bluuebunny/crossref_metadata_2025 huggingface dataset:
Original Research—Outcomes Assessment: Development and Validation of a 6-Item Version of the Female Sexual Function Index (FSFI) as a Diagnostic Tool for Female Sexual Dysfunction
Authors: Isidori Andrea M. , Pozza Carlotta , Esposito Katherine , Giugliano Dario , Morano Susanna , Vignozzi Linda , Corona Giovanni , Lenzi Andrea , Jannini Emmanuele A.
Published: December 2009

Introduction

A limiting step in the evaluation of female sexual dysfunction (FSD) is the availability of a rapid screening procedure. Often, practitioners avoid investigating sexual symptoms due to concerns of insufficient time or lack of proper tools to address FSD.

Aim

The purpose of this study was to prepare and validate an abridged form of the most popular psychometric diagnostic test (Female Sexual Function Index, FSFI-19) to provide a fast screener of FSD for easy use in outpatient visits, epidemiological studies, and assessment of treatment response.

Methods

We interviewed and administered the FSFI-19 to 200 women attending outpatient clinics for sexual and reproductive medicine. Forty women were excluded because they had no sexual activity or failed to attend the retest visit. Patients were evaluated on two subsequent visits to validate the abridged form of the questionnaire. Overall, 105 were found to suffer from a FSD.

Main Outcome Measure

We assessed, individually, the sensibility and sensitivity of all questions of the full-length FSFI. We then estimated the performance of each item with respect to the specific sexual domain they address. By selecting the best combination of performing items in each domain, we built an abridged, 6-item form of the FSFI.

Results

The Receiver Operating Characteristic curves of the FSFI-6 showed that women who scored ≤19 were classified as having FSD. Using the cut-off of 19, the sensitivity and specificity of the test were, 0.93 and 0.94, respectively. Reliability, internal consistency, and stability on retest were also good.

Conclusions

The abridged FSFI-6 is a valuable tool for screening women that are likely to suffer from FSD. In six simple questions, taking no more than 3 minutes, a score of less than 19 indicates the need for further investigations, including the full-length FSFI-19 and a dedicated interview. In conclusion, this is a novel tool that can help any doctor to disclose FSD rapidly and efficiently.
INFLUENCE OF PARTIAL REPLACEMENT OF OLIVE OIL ON FRYING PERFORMANCE OF PALM OLEIN
Authors: NAGHSHINEH M. , ARIFFIN A.A. , GHAZALI H.M. , MIRHOSSEINI H. , MOHAMMAD A.S. , KUNTOM A.
Published: November 2009

The influence of partial replacement of palm olein (POo) with olive oil (Oo), (25 and 50%, w/w) was investigated during five consecutive days of frying. The results indicated that frying performance of POo was significantly (P < 0.05) influenced by partial replacement with olive oil. The highest change in peroxide value (PV), anisidine value (AV), totox value (TV), total polar compound (TPC), viscosity and melting point was shown by the control sample; whereas the replacement of 50% (w/w) palm olein with 50% (w/w) olive oil exhibited the least changes in PV, AV, TV, TPC, viscosity and melting point during the frying process. This study suggests that the partial replacement of palm olein containing a high proportion of saturated fatty acids (i.e., palmitic acid) with olive oil containing a high content of monounsaturated fatty acid (i.e., oleic acid) can provide oil blends with higher chemical stability against oxidation. On the other hand, the prepared oil blend remained liquid at ambient temperature, thereby enhancing the physical stability induced by partial replacement with olive oil.

PRACTICAL APPLICATIONS

Partial replacement of palm olein with olive oil containing high oleic acid can improve the oxidative stability of palm olein during the frying process compared with regular palm olein. Therefore, the proposed oil blend can be suggested as a suitable replacement for pure palm olein for frying and cooking purposes by providing a higher oxidative stability besides the beneficial effects of olive oil on human health.
MONITORING OF VIRGIN COCONUT OIL (VCO) ADULTERATION WITH PALM OIL USING FOURIER TRANSFORM INFRARED SPECTROSCOPY
Authors: ROHMAN A. , CHE MAN Y.B.
Published: November 2009

Virgin coconut oil (VCO) may be adulterated with cheaper oils, such as palm oil (PO). Thus, the detection and quantification of VCO adulteration with PO was monitored using Fourier transform infrared (FTIR) spectroscopy, combined with chemometrics of partial least square (PLS) and discriminant analysis at frequency regions of 3,010–3,000, 1,660–1,650 and 1,120–1,105/cm. Attenuated total reflectance (ATR) measurements were made on pure VCO and that adulterated with varying concentrations of PO (0.5–50% w/w in VCO). PLS calibration exhibited a good relationship between actual and FTIR‐predicted values with coefficient of determination (R2) of 0.999 and standard error of calibration of 0.533. The cross validation was performed by removing one standard at a time, and the final R2 value of 0.996 and standard error of prediction of 0.953 were obtained. The discriminant analysis using seven principal components was able to classify pure VCO and that adulterated with PO.

PRACTICAL APPLICATIONS

The adulteration of virgin coconut oil (VCO) with cheaper oils is a serious matter, not only for food suppliers but also for consumers. Therefore, an analytical technique offering fast and reliable detection of such adulteration must be developed. Fourier transform infrared spectroscopy combined with chemometrics of partial least square for quantitative analysis of adulterant, and discriminant analysis for classification between VCO and that adulterated with palm oil, can be employed for detection of palm oil as an adulterant in VCO for quality assurance purposes.
Oral magnesium supplementation in adults with coronary heart disease or coronary heart disease risk
Authors: Mathers Tavia W. , Beckstrand Renea L.
Published: December 2009

Purpose: To review randomized control clinical trial (RCT) literature and prospective studies for the safety and efficacy of magnesium supplements in patients with coronary heart disease (CHD) or with CHD risk.

Data sources: Databases were searched using the keywords: magnesium, heart disease, endothelium, prevention, treatment, therapy, level, and supplement.

Conclusions: There were no reports of adverse effects from magnesium supplementation in any of the studies. Subjects reporting lower dietary magnesium intake had significantly lower serum magnesium concentrations than those reporting higher dietary magnesium intake and, in some cases, had a significantly higher frequency of supraventricular beats. There was a modest relationship between dietary magnesium intake and a reduced risk of CHD in male subjects; however, there was no noted decrease in the development of CHD disease in women who had high magnesium intake.

Implications for practice: Magnesium is vital for many functions in the body and magnesium supplementation is safe. There is a possible association between a modestly lower risk of CHD in men and increased magnesium intake; therefore, it is reasonable to encourage diets high in magnesium as a potential means to lower the risk of CHD.
Endothelin attenuates endothelium‐dependent platelet inhibition in man
Authors: Malmström R. E. , Settergren M. , Pernow J.
Published: November 2009

Aim: The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin‐1 (ET‐1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET‐1 on endothelium‐dependent vasodilatation and attenuation of platelet activation.

Methods: In 25 healthy male subjects (25 ± 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P‐selectin expression) in unstimulated and adenosine diphosphate (ADP)‐stimulated samples during administration of ET‐1, the endothelium‐dependent vasodilator acetylcholine and the NO synthase inhibitorl‐NMMA.

Results: Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP‐stimulated samples. In samples stimulated with 0.3 μmADP, fibrinogen binding decreased from 41 ± 4% to 31 ± 3% (P < 0.01,n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET‐1 andl‐NMMA. ET‐1 did not affect platelet activityper se, whereasl‐NMMA increased platelet P‐selectin expression. Both ET‐1 andl‐NMMA attenuated the acetylcholine‐induced inhibition of platelet activity.

Conclusions: Our study indicates that, further to inhibiting endothelium‐dependent vasodilatation, ET‐1 may also attenuate endothelium‐dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET‐1 activity, as suggested in endothelial dysfunction, may affect endothelium‐dependent platelet modulation and thereby have pathophysiological implications.
Gall bladder mucoceles and their association with endocrinopathies in dogs: a retrospective case‐control study
Authors: Mesich M. L. L. , Mayhew P. D. , Paek M. , Holt D. E. , Brown D. C.
Published: December 2009

Objective: To investigate the relationship between endocrinopathies and diagnosis of gall bladder mucocele in dogs via a retrospective case‐control study.

Methods: Records of 78 dogs with a surgical or ultrasonographic diagnosis of gall bladder mucocele were examined for the presence or absence of hyperadrenocorticism, hypothyroidism and diabetes mellitus. Two age‐ and breed‐matched controls for each gall bladder mucocele dog (156 total control dogs) were examined for the same concurrent diseases. A matched case‐control analysis was performed using conditional logistic regression.

Results: The odds of mucocele in dogs with hyperadrenocorticism were 29 times that of dogs without hyperadrenocorticism (P=0·001; 95 per cent CI 3·8, 219·9). No difference was found between dogs with and without diabetes mellitus. Although a significant association was found between gall bladder mucocele and hypothyroidism, potential observation bias was also identifi ed.

Clinical Significance: Hyperadrenocorticoid dogs that were presented for acute illness with laboratory evidence of hepatobiliary disease should undergo evaluation for the presence of a biliary mucocele. Dogs diagnosed with a gall bladder mucocele should be screened for concurrent hyperadrenocorticism if clinical suspicion exists.
Prognostic significance of specific magnetic resonance imaging features in canine nasal tumours treated by radiotherapy
Authors: Agthe P. , Caine A. R. , Gear R. N. A. , Dobson J. M. , Richardson K. J. , Herrtage M. E.
Published: December 2009

Objectives:To investigate the prognostic significance of the magnetic resonance (MR) findings of meningeal hyperintensity of the olfactory bulbs and tumour extension into the caudal nasal recess (CNR) in dogs with nasal tumours treated by radiotherapy.

Methods:MR images of 41 dogs with nasal tumours treated with radiotherapy were reviewed. The occurrence of neurological signs and survival of patients with and without meningeal hyperintensity of the olfactory bulbs and tumour extension into the CNR were analysed together with possible confounding factors including intracranial extension and patient age.

Results:There was no significant association between the presence of meningeal hyperintensity or CNR involvement and the occurrence of neurological signs. Although there was a tendency towards shorter survival in dogs with tumour extension into the CNR, multivariable analysis showed no significant difference in survival between dogs with/without CNR involvement, meningeal hyperintensity or intracranial tumour extension (P=0·12, 0·50 and 0·57, respectively).

ClinicalSignificance:In dogs with nasal tumours treated with radiotherapy, tumour extension into the cranium is not necessarily associated with shorter survival in patients without neurological signs at time of diagnosis. Although a definite influence of CNR involvement on case outcome could not be demonstrated, studies with a larger population are warranted.
Oesophageal and gastric endoscopic foreign body removal: complications and follow‐up of 102 dogs
Authors: Gianella P. , Pfammatter N. S. , Burgener I. A.
Published: December 2009

Objectives:To assess complication rate, risk factors for complications and outcome in dogs with oesophageal and gastric endoscopic foreign body (FB) removal.

Methods:Medical records of 102 dogs undergoing endoscopic removal of oesophageal and/or gastric FBs from March 2001 to November 2006 were retrospectively reviewed. All owners were contacted by telephone to provide follow‐up information.

Results:West Highland white terriers, Yorkshire terriers and Bernese mountain dogs were over‐represented compared to the hospital population. Endoscopy alone was successful in 92/102 dogs (90·2 per cent), whereas gastrotomy (but no oesophagotomy) was required in 10 dogs (9·8 per cent). Complications in 13/102 dogs (12·7 per cent) were perforation (8), oesophageal stricture (1), oesophageal diverticula (1), perioesophageal abscess (1), pneumothorax and pleural effusion (1) and respiratory arrest (1). Six dogs (all weighing <10 kg) had complications resulting in death or euthanasia. Bone FBs, bodyweight of less than 10 kg, and oesophageal or gastric FB in place for more than three days were significant risk factors for complications. Of the dogs available for follow‐up (75/96), 92 per cent had no complications after discharge.

ClinicalSignificance:Endoscopic FB removal is associated with a low overall complication rate with bone FBs and bodyweight of less than 10 kg as significant risk factors.
Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe
Authors: Dantas‐Torres F. , Lia R. P. , Barbuto M. , Casiraghi M. , Crovace A. , Caligiani L. , Genchi C. , Otranto D.
Published: December 2009

A five‐year‐old, entire female mixed‐breed dog was presented with corneal oedema and episcleral hyperaemia in the left eye. The ophthalmological examination revealed the presence of a free‐swimming nematode in the anterior chamber. Circulating microfilariae were not observed by a modified Knott test nor were adult antigens detected in serum by a commercial ELISA. The parasite was surgically removed from the dog's eye, but its anterior end was damaged during the surgery. Based on the morphology of the posterior end, the nematode was preliminarily identified as a male Dirofilaria immitis. The species identification was confirmed by PCR amplification and sequencing of the mitochondrial coxI and 12S rDNA genes, using a DNA barcoding approach. Although other cases of ocular dirofilariosis by D. immitis have been previously recorded in Australia and the United States, the case reported herein is the first in a dog from Europ.
Expression of KIT Receptor Tyrosine Kinase in Endothelial Cells of Juvenile Brain Tumors
Authors: Puputti Marjut , Tynninen Olli , Pernilä Paula , Salmi Marko , Jalkanen Sirpa , Paetau Anders , Sihto Harri , Joensuu Heikki
Published: December 2009

KIT receptor tyrosine kinase is expressed in tumor endothelial cells of adult glioblastomas, but its expression in pediatric brain tumor endothelial cells is unknown. We assessed expression of KIT, phosphorylated KIT, stem cell factor (SCF) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) in 35 juvenile pilocytic astrocytomas and 49 other pediatric brain tumors using immunohistochemistry, and KIT messenger RNA (mRNA) using in situ hybridization. KIT and phospho‐KIT were moderately or strongly expressed in tumor endothelia of 37% and 35% of pilocytic astrocytomas, respectively, whereas marked SCF and VEGFR‐2 expression was uncommon. KIT mRNA was detected in tumor endothelial cells. Tumor endothelial cell KIT expression was strongly (P < 0.01) associated with endothelial cell phospho‐KIT and SCF expression, and with tumor KIT (P = 0.0011) and VEGFR‐2 expression (P = 0.022). KIT and phospho‐KIT were present in endothelia of other pediatric brain tumors, notably ependymomas. Endothelial cell KIT expression was associated with a young age at diagnosis of pilocytic astrocytoma or ependymoma, and it was occasionally present in histologically normal tissue of the fetus and children. We conclude that KIT is commonly present in endothelial cells of juvenile brain tumors and thus may play a role in angiogenesis in these neoplasms.
Hydrogen silsesquioxane double patterning process for 12nm resolution x-ray zone plates
Authors: Chao Weilun , Kim Jihoon , Rekawa Senajith , Fischer Peter , Anderson Erik
Published: December 2009

Soft x-ray zone plate microscopy is a powerful nanoanalytic technique used for a wide variety of scientific and technological studies. Pushing its spatial resolution to 10nm and below is highly desired and feasible due to the short wavelength of soft x rays. Instruments using Fresnel zone plate lenses achieve a spatial resolution approximately equal to the smallest, outermost zone width. In this work, a double patterning zone plate fabrication process is developed. based on a high resolution resist, hydrogen silsesquioxane (HSQ), to bypass the limitations of conventional single exposure fabrication to pattern density, such as finite beam size, scattering in resist, and modest intrinsic resist contrast. To fabricate HSQ structures with zone widths on the order of 10nm on gold plating base, a surface conditioning process with (3-mercaptopropyl) trimethoxysilane, 3-MPT, is used, which forms a homogeneous hydroxylation surface on gold surface and provides good anchoring for the desired HSQ structures. Using the new HSQ double patterning process, coupled with an internally developed, subpixel alignment algorithm, the authors have successfully fabricated in-house gold zone plates of 12nm outer zones. Promising results for 10nm zone plates have also been obtained. With the 12nm zone plates, they have achieved a resolution of 12nm using the full-field soft x-ray microscope, XM-1.
Hydrogen silsesquioxane-based hybrid electron beam and optical lithography for high density circuit prototyping
Authors: Guillorn M. , Chang J. , Fuller N. , Patel J. , Darnon M. , Pyzyna A. , Joseph E. , Engelmann S. , Ott J. , Newbury J. , Klaus D. , Bucchignano J. , Joshi P. , Scerbo C. , Kratschmer E. , Graham W. , To B. , Parisi J. , Zhang Y. , Haensch W.
Published: December 2009

The authors report on a hybrid lithography process that integrates electron beam lithography (EBL) and optical photolithography. To maximize resolution and density, the EBL exposure is performed using a hydrogen silsesquioxane-based resist, while the photolithographic exposure is performed using standard positive or negative tone 248nm photoresists. Both exposures take place on a common underlayer consisting of an antireflective coating (ARC). During pattern transfer into the ARC layer, a composite image of the two lithographic exposures is formed creating a robust and versatile etch mask for further pattern transfer into the substrate. They demonstrate the utility of this technique by using it to pattern the active, gate, and wiring levels of complementary metal oxide semiconductor devices and circuits consisting of trigated Fin-based field effect transistors. These devices have a minimum active area pitch of 50nm, minimum gate pitch of 90nm, and achieve densities suitable for 15nm node static random access memory cells. Details of the exposure process and device fabrication are discussed along with electrical results from the resulting devices and circuits built using this technique.
Photoresist removal using an O2∕N2 medium pressure plasma jet with high speed wafer scanning: Unimplanted resist studies
Authors: Bhargava M. , Craver B. , Torres Jose L. , Guo H. , Vemula S. C. , Srivastava A. K. , Berry I. , Wolfe J. C.
Published: December 2009

The authors describe a plasma ashing system where a stationary jet of hot, activated gases removes photoresist from a scanning wafer. The jet is created by a reactant stream flowing through a 2.45GHz surface wave discharge in a 6mm quartz tube. For O2∕N2 plasmas in the medium pressure range from 20to100Torr, a luminous plasma jet emerges from the end of the discharge tube that transports both heat and reactive species to the wafer. A single scan results in a Gaussian track profile with a standard deviation of 7mm for the source-to-substrate distance of 9mm. A simple model of the ashing process, which assumes a thermally activated ash rate and Gaussian distributions for both power density and reactant flux, unifies the dependence of effective ash rate on the substrate temperature and scan speed at a constant power. The best fit activation energy at 2.5kW is 0.23eV, about half of the value found in conventional downstream ashing, implying that diffusion plays a significant role in limiting the ash rate. The peak thermal power density in a 2.5kW jet at 80Torr is 160W∕cm2, resulting in an effective instantaneous ash rate of 2.5mm∕min for a scan speed of 70cm∕s and 200°C chuck temperature. This implies that the time to clear a 1.2μm thick resist coating from a 300mm wafer is 18s.
Study on line edge roughness for electron beam acceleration voltages from 50to5kV
Authors: Rio D. , Constancias C. , Saied M. , Icard B. , Pain L.
Published: December 2009

Electrical characteristics of devices depend on the line edge roughness (LER). LER contributes to the off-state leakage budget and short-channel effect. Therefore, it has to be controlled during the lithography step since it strongly impacts the final component’s roughness. This work aims at the characterization of LER dependence on the beam acceleration voltage. Usually publications consider many lithography parameters that can influence LER. Different resists were exposed to beam acceleration voltage from 5to50kV. Thus, only two parameters that influence LER varied during experiments for a given resist: the exposure dose (i.e., shot noise) and the spot size. Then, simulations were carried out with those parameter variations. The impact of other LER contributors was considered as constant. Comparison between model and experimental results allows one to link resist sensitivity, exposure time, and LER.
Robust, efficient grating couplers for planar optical waveguides using no-photoacid generator SU-8 electron beam lithography
Authors: Bross A. L. , Lafyatis G. , Ayachitula R. , Morss A. , Hardman R. , Golden J.
Published: December 2009

450 nm pitch coupling gratings with greater than 10% input coupling efficiency have been fabricated on thin film planar optical waveguides using electron beam lithography on a novel SU-8 resist formulation that contains no photoacid generator (PAG). Even without the PAG component, we found the sensitivity of this material to be unexpectedly high, with a dose to clear of less than 10 μC/cm2 at 50kV for our substrates. Our optical waveguides consisted of 350 nm Ta2O5 ion beam sputtered onto superpolished fused silica substrates. The grating fabrication flow was designed to minimize process steps on the Ta2O5 surface and ensure that the original low surface roughness was sufficiently maintained after processing.
Why and How Bacteria Localize Proteins
Authors: Shapiro L. , McAdams H. H. , Losick R.
Published: November 2009

Despite their small size, bacteria have a remarkably intricate internal organization. Bacteria deploy proteins and protein complexes to particular locations and do so in a dynamic manner in lockstep with the organized deployment of their chromosome. The dynamic subcellular localization of protein complexes is an integral feature of regulatory processes of bacterial cells.
A High-Resolution Structure of the Pre-microRNA Nuclear Export Machinery
Authors: Okada Chimari , Yamashita Eiki , Lee Soo Jae , Shibata Satoshi , Katahira Jun , Nakagawa Atsushi , Yoneda Yoshihiro , Tsukihara Tomitake
Published: November 2009

Pre-MicroRNA Export Machinery

Micro (mi) RNAs play a role in the regulation of many biological processes. Long transcripts are initially processed in the nucleus to yield pre-miRNAs that are translocated through the nuclear pore complex and further processed to mature miRNAs in the cytoplasm.

Okada
et al.

(p.
1275
; see the Perspective by

Stewart

) describe the crystal structure of pre-miRNA complexed with the exportin Exp5 and the small nuclear GTPase RanGTP. The structure shows that Exp5 and RanGTP protect the miRNA from degradation by nucleases, as well as facilitate transport to the cytoplasm. RNA recognition is mainly through ionic interactions that are sequence independent, and model-building suggests that this nuclear export machinery could accommodate other small-structured RNAs.
Comparative Pharmacokinetics of Ceftaroline in Rats, Rabbits, and Monkeys following a Single Intravenous or Intramuscular Injection
Authors: Ge Yigong , Maynard David , Rickert Douglas E.
Published: December 2009

This study assessed the pharmacokinetic profiles for intramuscular and intravenous ceftaroline treatment for rats, rabbits, and monkeys. Ceftaroline, a novel cephalosporin with broad-spectrum activity against Gram-positive and Gram-negative pathogens, demonstrated favorable pharmacokinetic profiles following intramuscular administration in all 3 animal species, comparable to the levels for intravenous dosing. The areas under the plasma concentration-time curve obtained after intramuscular administration were increased in rats and similar in rabbits and monkeys, compared with the levels obtained after intravenous dosing (129%, 7.29%, and 12.7% greater in rats, rabbits, and monkeys, respectively). The data reported here support the development of an intramuscular formulation for ceftaroline.
Ex Vivo
Comparison of Microbicide Efficacies for Preventing HIV-1 Genomic Integration in Intraepithelial Vaginal Cells
Authors: McElrath M. Juliana , Ballweber Lamar , Terker Andrew , Kreger Allison , Sakchalathorn Polachai , Robinson Barry , Fialkow Michael , Lentz Gretchen , Hladik Florian
Published: December 2009

Vaginally applied microbicides hold promise as a strategy to prevent sexual HIV transmission. Several nonspecific microbicides, including the polyanion cellulose sulfate, have been evaluated in large-scale clinical trials but have failed to show significant efficacy. These findings have prompted a renewed search for preclinical testing systems that can predict negative outcomes of microbicide trials. Moreover, the pipeline of potential topical microbicides has been expanded to include antiretroviral agents, such as reverse transcriptase, fusion, and integrase inhibitors. Using a novel
ex vivo
model of vaginal HIV-1 infection, we compared the prophylactic potentials of two forms of the fusion inhibitor T-20, the CCR5 antagonist TAK-778, the integrase inhibitor 118-D-24, and cellulose sulfate (Ushercell). The T-20 peptide with free N- and C-terminal amino acids was the most efficacious compound, causing significantly greater inhibition of viral genomic integration in intraepithelial vaginal leukocytes, measured by an optimized real-time PCR assay, than the more water-soluble N-acetylated T-20 peptide (Fuzeon) (50% inhibitory concentration [IC
50
], 0.153 μM versus 51.2 μM [0.687 ng/ml versus 230 ng/ml];
P
< 0.0001). In contrast, no significant difference in IC
50
s was noted in peripheral blood cells (IC
50
, 13.58 μM versus 7.57 μM [61 ng/ml versus 34 ng/ml];
P
= 0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC
50
, 1.8 μg/ml). These results highlight the merit of our model for screening the mucosal efficacies of novel microbicides and their formulations and potentially rank ordering candidates for clinical evaluation.
Bortezomib Inhibits Hepatitis B Virus Replication in Transgenic Mice
Authors: Bandi Prasanthi , Garcia Mayra L. , Booth Carmen J. , Chisari Francis V. , Robek Michael D.
Published: December 2009

Pharmacological modulation of cellular proteins as a means to block virus replication has been proposed as an alternative antiviral strategy that may be less susceptible than others to the development of viral drug resistance. Recent evidence indicates that the ubiquitin-proteasome pathway interacts with different aspects of the hepatitis B virus (HBV) life cycle in cell culture models of virus replication. We therefore examined the effect of proteasome inhibition on HBV replicationin vivousing HBV transgenic mice. The proteasome inhibitor bortezomib (Velcade) inhibits proteasome activityin vivoand is used therapeutically for the clinical treatment of multiple myeloma. We found that a single intravenous dose of 1 mg of bortezomib/kg of body weight reduced virus replication for as long as 6 days. The inhibition of HBV by bortezomib was dose dependent and occurred at a step in replication subsequent to viral RNA and protein expression. The reduction in HBV replication did not result from nonspecific hepatocellular toxicity and was not mediated indirectly through the induction of an intrahepatic interferon response. Thus, pharmacological manipulation of the ubiquitin-proteasome pathway may represent an alternative therapeutic approach for the treatment of chronic HBV infection.
In Vitro
Adsorption of Gentamicin and Netilmicin by Polyacrylonitrile and Polyamide Hemofiltration Filters
Authors: K.N. Lam Philip , Tian Qi , Ip Margaret , Gomersall Charles D.
Published: December 2009

Adsorption of gentamicin and netilmicin by new polyacrylonitrile and polyamide hemofiltration filters was studied over 4 h, using a single-compartment
in vitro
continuous venovenous hemofiltration model. After the first dose (16.6 mg of gentamicin, 19.3 mg of netilmicin), 14.9 mg of gentamicin and 19.2 mg of netilmicin were adsorbed to polyacrylonitrile filters
in vitro
. Adsorption by polyacrylonitrile filters was rapid and irreversible and could be increased by repeated dosing. Adsorption by polyamide filters was substantially less.
Detection and Characterization of Heterogeneous Vancomycin-Intermediate
Staphylococcus aureus
Isolates in Canada: Results from the Canadian Nosocomial Infection Surveillance Program, 1995-2006
Authors: Adam Heather J. , Louie Lisa , Watt Christine , Gravel Denise , Bryce Elizabeth , Loeb Mark , Matlow Anne , McGeer Allison , Mulvey Michael R. , Simor Andrew E.
Published: December 2009

We describe the epidemiology of heterogeneously resistant
Staphylococcus aureus
(hVISA) identified in Canadian hospitals between 1995 and 2006. hVISA isolates were confirmed by the population analysis profiling-area under the curve method. Only 25 hVISA isolates (1.3% of all isolates) were detected. hVISA isolates were more likely to have been health care associated (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.9 to 14.2) and to have been recovered from patients hospitalized in central Canada (OR, 3.0; 95% CI, 1.2 to 7.4). There has been no evidence of vancomycin “MIC creep” in Canadian strains of methicillin (meticillin)-resistant
S. aureus
, and hVISA strains are currently uncommon.
Deep Sequencing of a Dimethylsulfoniopropionate-Degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data
Authors: Varaljay Vanessa A. , Howard Erinn C. , Sun Shulei , Moran Mary Ann
Published: December 2009

In silicodesign and testing of environmental primer pairs with metagenomic data are beneficial for capturing a greater proportion of the natural sequence heterogeneity in microbial functional genes, as well as for understanding limitations of existing primer sets that were designed from more restricted sequence data. PCR primer pairs targeting 10 environmental clades and subclades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of thousands ofdmdAsequences captured in marine metagenomic data sets. Using the bioinformatically optimized primers,dmdAgenes were amplified from composite free-living coastal bacterioplankton DNA (from 38 samples over 5 years and two locations) and sequenced using 454 technology. An average of 6,400 amplicons per primer pair represented more than 700 clusters of environmentaldmdAsequences across all primers, with clusters defined conservatively at >90% nucleotide sequence identity (∼95% amino acid identity). Degenerate and inosine-based primers did not perform better than specific primer pairs in determiningdmdArichness and sometimes captured a lower degree of richness of sequences from the same DNA sample. A comparison ofdmdAsequences in free-living versus particle-associated bacteria in southeastern U.S. coastal waters showed that sequence richness in somedmdAsubgroups differed significantly between size fractions, though most gene clusters were shared (52 to 91%) and most sequences were affiliated with the shared clusters (∼90%). The availability of metagenomic sequence data has significantly enhanced the design of quantitative PCR primer pairs for this key functional gene, providing robust access to the capabilities and activities of DMSP demethylating bacteriain situ.
Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by
Escherichia coli
O157:H7 Strains
Authors: Zhang Yongxiang , Laing Chad , Zhang Zhengzhong , Hallewell Jennyka , You Chunping , Ziebell Kim , Johnson Roger P. , Kropinski Andrew M. , Thomas James E. , Karmali Mohamed , Gannon Victor P. J.
Published: December 2009

Escherichia coli
O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between
E. coli
O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated “mobile genetic elements” such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the
stx
2
flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of
stx
2
) is replaced by a
pphA
(serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried
stx
2
, whereas all LII strains carried variant
stx
2c
and 4 of 14 LI/II strains had copies of both
stx
2
and variant
stx
2c
. Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more
stx
2
mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among
E. coli
O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of
E. coli
O157:H7 strains with human clinical disease.
Searching for a “Hidden” Prophage in a Marine Bacterium
Authors: Zhao Yanlin , Wang Kui , Ackermann Hans-Wolfgang , Halden Rolf U. , Jiao Nianzhi , Chen Feng
Published: December 2009

Prophages are common in many bacterial genomes. Distinguishing putatively viable prophages from nonviable sequences can be a challenge, since some prophages are remnants of once-functional prophages that have been rendered inactive by mutational changes. In some cases, a putative prophage may be missed due to the lack of recognizable prophage loci. The genome of a marine roseobacter,Roseovarius nubinhibensISM (hereinafter referred to as ISM), was recently sequenced and was reported to contain no intact prophage based on customary bioinformatic analysis. However, prophage induction experiments performed with this organism led to a different conclusion. In the laboratory, virus-like particles in the ISM culture increased more than 3 orders of magnitude following induction with mitomycin C. After careful examination of the ISM genome sequence, a putative prophage (ISM-pro1) was identified. Although this prophage contains only minimal phage-like genes, we demonstrated that this “hidden” prophage is inducible. Genomic analysis and reannotation showed that most of the ISM-pro1 open reading frames (ORFs) display the highest sequence similarity withRhodobacteralesbacterial genes and some ORFs are only distantly related to genes of other known phages or prophages. Comparative genomic analyses indicated that ISM-pro1-like prophages or prophage remnants are also present in otherRhodobacteralesgenomes. In addition, the lysis of ISM by this previously unrecognized prophage appeared to increase the production of gene transfer agents (GTAs). Our study suggests that a combination ofin silicogenomic analyses and experimental laboratory work is needed to fully understand the lysogenic features of a given bacterium.
Mechanism of Degradation of Immunogenic Gluten Epitopes from
Triticum turgidum
L. var.
durum
by Sourdough Lactobacilli and Fungal Proteases
Authors: De Angelis Maria , Cassone Angela , Rizzello Carlo G. , Gagliardi Francesca , Minervini Fabio , Calasso Maria , Di Cagno Raffaella , Francavilla Ruggero , Gobbetti Marco
Published: December 2009

As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37°C) of various cultivars of
Triticum turgidum
L. var.
durum
to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of
Triticum
species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of α9-gliadin, 62-75 of A-gliadin, and 134-153 of γ-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to
ca
. 2 ppm. The
in vivo
digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.
Biosynthesis and Degradation of H
2
O
2
by Vaginal Lactobacilli
Authors: Martín Rebeca , Suárez Juan E.
Published: December 2009

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal
Lactobacillus
strains to generate H
2
O
2
, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H
2
O
2
concentrations. Generation of H
2
O
2
was found to be especially common and strong for
L. jensenii
strains, while it was variable among
L. crispatus
and
L. gasseri
strains. Biosynthesis of H
2
O
2
only occurred upon agitation of the cultures, but the H
2
O
2
-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H
2
O
2
production, while Cu
2+
inhibited the growth of
Lactobacillus jensenii
CECT 4306, which was chosen as a model strain. Cultures with Fe
3+
, hemin, and hemoglobin did not accumulate H
2
O
2
. Fe
3+
activated an extracellular peroxidase that destroyed the H
2
O
2
being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe
3+
ions being a necessary cofactor of the reaction.
Contributions of the Pre- and Pro-Regions of a
Staphylococcus hyicus
Lipase to Secretion of a Heterologous Protein by
Bacillus subtilis
Authors: Kouwen Thijs R. H. M. , Nielsen Allan K. , Denham Emma L. , Dubois Jean-Yves F. , Dorenbos Ronald , Rasmussen Michael D. , Quax Wim J. , Freudl Roland , van Dijl Jan Maarten
Published: December 2009

Bacillus subtilis
is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of
B. subtilis
as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre
lip
) and pro (pro
lip
) sequences of a
Staphylococcus hyicus
lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of
Escherichia coli
, by
B. subtilis
. The results indicate that the presence of the pro
lip
-peptide, in combination with the lipase signal peptide (pre
lip
), contributes significantly to the efficient secretion of PhoA by
B. subtilis
and that pre
lip
directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the
S. hyicus
lipase are very useful tools for secretion of heterologous proteins in
B. subtilis
.
Contributions ofFrancisella tularensissubsp.novicidaChitinases and Sec Secretion System to Biofilm Formation on Chitin
Authors: Margolis Jeffrey J. , El-Etr Sahar , Joubert Lydia-Marie , Moore Emily , Robison Richard , Rasley Amy , Spormann Alfred M. , Monack Denise M.
Published: December 2009

Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studyingF. tularensispathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology ofFrancisellaspecies.F. tularensishas been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show thatF. tularensissubsp.novicidaforms biofilms during the colonization of chitin surfaces. The ability ofF. tularensisto persist using chitin as a sole carbon source is dependent on chitinases, since mutants lackingchiAorchiBare attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1andsecB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-typeF. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation byF. tularensispromotes persistence on chitin surfaces. Further study of the interaction ofF. tularensiswith the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.
Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing
Escherichia coli
Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics
Authors: Chaucheyras-Durand Frédérique , Faqir Fahima , Ameilbonne Aurélie , Rozand Christine , Martin Christine
Published: December 2009

Healthy ruminants are the main reservoir of Shiga toxin-producing
Escherichia coli
(STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic
Saccharomyces cerevisiae
CNCM I-1077 or
Lactobacillus acidophilus
BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition,
S. cerevisiae
did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.
Alanine Scanning Analyses of the Three Major Loops in Domain II of
Bacillus thuringiensis
Mosquitocidal Toxin Cry4Aa
Authors: Howlader Mohammad Tofazzal Hossain , Kagawa Yasuhiro , Miyakawa Ai , Yamamoto Ayaka , Taniguchi Tetsuya , Hayakawa Tohru , Sakai Hiroshi
Published: December 2009

Cry4Aa produced by
Bacillus thuringiensis
is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using
Culex pipiens
larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.