized starting from 2-formyl-meso-tetraphenylporphyrin, and the corresponding Zn(II) complex, and different N-alkyl derivatives of 2- and 4-methylpyridine and 2- and 4-methylquinoline. The new compounds were obtained in a one-step process via base catalyzed aldol-type condensation reactions. Electrospray ionization mass spectrometry (ESI-MS) and ultraviolet-visible (UV-vis) spectroscopy were used to investigate the binding mode of the synthesized cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenylporphyrin derivatives with a short GC duplex oligonucleotide. Analysis of the obtained mass spectrometry results indicates the probable occurrence of outside binding. UV-vis spectroscopy data also points to non-intercalation. The potential photosensitizing capacity of these compounds was also ascertained from preliminary photophysical studies.