bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis, Characterization, and Microwave Dielectric Properties of Sr2−xLa2Mg1+xW2O12 (x=0, 1) Ceramics
Authors: Venugopal Vineeth , Anjana Prabhakaran Sreekumari , Parkash Om , Kumar Devendra , Sebastian Mailadil Thomas
Published: May 2010

SrLa2Mg2W2O12 and Sr2La2MgW2O12 ceramics were prepared by the conventional solid‐state ceramic route and their dielectric properties were investigated in the radio and microwave frequency regions. SrLa2Mg2W2O12 sintered at 1500°C has ɛr=25.2, Qu×f=15 900 GHz (at 4.9 GHz), and τf=0 ppm/°C. Sr2La2MgW2O12 sintered at 1525°C has ɛr=24.7, Qu×f=35 000 GHz (at 4.7 GHz), and τf=−83 ppm/°C. The dielectric properties of these ceramics are reported for the first time.
Limited prognostic value of tissue protein expression levels of BCl‐2 in Danish ovarian cancer patients: from the Danish ‘MALOVA’ ovarian cancer study
Authors: Høgdall ESTRID V.S. , Christensen Lise , Kjaer Susanne K. , Blaakaer Jan , Christensen Ib Jarle , Høgdall Claus K.
Published: May 2010

Høgdall EVS, Christensen L, Kjaer SK, Blaakaer J, Christensen IJ, Høgdall CK. Limited prognostic value of tissue protein expression levels of BCl‐2 in Danish ovarian cancer patients. APMIS 2010; 118: 557–64.

The purpose of the study was to determine the expression of BCl‐2 in epithelial ovarian tumors and to correlate expression levels with selected clinicopathologic parameters, time to progression and prognosis of the disease. Using tissue arrays (TA), we analyzed BCl‐2 expression in tissues from 191 women diagnosed with low malignant potential ovarian tumors (LMP) and from 582 patients diagnosed with ovarian cancer (OC). Using 30% as cutoff level for BCl‐2 overexpression, 5% of LMPs were positive with a higher proportion of serous ovarian tumor of LMP, compared to mucinous ovarian tumor of LMP (p = 0.02). Women with a BCl‐2‐positive LMP tumor were older than women with a BCl‐2 negative tumor (p = 0.02). Ten percent of OCs were positive for BCl‐2 expression (≥30%). No significant association was found between BCl‐2 expression levels and histologic type of tumors (serous vs mucinous, p = 0.19). A 30% cutoff value or a percentage scale showed that BCl‐2 expression had no prognostic value, both in univariate and in multivariate survival analyses. No difference in time to progression was observed between patients with BCl‐2‐positive and negative tumors. These data suggest that BCl‐2 expression may not be of important clinical value in the treatment of Danish OC patients.
Resistin Concentration is Increased in the Peritoneal Fluid of Women with Endometriosis
Authors: Yi Kyong Wook , Shin Jung‐Ho , Park Hyun Tae , Kim Tak , Kim Sun Haeng , Hur Jun‐Young
Published: May 2010

Citation Yi KW, Shin J‐H, Park HT, Kim T, Kim SH, Hur J‐Y. Resistin concentration is increased in the peritoneal fluid of women with endometriosis. Am J Reprod Immunol 2010

Problem  The aim of this study was to investigate the concentration of resistin and adiponectin in the peritoneal fluid (PF) of patients with endometriosis.

Method of study  PF sampling was obtained from women with (n = 48) and without endometriosis (n = 36), and the anthropometric indices of the patients were measured. Resistin and adiponectin concentrations in the PF were determined using the enzyme‐linked immunosorbent assay.

Results  The mean concentration of PF resistin was significantly higher in women with endometriosis compared to the controls. PF resistin concentrations were not associated with any of the anthropometric indices. The PF adiponectin did not differ between the two groups, but showed a significant association with the weight, body mass index, and hip circumference. After adjusting for these factors, PF adiponectin expression was not associated with endometriosis.

Conclusion  The findings of this study suggest a potent role for resistin in endometriosis. Further studies are needed to elucidate the biological implications of resistin in endometriosis.
Keratinocyte Growth Factor Stimulates Macrophage Inflammatory Protein 3α and Keratinocyte‐derived Chemokine Secretion by Mouse Uterine Epithelial Cells
Authors: Haddad Severina N. , Wira Charles R.
Published: May 2010

Citation Haddad SN, Wira CR. Keratinocyte growth factor stimulates macrophage inflammatory protein 3α and keratinocyte‐derived chemokine secretion by mouse uterine epithelial cells. Am J Reprod Immunol 2010; 64: 197–211

Problem  Communication between uterine epithelial cells and the underlying stromal fibroblasts is critical for proper endometrial function. Stromal fibroblast‐derived growth factors have been shown to regulate epithelial immune functions. The purpose of this study was to determine whether keratinocyte growth factor (KGF) regulates uterine epithelial cell chemokine and antimicrobial secretion.

Method of study  Uterine epithelial cells were isolated from Balb/c mice and cultured in either 96‐well plates or transwell inserts. Epithelial cells were treated with KGF, epidermal growth factor (EGF), or hepatocyte growth factor (HGF). Macrophage inflammatory protein 3α (MIP3α) and keratinocyte‐derived chemokine (KC) levels were measured by ELISA.

Results  Keratinocyte growth factor stimulated the secretion of MIP3α and KC. The effects on MIP3α by KGF were specific because EGF and HGF had no effect. In contrast, KGF, EGF, and HGF had similar effects on KC. Furthermore, KGF administered to the apical side of epithelial cells had no effect on MIP3α or KC secretion, indicating that the KGF receptor is located on the basolateral surface of uterine epithelial cells.

Conclusion  We demonstrate that KGF plays a role in uterine epithelial cell secretion of MIP3α and KC, key immune mediators involved in the protection of mucosal surfaces in the female reproductive tract.
Does caffeine impair cerebral oxygenation and blood flow velocity in preterm infants?
Authors: Tracy MB , Klimek J , Hinder M , Ponnampalam G , Tracy SK
Published: May 2010

Aim:  The aim of the study is to assess the effects of an intravenous 10 mg/kg loading dose of caffeine base in cerebral oxygenation, cerebral Doppler blood flow velocity and cardiac output in preterm infants.

Methods:  Preterm neonates <34 weeks gestation were investigated at 1 and 4 h following the loading dose of caffeine using Doppler cerebral sonography, cardiac echocardiography and cerebral spatially resolved near‐infrared spectroscopy.

Results:  Forty infants were studied with a mean gestational age (mean ± standard deviation) of 27.7 (±2.5) weeks, birth weight of 1155 (±431) g and a postnatal age of 2.8 (±2.2) days. Mean Anterior Cerebral Artery peak and time average mean blood flow velocity fell significantly by 14% and 17.7%, respectively at 1 h post‐caffeine loading dose, which recovered partially by 4 h. Cerebral Tissue Oxygenation Index fell from pre‐dose levels by 9.5% at 1 h with partial recovery to 4.9% reduced at 4 h post‐dose. There were no significant changes in left or right ventricular output, transcutaneous oxygen saturation, transcutaneous PCO2 or total vascular resistance.

Conclusions:  A loading dose of 10 mg/kg caffeine base resulted in significant reduction at 1 h post‐dose in cerebral oxygenation and cerebral blood flow velocity with partial recovery at 4 h.
Twelve‐month neurodevelopmental outcome in preterm infants with and without intrauterine growth restriction
Authors: Padilla Nelly , Perapoch Josep , Carrascosa Antonio , Acosta‐Rojas Ruthy , Botet Francesc , Gratacós Eduard
Published: May 2010

Aim:  To evaluate the neurodevelopmental outcome at 12 months’ corrected age in preterm infants with and without severe intrauterine growth restriction.

Methods:  This prospective follow‐up study included 37 infants with severe intrauterine growth restriction and 36 appropriate‐for‐gestational‐age infants born between 26 and 34 weeks. Neonatal and infant data were prospectively recorded. Infants were assessed at 12 ± 2 months’ corrected age with the Hammersmith Infant Neurological Examination and the Bayley Scale for Infant Development version‐II.

Results:  Both groups were similar in demographic characteristics and perinatal status. No significant differences in neurodevelopmental performance were found. The mental development index was 98.8 (SD 9.0) vs 98.4 (SD 13.1) (p = 0.9) and the psychomotor development index was 91.7 (SD 9.9) vs 95.5 (SD 13.4) (p = 0.2) for the study and reference groups respectively. Neurological assessment showed no significant differences between the two groups.

Conclusion:  Although the study group showed a non‐significant trend towards a lower score in the psychomotor development index than the reference group, significant differences at 12 months could not be demonstrated. IUGR infants continued to have significantly lower weight, length and head circumference at 1 year.
ADHD symptoms and maturity – a follow‐up study in school children
Authors: Gustafsson P , Holmström E , Besjakov J , Karlsson MK
Published: May 2010

Aim:  To test the hypothesis that there is a subgroup of children with attention deficit hyperactivity disorder (ADHD) who show a decline in ADHD‐symptoms that is associated with signs of biological maturation, a phenomenon referred to as a ‘maturation catch‐up’.

Methods:  The parents of 147 children who were given an examination in grades one and two 1999–2000 that included assessment of ADHD‐symptoms and estimation of skeletal bone‐age by use of hand radiographs (which was repeated in the eighth grade), were contacted 2008–2009 and were asked to answer questions concerning ADHD‐symptoms and behavioural maturity in their children. The response frequency was 67%. A complete dataset was achieved in 57 children. A reduction of Conners scores for ADHD‐symptoms of eight or more between the evaluations was defined as a marked reduction in symptoms.

Results:  When the children with a marked symptom reduction (n = 6) were compared with children without such a reduction (n = 51), we found a significant difference in skeletal maturation (p < 0.05).

Conclusion:  This study gives support to the theory that there is a group of children with ADHD‐symptoms who have a biological maturational‐lag who will show a decrease in their ADHD‐symptoms as they show a maturation catch‐up with increasing age.
Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin
Authors: Surkov Serhiy , Nilsson Hanna , Rasmussen Louise C. V. , Sperling‐Petersen Hans U. , Isaksson Leif A.
Published: May 2010

Translation initiation factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation initiation on the ribosome remains unclear, even though the factor has several known functions, one of them being RNA chaperone activity. In this study, we analyzed translational gene expression in vivo in two cold‐sensitive chromosomal mutant variants of IF1 with amino acid substitutions, R40D and R69L, using two different reporter gene systems. The strains with the mutant IF1 gave higher reporter gene expression than the control strain. The extent of this effect was dependent on the composition of the translation initiation region. The Shine–Dalgarno (SD) sequence, AU‐rich elements upstream of the SD sequence and the region between the SD sequence and the initiation codon are important for the magnitude of this effect. The data suggest that the wild‐type form of IF1 has a translation initiation region‐dependent inhibitory effect on translation initiation. Kasugamycin is an antibiotic that blocks translation initiation. Addition of kasugamycin to growing wild‐type cells increases reporter gene expression in a very similar way to the altered IF1, suggesting that the infA mutations and kasugamycin affect some related step in translation initiation. Genetic knockout of three proteins (YggJ, BipA, and CspA) that are known to interact with RNA causes partial suppression of the IF1‐dependent cold sensitivity.
Bacitracin is not a specific inhibitor of protein disulfide isomerase
Authors: Karala Anna‐Riikka , Ruddock Lloyd W.
Published: May 2010

To successfully dissect molecular pathways in vivo, there is often a need to use specific inhibitors. Bacitracin is very widely used as an inhibitor of protein disulfide isomerase (PDI) in vivo. However, the specificity of action of an inhibitor for a protein‐folding catalyst cannot be determined in vivo. Furthermore, in vitro evidence for the specificity of bacitracin for PDI is scarce, and the mechanism of inhibition is unknown. Here, we present in vitro data showing that 1 mm bacitracin has no significant effect on the ability of PDI to introduce or isomerize disulfide bonds in a folding protein or on its ability to act as a chaperone. Where bacitracin has an effect on PDI activity, the effect is relatively minor and appears to be via competition of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI, it has significant effects on both noncatalyzed protein folding and on other molecular chaperones. These results suggest that the use of bacitracin as a specific inhibitor of PDI in cellular systems requires urgent re‐evaluation.
Association of the +874 T/A interferon gamma polymorphism with infections in sickle cell disease
Authors: Joannes M. O. , Loko G. , Deloumeaux J. , Chout R. , Marianne‐Pepin T.
Published: May 2010

Infectious complications are a leading cause of morbidity and mortality in patients with sickle cell disease. Several mechanisms are supposed to contribute to this susceptibility. The exact reasons for the susceptibility of sickle cell patients to infection are not clear and are still a matter of debate. Interferon gamma (IFNγ) is a key cytokine involved mainly in the defence against intracellular pathogens. We investigated a possible association of an IFNγ +874 T/A polymorphism and infectious complications in sickle cell patients. Seventy‐two sickle cell patients were typed for +874 T/A IFNγ polymorphism. Genotype frequencies were different between cases and controls. Indeed, the T allele frequency was significantly higher in patients with infections than in patients without infections (P = 0.014). Our results suggest that the +874 T/A IFNγ polymorphism is associated with infectious complications in sickle cell patients. T allele could be involved in infections in sickle cell patients.
Arsenite induces oxidative injury in rat brain
Authors: Fan Su‐Feng , Chao Pei‐Ling , Lin Anya Maan‐Yuh
Published: May 2010

Sodium arsenite (arsenite)‐induced neurotoxicity and its interaction with ferrous citrate (iron) was investigated in rat brain.In vitrodata showed that arsenite (1–10 μmol/L) concentration dependently increased lipid peroxidation and the potency of arsenite was less than that of iron. The oxidative activity of arsenite, sodium arsenate (arsenate), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were evaluated by inducing lipid peroxidation in cortical homogenates, and the potency for this effect was as follows: arsenite > arsenate > MMA and DMA. Several well‐known antioxidants, including glutathione, melatonin, and β‐estradiol inhibited arsenite‐induced lipid peroxidation in a concentration‐dependent manner. Ourin vivostudy employed intranigral infusion of arsenite (5 nmol) in the substantia nigra (SN) of anesthetized rats. Four hours to 7 days after infusion, lipid peroxidation was elevated while glutathione was depleted in the infused SN. The dopamine content in the striatum ipsilateral to arsenite‐infused SN was first elevated 24 h and then decreased 7 days after intranigral infusion of arsenite. Using pretreatment ofl‐buthionine‐[S,R]‐sulfoximine (l‐BSO, i.c.v.) to reduce glutathione content in rat brain, arsenite‐induced oxidative injury was augmented. Low doses of arsenite (1.5 nmol) and iron (3 nmol) alone induced minimal oxidative injury; however, co‐infusion of arsenite and iron augmented neurotoxicity, including elevated lipid peroxidation and reduced striatal dopamine content. Moreover, expression of heme oxygenase‐1, α‐synuclein aggregation, and DNA fragmentation were significantly enhanced in SN co‐infused with low doses of arsenite and iron. Taken together, our study demonstrates that arsenite was less potent than iron in inducing oxidative stress. Furthermore, concomitant arsenite and iron potentiated oxidative injury in the nigrostriatal dopaminergic system, indicating that interaction of metals plays a more clinically‐relevant role in pathophysiology of central nervous system neurodegeneration.
Evolutionary ecology of insect adaptation to Bt crops
Authors: Carrière Yves , Crowder David W. , Tabashnik Bruce E.
Published: May 2010

Transgenic crops producing Bacillus thuringiensis (Bt) toxins are used worldwide to control major pests of corn and cotton. Development of strategies to delay the evolution of pest resistance to Bt crops requires an understanding of factors affecting responses to natural selection, which include variation in survival on Bt crops, heritability of resistance, and fitness advantages associated with resistance mutations. The two main strategies adopted for delaying resistance are the refuge and pyramid strategies. Both can reduce heritability of resistance, but pyramids can also delay resistance by reducing genetic variation for resistance. Seasonal declines in the concentration of Bt toxins in transgenic cultivars, however, can increase the heritability of resistance. The fitness advantages associated with resistance mutations can be reduced by agronomic practices, including increasing refuge size, manipulating refuges to increase fitness costs, and manipulating Bt cultivars to reduce fitness of resistant individuals. Manipulating costs and fitness of resistant individuals on transgenic insecticidal crops may be especially important for thwarting evolution of resistance in haplodiploid and parthenogenetic pests. Field‐evolved resistance to Bt crops in only five pests during the last 14 years suggests that the refuge strategy has successfully delayed resistance, but the accumulation of resistant pests could accelerate.
Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2
Authors: Ohbayashi Norihiko , Mamishi Setareh , Ishibashi Koutaro , Maruta Yuto , Pourakbari Babak , Tamizifar Banafshe , Mohammadpour Masoud , Fukuda Mitsunori , Parvaneh Nima
Published: May 2010

Human Griscelli syndrome type 2 (GS‐2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2‐a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS‐2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2‐a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2‐a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2‐a, Slp4‐a/granuphilin‐a, and Munc13‐4. The Rab27A(K22R) mutant normally binds Munc13‐4, but not Slp2‐a or Slp4‐a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2‐a and Munc13‐4 but normally binds Slp4‐a.
Topoisomerase I amplification in melanoma is associated with more advanced tumours and poor prognosis
Authors: Ryan Denise , Rafferty Mairin , Hegarty Shauna , O’Leary Patrick , Faller William , Gremel Gabriela , Bergqvist Michael , Agnarsdottir Margret , Strömberg Sara , Kampf Caroline , Pontén Fredrik , Millikan Robert C. , Dervan Peter A. , Gallagher William M.
Published: May 2010

In this study, we used array‐comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array‐comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1‐targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.
Changes in liver function parameters after percutaneous radiofrequency ablation therapy in patients with hepatocellular carcinoma
Authors: Kuroda Hidekatsu , Kasai Kazuhiro , Kakisaka Keisuke , Yasumi Yuki , Kataoka Koujiro , Ushio Akira , Miyamoto Yasuhiro , Sawara Kei , Oikawa Kanta , Kondo Koryo , Miura Yoshiaki , Endo Ryujin , Takikawa Yasuhiro , Suzuki Kazuyuki
Published: May 2010

Aim:  To evaluate changes in liver function parameters and risk factors 1 year after percutaneous radiofrequency ablation (RFA) therapy in patients with hepatocellular carcinoma (HCC).

Methods:  Subjects in this retrospective study comprised 45 patients with HCC who underwent RFA therapy (RFA alone, n = 25; transcatheter arterial embolization therapy before RFA, n = 20) and showed no recurrence of HCC 1 year after RFA. Serial changes in serum total bilirubin, albumin, prothrombin time and Child–Pugh score (CPs) were evaluated before and after RFA. In addition, Cox proportional hazards regression analysis was used to clarify risk factors for aggravation of liver function after RFA therapy.

Results:  Serum albumin levels showed a significant decrease from before (3.6 ± 0.4 g/dL) to 12 months after RFA therapy (3.2 ± 0.4 g/dL; P ≤ 0.05). CPs was significantly increased from before (6.4 ± 1.4) to both 6 months (6.8 ± 1.9; P ≤ 0.05) and 12 months after RFA (6.9 ± 2.0; P ≤ 0.05). Based on stepwise multivariate analysis, CPs of 9 or more before RFA was selected as a significant risk factor for long‐term aggravation of liver function after RFA.

Conclusion:  Liver function parameters, particularly serum albumin level, gradually and dominantly decreased in HCC patients with grade B and C according to the CPs classification over the course of 1 year after RFA therapy. A CPs of 9 or more represents a major risk factor for the aggravation of liver function after RFA therapy.
Hollow-Fiber Ultrafiltration and PCR Detection of Human-Associated Genetic Markers from Various Types of Surface Water in Florida
Authors: Leskinen Stephaney D. , Brownell Miriam , Lim Daniel V. , Harwood Valerie J.
Published: May 2010

Hollow-fiber ultrafiltration (HFUF) and PCR were combined to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF allowed marker detection when membrane filtration did not, demonstrating HFUF's ability to facilitate detection of diluted targets by PCR in a variety of water types.
Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain ofSaccharomyces cerevisiae
Authors: Zara Severino , Gross Michael K. , Zara Giacomo , Budroni Marilena , Bakalinsky Alan T.
Published: May 2010

Flor strains ofSaccharomyces cerevisiaeform a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.
RNA-Based Investigation of Ammonia-Oxidizing Archaea in Hot Springs of Yunnan Province, China
Authors: Jiang Hongchen , Huang Qiuyuan , Dong Hailiang , Wang Peng , Wang Fengping , Li Wenjun , Zhang Chuanlun
Published: May 2010

Using RNA-based techniques and hot spring samples collected from Yunnan Province, China, we show that the
amoA
gene of aerobic ammonia-oxidizing archaea can be transcribed at temperatures higher than 74°C and up to 94°C, suggesting that archaeal nitrification can potentially occur at near boiling temperatures.
Terrestrial Small Mammals as Reservoirs of
Mycobacterium ulcerans
in Benin
Authors: Durnez Lies , Suykerbuyk Patrick , Nicolas Violaine , Barrière Patrick , Verheyen Erik , Johnson Christian R. , Leirs Herwig , Portaels Françoise
Published: May 2010

Mycobacterium ulcerans
, the causative agent of Buruli ulcer (BU), is considered an environmental pathogen. Different mycobacteria were detected in 68 (12%) out of 565 small mammals collected in areas in Benin where BU is endemic. Although
M. ulcerans
was not found, we suggest that more research on
M. ulcerans
in African (small) mammals is needed.
High-Yield Intra- and Extracellular Protein Production Using
Bacillus megaterium
Authors: Stammen Simon , Müller Britta Katrin , Korneli Claudia , Biedendieck Rebekka , Gamer Martin , Franco-Lara Ezequiel , Jahn Dieter
Published: May 2010

The
Bacillus megaterium
protein production system based on the inducible promoter of the
xyl
operon (P

xylA

) was systematically optimized. Multiple changes in basic promoter elements, such as the −10 and −35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g
CDW
) or 124 mg liter
−1
. In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter
−1
, corresponding to 36.8 mg protein per g
CDW
. Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted
in silico
using the
B. megaterium
genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein
Thermobifida fusca
hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter
−1
(5.3 to 6.6 mg liter
−1
), was observed for strains where the Tfh export was facilitated by a codon-optimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter
−1
, 7.7 mg liter
−1
, was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.
Diversity of Oligotrichia and Choreotrichia Ciliates in Coastal Marine Sediments and in Overlying Plankton
Authors: Doherty Mary , Tamura Maiko , Vriezen Jan A. C. , McManus George B. , Katz Laura A.
Published: May 2010

Elucidating the relationship between ciliate communities in the benthos and the plankton is critical to understanding ciliate diversity in marine systems. Although data for many lineages are sparse, at least some members of the dominant marine ciliate clades Oligotrichia and Choreotrichia can be found in both plankton and benthos, in the latter either as cysts or active forms. In this study, we developed a molecular approach to address the relationship between the diversity of ciliates in the plankton and those of the underlying benthos in the same locations. Samples from plankton and sediments were compared across three sites along the New England coast, and additional subsamples were analyzed to assess reproducibility of methods. We found that sediment and plankton subsamples differed in their robustness to repeated subsampling. Sediment subsamples (i.e., 1-g aliquots from a single ∼20-g sample) gave variable estimates of diversity, while plankton subsamples produced consistent results. These results indicate the need for additional study to determine the spatial scale over which diversity varies in marine sediments. Clustering of phylogenetic types indicates that benthic assemblages of oligotrichs and choreotrichs appear to be more like those from spatially remote benthic communities than the ciliate communities sampled in the water above them.
Random Mutagenesis of
Clostridium cellulolyticum
by Using a Tn
1545
Derivative
Authors: Blouzard Jean-Charles , Valette Odile , Tardif Chantal , de Philip Pascale
Published: May 2010

Further understanding of the plant cell wall degradation system of
Clostridium cellulolyticum
and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn
1545
-derived delivery tool which allows monocopy random insertion within the genome.
Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples
Authors: Kirchner Sebastian , Krämer K. Melanie , Schulze Martin , Pauly Diana , Jacob Daniela , Gessler Frank , Nitsche Andreas , Dorner Brigitte G. , Dorner Martin B.
Published: May 2010

Botulinum neurotoxins are produced by the anaerobic bacteriumClostridium botulinumand are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenicC. botulinumserotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of theC. botulinumserotypes A, B, C, D, E, and F. Upon analysis of 42C. botulinumand 57 non-C. botulinumstrains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103and 105CFU/ml, respectively. Furthermore, we were able to detect C.botulinumin samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens forC. botulinumA, B, E, and F.
Role of Absolute Humidity in the Inactivation of Influenza Viruses on Stainless Steel Surfaces at Elevated Temperatures
Authors: McDevitt James , Rudnick Stephen , First Melvin , Spengler John
Published: May 2010

Influenza virus has been found to persist in the environment for hours to days, allowing for secondary transmission of influenza via inanimate objects known as fomites. We evaluated the efficacy of heat and moisture for the decontamination of surfaces for the purpose of preventing of the spread of influenza. Aqueous suspensions of influenza A virus were deposited onto stainless steel coupons, allowed to dry under ambient conditions, and exposed to temperatures of 55°C, 60°C, or 65°C and relative humidity (RH) of 25%, 50%, or 75% for up to 1 h. Quantitative virus assays were performed on the solution used to wash the viruses from these coupons, and results were compared with the solution used to wash coupons treated similarly but left under ambient conditions. Inactivation of influenza virus on surfaces increased with increasing temperature, RH, and exposure time. Reductions of greater than 5 logs of influenza virus on surfaces were achieved at temperatures of 60 and 65°C, exposure times of 30 and 60 min, and RH of 50 and 75%. Our data also suggest that absolute humidity is a better predictor of surface inactivation than RH and allows the prediction of survival using two parameters rather than three. Modest amounts of heat and adequate moisture can provide effective disinfection of surfaces while not harming surfaces, electrical systems, or mechanical components, leaving no harmful residues behind after treatment and requiring a relatively short amount of time.
Practical Method for Extraction of PCR-Quality DNA from Environmental Soil Samples
Authors: Fitzpatrick Kelly A. , Kersh Gilbert J. , Massung Robert F.
Published: May 2010

Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated.
Coxiella burnetii
DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.
Application of Paramagnetically Tagged Molecules for Magnetic Resonance Imaging of Biofilm Mass Transport Processes
Authors: Ramanan B. , Holmes W. M. , Sloan W. T. , Phoenix V. R.
Published: May 2010

Molecules become readily visible by magnetic resonance imaging (MRI) when labeled with a paramagnetic tag. Consequently, MRI can be used to image their transport through porous media. In this study, we demonstrated that this method could be applied to image mass transport processes in biofilms. The transport of a complex of gadolinium and diethylenetriamine pentaacetic acid (Gd-DTPA), a commercially available paramagnetic molecule, was imaged both in agar (as a homogeneous test system) and in a phototrophic biofilm. The images collected were
T
1
weighted, where
T
1
is an MRI property of the biofilm and is dependent on Gd-DTPA concentration. A calibration protocol was applied to convert
T
1
parameter maps into concentration maps, thus revealing the spatially resolved concentrations of this tracer at different time intervals. Comparing the data obtained from the agar experiment with data from a one-dimensional diffusion model revealed that transport of Gd-DTPA in agar was purely via diffusion, with a diffusion coefficient of 7.2 × 10
−10
m
2
s
−1
. In contrast, comparison of data from the phototrophic biofilm experiment with data from a two-dimensional diffusion model revealed that transport of Gd-DTPA inside the biofilm was by both diffusion and advection, equivalent to a diffusion coefficient of 1.04 × 10
−9
m
2
s
−1
. This technology can be used to further explore mass transport processes in biofilms, either by using the wide range of commercially available paramagnetically tagged molecules and nanoparticles or by using bespoke tagged molecules.
Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of
Clostridium botulinum
Neurotoxins and
Escherichia coli
O157
Authors: Brunt Jason , Webb Martin D. , Peck Michael W.
Published: May 2010

Existing methods for detection of food-borne pathogens and their toxins are frequently time-consuming, require specialized equipment, and involve lengthy culture procedures and/or animal testing and are thus unsuitable for a rapid response to an emergency public health situation. A series of simple and rapid affinity immunochromatography column (AICC) assays were developed to detect
Clostridium botulinum
neurotoxin types A, B, E, and F and
Escherichia coli
O157 in food matrices. Specifically, for milk, grape juice with peach juice, and bottled water, the detection limit for the botulinum neurotoxin type A complex was 0.5 ng. Use of this method with a 10-ml sample would therefore result in a detection limit of 50 pg ml
−l
. Thus, this assay is approximately 2 orders of magnitude more sensitive than a comparable lateral-flow assay. For botulinum neurotoxin complex types B, E, and F, the minimum detection limit was 5 ng to 50 ng. Sensitive detection of
E. coli
O157 was achieved, and the detection limit was 500 cells. The AICC test was also shown to be specific, rapid, and user friendly. This test takes only 15 to 30 min to complete without any specialized equipment and thus is suitable for use in the field. It has the potential to replace existing methods for presumptive detection of botulinum neurotoxin types A, B, E, and F and
E. coli
O157 in contaminated matrices without a requirement for preenrichment.
Commonalities and Differences in Regulation of
N
-Acyl Homoserine Lactone Quorum Sensing in the Beneficial Plant-Associated
Burkholderia
Species Cluster
Authors: Suárez-Moreno Zulma Rocío , Devescovi Giulia , Myers Mike , Hallack Letícia , Mendonça-Previato Lucia , Caballero-Mellado Jesús , Venturi Vittorio
Published: May 2010

The genus
Burkholderia
includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as
Burkholderia glumae
,
B. pseudomallei
, and
B. mallei
and 17 well-studied species of the
Burkholderia cepacia
complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that
Burkholderia kururiensis
, a member of the latter cluster, possesses an
N
-acyl homoserine lactone (AHL) quorum-sensing (QS) system designated “BraI/R,” which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in
B. xenovorans
and
B. unamae
and were designated the BraI/R
XEN
and BraI/R
UNA
systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species
B. kururiensis
,
B. unamae
, and
B. xenovorans
, highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in
B. xenovorans
LB400
T
. The two AHL QS systems of
B. xenovorans
are not transcriptionally regulating each other, whereas BxeR solo negatively regulated
xenI2
. The XenI2/R2 and BxeR solo proteins are not widespread in the
Burkholderia
species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the
Burkholderia
plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.
Functional Characterization of
MAT1
-
1
-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators
Authors: Klix V. , Nowrousian M. , Ringelberg C. , Loros J. J. , Dunlap J. C. , Pöggeler S.
Published: May 2010

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes,
SmtA
-
1
(
MAT1
-
1
-
1
),
SmtA
-
2
(
MAT1
-
1
-
2
), and
SmtA
-
3
(
MAT1
-
1
-
3
), contained in the
MAT1
-
1
part of the mating-type locus of the homothallic ascomycete species
Sordaria macrospora
. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene
SmtA
-
2
is essential for sexual reproduction, whereas the α domain protein-encoding genes
SmtA
-
1
and
SmtA
-
3
play no role in fruiting-body development. By means of cross-species microarray analysis using
Neurospora crassa
oligonucleotide microarrays hybridized with
S. macrospora
targets and quantitative real-time PCR, we identified genes expressed under the control of
SmtA
-
1
and
SmtA
-
2
. Both genes are involved in the regulation of gene expression, including that of pheromone genes.
Adaptations of Candida albicans for Growth in the Mammalian Intestinal Tract
Authors: Rosenbach Ari , Dignard Daniel , Pierce Jessica V. , Whiteway Malcolm , Kumamoto Carol A.
Published: May 2010

Although the fungus
Candida albicans
is a commensal colonizer of humans, the organism is also an important opportunistic pathogen. Most infections caused by
C. albicans
arise from organisms that were previously colonizing the host as commensals, and therefore successful establishment of colonization is a prerequisite for pathogenicity. To elucidate fungal activities that promote colonization, an analysis of the transcription profile of
C. albicans
cells recovered from the intestinal tracts of mice was performed. The results showed that within the
C. albicans
colonizing population, cells expressed genes characteristic of the laboratory-grown exponential phase and genes characteristic of post-exponential-phase cells. Thus, gene expression both promoted the ability to grow rapidly (a characteristic of exponential-phase cells) and enhanced the ability to resist stresses (a characteristic of post-exponential-phase cells). Similarities in gene expression in commensal colonizing cells and cells invading host tissue during disease were found, showing that
C. albicans
cells adopt a particular cell surface when growing within a host in both situations. In addition, transcription factors Cph2p and Tec1p were shown to regulate
C. albicans
gene expression during intestinal colonization.
Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton
Authors: Sweeney Kristin R. , Morrissette Naomi S. , LaChapelle Stephanie , Blader Ira J.
Published: May 2010

Toxoplasma gondiiis an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used byToxoplasmato rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.
The Frequency of Persisters in
Escherichia coli
Reflects the Kinetics of Awakening from Dormancy
Authors: Jõers Arvi , Kaldalu Niilo , Tenson Tanel
Published: May 2010

A genetically homogenous bacterial population may contain physiologically distinct subpopulations. In one such case, a minor part of an otherwise antibiotic-sensitive bacterial population maintains a nondividing state even in a growth-supporting environment and is therefore not killed by bactericidal antibiotics. This phenomenon, called persistence, can lead to failure of antibiotic treatment. We followed the development of sensitivity to killing by ampicillin and norfloxacin when
Escherichia coli
cells were transferred from a stationary-phase culture into fresh growth medium. In parallel, we monitored growth resumption by individual bacteria. We found that bacteria in a population resumed growth and became sensitive to antibiotics at different times after transfer to fresh medium. Moreover, both growing and dormant bacteria coexisted in the same culture for many hours. The kinetics of awakening was strongly influenced by growth conditions: inocula taken from the same stationary-phase culture led to very different persister frequencies when they were transferred into different fresh media. Bactericidal antibiotics kill cells that have woken up, but the later-awakening subpopulation is tolerant to them and can be identified as persisters when the antibiotic is removed. Our observations demonstrate that persister count is a dynamic measure and that the persister frequency of a particular culture is not a fixed value.
Analysis of the Mechanism of Action of the Antisense RNA That Controls the Replication of the
repABC
Plasmid p42d
Authors: Cervantes-Rivera Ramón , Romero-López Cristina , Berzal-Herranz Alfredo , Cevallos Miguel A.
Published: May 2010

Replication and segregation of the
Rhizobium etli
symbiotic plasmid (pRetCFN42d) depend on the presence of a
repABC
operon, which carries all the plasmid-encoded elements required for these functions. All
repABC
operons share three protein-encoding genes (
repA
,
repB
, and
repC
), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (
parS
). The products of
repA
and
repB
, in conjunction with the
parS
region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon,
repC
, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of
repC
. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5′ end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in
R. etli
.
Functional Characterization of a Phospholipase A
2
Homolog from
Rickettsia typhi
Authors: Rahman M. Sayeedur , Ammerman Nicole C. , Sears Khandra T. , Ceraul Shane M. , Azad Abdu F.
Published: May 2010

Phospholipase A
2
(PLA
2
) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA
2
activity has not been identified or functionally characterized. Here, we report that the
Rickettsia typhi
genome possesses two genes encoding patatin-like PLA
2
proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA
2
activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of
R. typhi
in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during
R. typhi
infection of host cells.
In silico
, RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA
2
inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from
Rickettsia typhi
with functional phospholipase A activity.