bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Diamond‐Like Carbon Thin Film Coated Acrylic Resin on Candida albicans Biofilm Formation
Authors: Queiroz José Renato Cavalcanti , Fissmer Sara Fernanda , Koga‐Ito Cristiane Yumi , Salvia Ana C. R. D. , Massi Marcos , Sobrinho Argermiro Soares da Silva , Júnior Lafayette Nogueira
Published: April 2013

Purpose

The purpose of this study was to evaluate the effect of diamond‐like carbon thin films doped and undoped with silver nanoparticles coating poly(methyl methacrylate) (PMMA) on Candida albicans biofilm formation. The control of biofilm formation is important to prevent oral diseases in denture users.

Materials and Methods

Forty‐five PMMA disks were obtained, finished, cleaned in an ultrasonic bath, and divided into three groups: Gc, no surface coating (control group); Gdlc, coated with diamond‐like carbon film; and Gag, coated with diamond‐like carbon film doped with silver nanoparticles. The films were deposited using a reactive magnetron sputtering system (physical vapor deposition process). The specimens were characterized by optical profilometry, atomic force microscopy, and Rutherford backscattering spectroscopy analyses that determined differences in chemical composition and morphological structure. Following sterilization of the specimens by γ‐ray irradiation, C. albicans (ATCC 18804) biofilms were formed by immersion in 2 ml of Sabouraud dextrose broth inoculated with a standardized fungal suspension. After 24 hours, the number of colony forming units (cfu) per specimen was counted. Data concerning biofilm formation were analyzed using ANOVA and the Tukey test (p < 0.05).

Results

C. albicans biofilm formation was significantly influenced by the films (p < 0.00001), reducing the number of cfu, while not affecting the roughness parameters (p > 0.05). The Tukey test showed no significant difference between Gdlc and Gag. Films deposited were extremely thin (∼50 nm). The silver particles presented a diameter between 60 and 120 nm and regular distribution throughout the film surface (to Gag).

Conclusion

Diamond‐like carbon films, doped or undoped with silver nanoparticles, coating the base of PMMA‐based dentures could be an alternative procedure for preventing candidosis in denture users.
Describing the growth and rapid weight gain of urban Australian Aboriginal infants
Authors: Webster Vana , Denney‐Wilson Elizabeth , Knight Jennifer , Comino Elizabeth ,   Gudaga Research Team
Published: April 2013

Aim

The aims of this paper are to describe the growth of urban Australian Aboriginal infants from birth to 24 months of age and to identify the proportion of these infants experiencing rapid weight gain (RWG) and overweight/obesity.

Methods

The Gudaga Study is a longitudinal birth cohort of 159 Australian Aboriginal children born on the urban fringe of Sydney. Birthweight and length were extracted from hospital data. Children with a birthweight >1500 grams were included in the analysis (n = 157). Weight, length and head circumference were measured at 2–3 weeks and then six‐monthly until 24 months of age. Age‐ and gender‐specific Z‐scores were determined from the Centers for Disease Control (CDC) 2000 growth charts for weight, length, head circumference and body mass index (BMI). The proportion of children experiencing RWG (an increase in weight‐for‐age Z‐scores ≥0.67 between birth and 12 months) was calculated. The association between RWG and ≥85th CDC percentile for BMI at 24 months was tested using Pearson's χ2.

Results

The mean weight of Gudaga infants was less than the CDC mean length‐for‐age at birth and 2–3 weeks of age but greater than CDC mean length‐for‐age and weight‐for‐age at 18 and 24 months of age. Overall, 42 infants (34.4%) experienced RWG, and 45 infants (36.9%) were overweight/obese at 24 months of age. A greater proportion of those who experienced RWG (61.9%) were overweight/obese at 24 months than those who did not experience RWG (23.8%).

Conclusion

Our study suggests a concerning proportion of urban Indigenous infants experience RWG and overweight/obesity in early childhood.
The evolution of co‐orbiting material in the orbit of 2201 Oljato from 1980 to 2012 as deduced from Pioneer Venus Orbiter and Venus Express magnetic records
Authors: Lai Hairong , Russell Christopher T. , Wei Hanying , Zhang Tielong
Published: April 2013

Asteroid 2201 Oljato passed through perihelion inside the orbit of Venus near the time of its conjunction with Venus in 1980, 1983, and 1986. During those three years, many interplanetary field enhancements (IFEs) were observed by the Pioneer Venus Orbiter (PVO) in the longitude sector where the orbit of Oljato lies inside Venus' orbit. We attribute IFEs to clouds of fine‐scale, possibly highly charged dust picked up by the solar wind after an interplanetary collision between objects in the diameter range of 10–1000 m. We interpret the increase rate in IFEs at PVO in these years as due to material in Oljato's orbit colliding with material in, or near to, Venus' orbital plane and producing a dust‐anchored structure in the interplanetary magnetic field. In March 2012, almost 30 yr later, with Venus Express (VEX) now in orbit, the Oljato‐Venus geometry is similar to the one in 1980. Here, we compare IFEs detected by VEX and PVO using the same IFE identification criteria. We find an evolution with time of the IFE rate. In contrast to the results in the 1980s, the recent VEX observations reveal that at solar longitudes in which the Oljato orbit is inside that of Venus, the IFE rate is reduced to the level even below the rate seen at solar longitudes where Oljato's orbit is outside that of Venus. This observation implies that Oljato not only lost its co‐orbiting material but also disrupted the “target material,” with which the co‐orbiting material was colliding, near Venus.
Novel receptor‐like kinases in cacao contain PR‐1 extracellular domains
Authors: Teixeira Paulo José Pereira Lima , Costa Gustavo Gilson Lacerda , Fiorin Gabriel Lorencini , Pereira Gonçalo Amarante Guimarães , Mondego Jorge Maurício Costa
Published: April 2013

Members of the pathogenesis‐related protein 1 (PR‐1) family are well‐known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR‐1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor‐like kinases (RLKs). These proteins (TcPR‐1f and TcPR‐1g) were named PR‐1 receptor kinases (PR‐1RKs). Phylogenetic analysis of RLKs and PR‐1 proteins from cacao indicated that PR‐1RKs originated from a fusion between sequences encoding PR‐1 and the kinase domain of a LecRLK (Lectin Receptor‐Like Kinase). Retrotransposition marks surround TcPR‐1f, suggesting that retrotransposition was involved in the origin of PR‐1RKs. Genes with a similar domain architecture to cacao PR‐1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR‐1g expression was up‐regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR‐1RKs during cacao defence responses. We hypothesize that PR‐1RKs transduce a defence signal by interacting with a PR‐1 ligand.
Inflammatory tinea pedis with bacterial superinfection effectively treated with isoconazole nitrate and diflucortolone valerate combination therapy
Authors: Friedrich Markus
Published: April 2013

Undetected tinea pedis in a patient with diabetes can lead to serious bacterial infections with potentially serious consequences, such as foot amputations. Here we report on a 60‐year‐old patient with diabetes presenting with pain, severe pruritus, and malodour in the foot's interdigital area, and subsequently, diagnosed with inflammatory tinea pedis with bacterial superinfection. The patient was successfully treated with Travocort cream containing isoconazole nitrate 1% and diflucortolone valerate 0.1%; marked improvement occurred within 5 days.
Pyelonephritis: What are the present day causative organisms and antibiotic susceptibilities?
Authors: Prabhu Ajay , Taylor Peter , Konecny Pam , Brown Mark A
Published: April 2013

Aim

Infections of the lower urinary tract and Acute Pyelonephritis are commonly encountered in clinical practice. Widespread usage of antibiotics and changing susceptibility profiles of uropathogens requires regular review of treatment guidelines to meet these challenges. We aimed to better understand the prevalence of uropathogens and emerging antibiotic resistance in patients with pyelonephritis requiring hospital admission.

Methods

In this single centre, 12‐year retrospective observational study, we reviewed case notes and urine culture results of 249 patients admitted with Acute Pyelonephritis under the care of the Nephrology Department, along with 46 660 urine samples with positive isolates from the Emergency Department (ED) during the same period. The prevalence of uropathogens, their antibiotic susceptibilities and emerging resistance patterns to commonly used antibiotics were studied. Antibiotic susceptibilities were also reviewed in line with the currently recommended national guidelines for empiric therapy.

Results

We found the most prevalent uropathogen to be Escherichia coli. Approximately 50% of E. coli infections were resistant to ampicillin. First and third generation cephalosporin resistance was <5%, however, the latter has increased over the last decade and is more prevalent in the elderly. Enterococcus faecalis was associated with less than 10% of cases of lower urinary tract infections and no case of pyelonephritis.

Conclusion

Antibiotic resistance of uropathogens to commonly used antibiotics is increasing with time and there is a need for hospitals to review their recommended guidelines for empiric therapy in line with local patterns of uropathogens and antibiotic susceptibilities.
Translocation of imperiled species under changing climates
Authors: Schwartz Mark W. , Martin Tara G.
Published: April 2013

Conservation translocation of species varies from restoring historic populations to managing the relocation of imperiled species to new locations. We review the literature in three areas—translocation, managed relocation, and conservation decision making—to inform conservation translocation under changing climates. First, climate change increases the potential for conflict over both the efficacy and the acceptability of conservation translocation. The emerging literature on managed relocation highlights this discourse. Second, conservation translocation works in concert with other strategies. The emerging literature in structured decision making provides a framework for prioritizing conservation actions—considering many possible alternatives that are evaluated based on expected benefit, risk, and social–political feasibility. Finally, the translocation literature has historically been primarily concerned with risks associated with the target species. In contrast, the managed relocation literature raises concerns about the ecological risk to the recipient ecosystem. Engaging in a structured decision process that explicitly focuses on stakeholder engagement, problem definition and specification of goals from the outset will allow creative solutions to be developed and evaluated based on their expected effectiveness.
Effects of a liquid diet on the response properties of temporomandibular joint nociceptive neurons in the trigeminal subnucleus caudalis of growing rats
Authors: Hiranuma M. , Kokai S. , Fujita K. , Ishida T. , Shibata M. , Naito S. , Yabushita T. , Ono T.
Published: April 2013

Structured Abstract

Objective

To investigate whether low mechanical loading on the temporomandibular joint (TMJ) when ingesting a liquid diet affects the response properties of neurons in the trigeminal spinal tract subnucleus caudalis (Sp5C) in growing rats.

Materials and Methods

Shortly after weaning, 2‐week‐old male rats were fed chow pellets (control) or a liquid diet (experimental). Firing activities of single sensory units were recorded from the Sp5C at 4, 5, 7, and 9 weeks. Neurons were functionally classified by their responsiveness to TMJ stimuli. The responses of Class II and III neurons to TMJ stimuli were investigated.

Results

In both neuron classes, the firing threshold in the experimental group was significantly lower than in the control group at all time points, but remained static in the control group throughout the experimental period, whereas it peaked in the experimental group at 4 weeks, decreased at 5 weeks, and remained stable thereafter until 9 weeks. Similarly, the initial firing frequency was significantly higher in the experimental group than in the control group, but remained static in the control group throughout the experimental period, whereas in the experimental group, it was at its lowest at 4 weeks, increased at 5 weeks, and stayed stable thereafter until 9 weeks.

Conclusion

Differences in TMJ loading arising from variable diet consistency during growth may affect the functional characteristics of Sp5C neurons.
Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity
Authors: PODGÓRSKA ANNA , GIECZEWSKA KATARZYNA , ŁUKAWSKA‐KUŹMA KATARZYNA , RASMUSSON ALLAN G. , GARDESTRÖM PER , SZAL BOŻENA
Published: April 2013

Ammonium nutrition has been suggested to be associated with alterations in the oxidation‐reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)+ ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over‐reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)+ ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.
Functional characterisation of wheat Pgip genes reveals their involvement in the local response to wounding
Authors: Janni M. , Bozzini T. , Moscetti I. , Volpi C. , D'Ovidio R.
Published: April 2013

Polygalacturonase‐inhibiting proteins (PGIPs) are cell wall leucine‐rich repeat (LRR) proteins involved in plant defence. The hexaploid wheat (Triticum aestivum, genome AABBDD) genome contains one Pgip gene per genome. Tapgip1 (B genome) and Tapgip2 (D genome) are expressed in all tissues, whereas Tapgip3 (A genome) is inactive because of a long terminal repeat, Copia retrotransposon insertion within the coding region. To verify whether Tapgip1 and Tapgip2 encode active PGIPs and are involved in the wheat defence response, we expressed them transiently and analysed their expression under stress conditions. Neither TaPGIP1 nor TaPGIP2 showed inhibition activity in vitro against fungal polygalacturonases. Moreover, a wheat genotype (T. turgidum ssp. dicoccoides) lacking active homologues of Tapgip1 or Tapgip2 possesses PGIP activity. At transcript level, Tapgip1 and Tapgip2 were both up‐regulated after fungal infection and strongly induced following wounding. This latter result has been confirmed in transgenic wheat plants expressing the β‐glucuronidase (GUS) gene under control of the 5′‐flanking region of Tdpgip1, a homologue of Tapgip1 with an identical sequence. Strong and transient GUS staining was mainly restricted to the damaged tissues and was not observed in adjacent tissues. Taken together, these results suggest that Tapgips and their homologues are involved in the wheat defence response by acting at the site of the lesion caused by pathogen infection.
Reactive oxygen species are involved in regulation of pollen wall cytomechanics
Authors: Smirnova A. V. , Matveyeva N. P. , Yermakov I. P.
Published: April 2013

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H2O2 and the hydroxyl radical (•OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H2O2, •OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates •OH. The inhibiting concentrations of exogenous H2O2 and •OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. •OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H2O2, as well as partial removal of endogenous •OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H2O2‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. •OH is involved in local loosening of the intine in the germination pore region, while H2O2 is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.
Analysis of the role of the pyruvate decarboxylase gene family in Arabidopsis thaliana under low‐oxygen conditions
Authors: Mithran M. , Paparelli E. , Novi G. , Perata P. , Loreti E.
Published: April 2013

Plants under low‐oxygen conditions adapt their metabolism by inducing the fermentative pathway, with ethanol as the predominant end product. Activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required for this pathway. While a single gene encodes ADH in Arabidopsis, a family of four genes codes PDC. The availability of microarray data sets enabled the relative importance of the four PDC genes under low oxygen to be assessed, and revealed that, contrary to previous published evidence, not only PDC1 but also PDC2 plays a role under hypoxic conditions. We observed a high level of expression, both at transcript and protein levels of PDCs, even under aerobic conditions when ADH is almost absent. This suggests that PDC has a role under aerobic conditions, which is not coupled to fermentative metabolism. The expression of both PDC1 and PDC2 is strongly up‐regulated under low oxygen. PDC1 is predominantly present in roots, while PDC2 appears to be leaf‐specific. We showed that mutations in both PDC1 and PDC2 result in lower tolerance to submergence.
Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants
Authors: López‐Climent M. F. , Arbona V. , Pérez‐Clemente R. M. , Zandalinas S. I. , Gómez‐Cadenas A.
Published: April 2013

Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd2+). To test the effects of a calcium (Ca2+) treatment on Cd2+ accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd2+, and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd2+ excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca2+ treatment on Cd2+ accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca2+ had two positive effects on citrus physiology: it reduced Cd+2 uptake into roots and also increased GSH content (even in the absence of Cd2+). Overall, the data indicate that although Cd2+ exclusion is a powerful mechanism to avoid heavy metal build‐up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance.
Comparative demography of two co‐occurring Linum species with different distribution patterns
Authors: Münzbergová Z.
Published: April 2013

Understanding similarities and differences in population dynamics of closely related species is a key prerequisite in attempts to apply knowledge obtained in one species to another species, e.g., for the purpose of predicting future fate of populations of various rare species. It can be expected that species will have similar population dynamics if they are closely related and share similar habitats. Contrasting population sizes and distribution patterns may, however, indicate that the population dynamics will be different. To understand similarities and differences in population dynamics of closely related species, I studied demography of two congeneric endangered species, Linum flavum and L. tenuifolium co‐occurring in dry grasslands. Linum flavum occurs with a lower number of large populations, while L. tenuifolium occurs as a large number of small populations. The results showed that L. flavum had higher population growth rates, relied more on survival and growth and its populations were more persistent. In contrast, populations of L. tenuifolium were more prone to extinction and frequent recolonisation was necessary for their survival in the landscape. This was in accordance with observed population sizes of the two species and their frequency in the landscape. The results indicate that despite being closely related and occurring in the same habitat types, the two Linum species have different growth strategies. The strong differences in population dynamics between the two species suggest that similarity in population sizes and frequency of the species in the landscape may be more important when attempting to transfer knowledge between species than is taxonomic relatedness.
Observation of polyphosphate bodies and DNA during the cell division cycle of Synechococcus elongatus PCC 7942
Authors: Seki Y. , Nitta K. , Kaneko Y.
Published: April 2013

Although most cyanobacterial cells contain prominent polyphosphate bodies in the central cytoplasmic area enclosed by the peripheral thylakoid membranes, their roles are not fully understood. Storing phosphate for nucleotide production might be one of their important roles in survival of the cells. As a step towards identifying a possible contribution of the polyphosphate bodies to DNA synthesis, the relationship between polyphosphate bodies and DNA throughout cell division cycle of Synechococcus elongatus PCC 7942 cells cultured under light/dark cycles was investigated with light and electron microscopy. During the dark period, the average size of polyphosphate bodies increased gradually without significant change in their number and distribution. However, during the light period, the number of polyphosphate bodies increased, while the size of each polyphosphate body decreased and cells elongated until the end of the light period, when most cells divided. The ratio of the content of polyphosphate bodies to cell length increased gradually during the dark period and decreased during the light period. Hoechst 33342‐stained DNA appeared diffuse during the dark period, but in the light period it became condensed and eventually formed a wavy, rope‐like structure prior to cell division. Close association between fibres containing DNA and polyphosphate bodies was demonstrated by TEM using DNA‐specific staining and BrdU labelling. These regular coordinated changes of polyphosphate bodies and DNA shape during the cell division cycle, together with their intimate interaction, imply a role of polyphosphate bodies in supplying material for DNA.
Do Employees Care About Their Relative Income Position? Behavioral Evidence Focusing on Performance in Professional Team Sport
Authors: Frey Bruno S. , Schaffner Markus , Schmidt Sascha L. , Torgler Benno
Published: April 2013

Objective

Do employees care about their relative (economic) position in comparison to their co‐workers in an organization? And if so, does it raise or lower their performance? While the topic is widely discussed in the literature, behavioral evidence on these important questions is relatively rare.

Methods

This article explores the pay‐performance relationship using a sports data set. The strength of analyzing such data is that sports tournaments take place in a very controlled environment that helps to isolate a relative income effect.

Results

Using two large unique data sets that cover 26 seasons in basketball and eight seasons in soccer (Bundesliga), we find considerable support for the idea that a relative income disadvantage is correlated with a decrease in individual performance. In addition, there does not seem to be any tolerance for income disparity based on the hope that such differences may signal that better times are ahead.

Conclusions

This suggests the need to consider the impact of the relative income position when designing pay‐for‐performance mechanisms within firms and teams.
GABI‐DUPLO: a collection of double mutants to overcome genetic redundancy in Arabidopsis thaliana
Authors: Bolle Cordelia , Huep Gunnar , Kleinbölting Nils , Haberer Georg , Mayer Klaus , Leister Dario , Weisshaar Bernd
Published: April 2013

Owing to duplication events in its progenitor, more than 90% of the genes in the Arabidopsis thaliana genome are members of multigene families. A set of 2108 gene families, each consisting of precisely two unlinked paralogous genes, was identified in the nuclear genome of A. thaliana on the basis of sequence similarity. A systematic method for the creation of double knock‐out lines for such gene pairs, designated as DUPLO lines, was established and 200 lines are now publicly available. Their initial phenotypic characterisation led to the identification of seven lines with defects that emerge only in the adult stage. A further six lines display seedling lethality and 23 lines were lethal before germination. Another 14 lines are known to show phenotypes under non‐standard conditions or at the molecular level. Knock‐out of gene pairs with very similar coding sequences or expression profiles is more likely to produce a mutant phenotype than inactivation of gene pairs with dissimilar profiles or sequences. High coding sequence similarity and highly similar expression profiles are only weakly correlated, implying that promoter and coding regions of these gene pairs display different degrees of diversification.
Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase
Authors: De Paepe Annelies , De Buck Sylvie , Nolf Jonah , Van Lerberge Els , Depicker Ann
Published: April 2013

Random T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site‐specific integration (SSI) of this T–DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium‐mediated integration, allowing the serial integration of transgenic DNA sequences in plants.
A Novel Assay for Measurement of Membrane‐Protein Surface Expression using a β‐lactamase Reporter
Authors: Lam Vincent M. , Beerepoot Pieter , Angers Stephane , Salahpour Ali
Published: April 2013

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one‐step and potentially high‐throughput assay, using the β‐lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell‐impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well‐established paradigms of internalization and molecular chaperoning, applied to two G‐protein‐coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression.
Inducing a junction in n-type InxGa(1−x)N
Authors: Williams Joshua J. , Williamson Todd L. , Hoffbauer Mark A. , Fischer Alec M. , Goodnick Stephen M. , Faleev Nikolai N. , Ghosh Kunal , Honsberg Christiana B.
Published: April 2013

The pseudo-binary alloy of indium(x)gallium(1−x)nitride has a compositionally dependent bandgap ranging from 0.65 to 3.42 eV, making it desirable for light emitting diodes and solar cell devices. Through modeling and film growth, the authors investigate the use of InxGa1−xN as an active layer in an induced junction. In an induced junction, electrostatics are used to create strong band bending at the surface of a doped material and invert the bands. The authors report modeling results, as well as preliminary film quality experiments for an induced junction in InGaN by space charge effects of neighboring materials, piezoelectric effects, and spontaneous polarization.
Distributional training of speech sounds can be done with continuous distributions
Authors: Wanrooij Karin , Boersma Paul
Published: April 2013

In previous research on distributional training of non-native speech sounds, distributions were always discontinuous: typically, each of only eight different stimuli was repeated multiple times. The current study examines distributional training with continuous distributions, in which all presented tokens are acoustically different. Adult Spanish learners of Dutch were trained on either a discontinuous or a continuous bimodal distribution of the Dutch vowel contrast /ɑ/–/aː/. Both groups improved their perception of the contrast; this shows that continuous training works equally well as discontinuous training. Using the more natural continuous distributions is therefore recommended for future distributional learning experiments.
In Search of a (Functional) Cure
Authors: Sheth Anandi N.
Published: April 2013

HIV-infected adults who are able to control viral replication after therapy have small latent reservoirs in long-living cells.
Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity
Authors: Lewandowski Thomas , Huang Jianzhong , Fan Frank , Rogers Shannon , Gentry Daniel , Holland Reannon , DeMarsh Peter , Aubart Kelly , Zalacain Magdalena
Published: April 2013

Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in
Staphylococcus aureus
. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with
S. aureus
pathogenicity. Consequently,
S. aureus
FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type
S. aureus
. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection.
Pharmacokinetics and Pharmacodynamics of Fluconazole for Cryptococcal Meningoencephalitis: Implications for Antifungal Therapy andIn VitroSusceptibility Breakpoints
Authors: Sudan Ajay , Livermore Joanne , Howard Susan J. , Al-Nakeeb Zaid , Sharp Andrew , Goodwin Joanne , Gregson Lea , Warn Peter A. , Felton Tim W. , Perfect John R. , Harrison Thomas S. , Hope William W.
Published: April 2013

Fluconazole is frequently the only antifungal agent that is available for induction therapy for cryptococcal meningitis. There is relatively little understanding of the pharmacokinetics and pharmacodynamics (PK-PD) of fluconazole in this setting. PK-PD relationships were estimated with 4 clinical isolates ofCryptococcus neoformans. MICs were determined using Clinical and Laboratory Standards Institute (CLSI) methodology. A nonimmunosuppressed murine model of cryptococcal meningitis was used. Mice received two different doses of fluconazole (125 mg/kg of body weight/day and 250 mg/kg of body weight/day) orally for 9 days; a control group of mice was not given fluconazole. Fluconazole concentrations in plasma and in the cerebrum were determined using high-performance liquid chromatography (HPLC). The cryptococcal density in the brain was estimated using quantitative cultures. A mathematical model was fitted to the PK-PD data. The experimental results were extrapolated to humans (bridging study). The PK were linear. A dose-dependent decline in fungal burden was observed, with near-maximal activity evident with dosages of 250 mg/kg/day. The MIC was important for understanding the exposure-response relationships. The mean AUC/MIC ratio associated with stasis was 389. The results of the bridging study suggested that only 66.7% of patients receiving 1,200 mg/kg would achieve or exceed an AUC/MIC ratio of 389. The potential breakpoints for fluconazole againstCryptococcus neoformansfollow: susceptible, ≤2 mg/liter; resistant, >2 mg/liter. Fluconazole may be an inferior agent for induction therapy because many patients cannot achieve the pharmacodynamic target. Clinical breakpoints are likely to be significantly lower than epidemiological cutoff values. The MIC may guide the appropriate use of fluconazole. If fluconazole is the only option for induction therapy, then the highest possible dose should be used.
Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the
murA1
Gene
Authors: Engel Hansjürg , Gutiérrez-Fernández Javier , Flückiger Christine , Martínez-Ripoll Martín , Mühlemann Kathrin , Hermoso Juan A. , Hilty Markus , Hathaway Lucy J.
Published: April 2013

Fosfomycin targets the first step of peptidoglycan biosynthesis in
Streptococcus pneumoniae
catalyzed by UDP-
N
-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in
S. pneumoniae
. We found that of 11 strains tested, all but 1 (Hungary
19A
) displayed heteroresistance and that deletion of
murA1
abolished heteroresistance. Hungary
19A
differs from the other strains by a single amino acid substitution in MurA1 (Ala
364
Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary
19A
were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of
S. pneumoniae
and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.
Analysis of Anti-Clostridium difficile Activity of Thuricin CD, Vancomycin, Metronidazole, Ramoplanin, and Actagardine, both Singly and in Paired Combinations
Authors: Mathur Harsh , O'Connor Paula M. , Hill Colin , Cotter Paul D. , Ross R. Paul
Published: April 2013

Due to the ongoing problem of recurrence of
Clostridium difficile
-associated diarrhea following antibiotic treatment, there is an urgent need for alternative treatment options. We assessed the MICs of five antimicrobials singly and in combinations against a range of
C. difficile
clinical isolates. Ramoplanin-actagardine combinations were particularly effective, with partial synergistic/additive effects observed against 61.5% of
C. difficile
strains tested.
Complex Patterns of Protease Inhibitor Resistance among Antiretroviral Treatment-Experienced HIV-2 Patients from Senegal: Implications for Second-Line Therapy
Authors: Raugi Dana N. , Smith Robert A. , Ba Selly , Toure Macoumba , Traore Fatou , Sall Fatima , Pan Charlotte , Blankenship Lindsey , Montano Alexandra , Olson Julia , Dia Badiane Ndeye Mery , Mullins James I. , Kiviat Nancy B. , Hawes Stephen E. , Sow Papa Salif , Gottlieb Geoffrey S.
Published: April 2013

Protease inhibitor (PI)-based antiretroviral therapy (ART) can effectively suppress HIV-2 plasma load and increase CD4 counts; however, not all PIs are equally active against HIV-2, and few data exist to support second-line therapy decisions. To identify therapeutic options for HIV-2 patients failing ART, we evaluated the frequency of PI resistance-associated amino acid changes in HIV-2 sequences from a cohort of 43 Senegalese individuals receiving unboosted indinavir (n= 18 subjects)-, lopinavir/ritonavir (n= 4)-, or indinavir and then lopinavir/ritonavir (n= 21)-containing ART. Common protease substitutions included V10I, V47A, I54M, V71I, I82F, I84V, L90M, and L99F, and most patients harbored viruses containing multiple changes. Based on genotypic data, we constructed a panel of 15 site-directed mutants of HIV-2ROD9containing single- or multiple-treatment-associated amino acid changes in the protease-encoding region ofpol. We then quantified the susceptibilities of the mutants to the HIV-2 “active” PIs saquinavir, lopinavir, and darunavir using a single-cycle assay. Relative to wild-type HIV-2, the V47A mutant was resistant to lopinavir (6.3-fold increase in the mean 50% effective concentration [EC50]), the I54M variant was resistant to darunavir and lopinavir (6.2- and 2.7-fold increases, respectively), and the L90M mutant was resistant to saquinavir (3.6-fold increase). In addition, the triple mutant that included I54M plus I84V plus L90M was resistant to all three PIs (31-, 10-, and 3.8-fold increases in the mean EC50for darunavir, saquinavir, and lopinavir, respectively). Taken together, our data demonstrate that PI-treated HIV-2 patients frequently harbor viruses that exhibit complex patterns of PI cross-resistance. These findings suggest that sequential PI-based regimens for HIV-2 treatment may be ineffective.
Novel Bacteriophage Lysin with Broad Lytic Activity Protects against Mixed Infection by Streptococcus pyogenes and Methicillin-Resistant Staphylococcus aureus
Authors: Gilmer Daniel B. , Schmitz Jonathan E. , Euler Chad W. , Fischetti Vincent A.
Published: April 2013

Methicillin-resistant
Staphylococcus aureus
(MRSA) and
Streptococcus pyogenes
(group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a
Streptococcus suis
phage, with broad lytic activity against MRSA, vancomycin-intermediate
S. aureus
(VISA),
Streptococcus suis
,
Listeria
,
Staphylococcus simulans
,
Staphylococcus epidermidis
,
Streptococcus equi
,
Streptococcus agalactiae
(group B streptococcus [GBS]),
S. pyogenes
,
Streptococcus sanguinis
, group G streptococci (GGS), group E streptococci (GES), and
Streptococcus pneumoniae
. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50°C for 30 min, 37°C for >24 h, 4°C for 15 days, and −80°C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 μg/ml
in vitro
reduced MRSA and
S. pyogenes
growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 μg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and
S. pyogenes
infection. Serially increasing exposure of MRSA and
S. pyogenes
to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.
Impact of Meropenem in Combination with Tobramycin in a Murine Model of Pseudomonas aeruginosa Pneumonia
Authors: Louie Arnold , Liu Weiguo , Fikes Steven , Brown David , Drusano G. L.
Published: April 2013

Pseudomonas aeruginosa
pneumonia remains a difficult therapeutic problem. Optimal doses and modes of administration of single agents often do not result in acceptable outcomes. Further, emergence of resistance occurs frequently in this setting with single-agent chemotherapy. The purpose of these experiments was to evaluate combination chemotherapy with meropenem plus tobramycin for
P. aeruginosa
in a murine pneumonia model. Neutropenia was induced by cyclophosphamide. Pharmacokinetics of meropenem and tobramycin were determined using a population pharmacokinetic approach. Both drugs were given at 4-h intervals. Meropenem was administered as total daily doses of 30 to 600 mg/kg of body weight, while tobramycin doses ranged from 50 to 400 mg/kg. Combination therapy evaluated all combinations of 50, 100, and 150 mg/kg/day of tobramycin doses with 60 or 300 mg/kg/day of meropenem. Total and drug-resistant organisms were enumerated. Meropenem alone had a near-maximal effect at 60 mg/kg/day (3.18 log
10
[CFU/g] kill from stasis). The time > MIC in epithelial lining fluid (ELF) at this dose was 35.25% of 24 h. For tobramycin alone, the near-maximal effect was at 150 mg/kg/day and the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) in ELF was 240.3. Resistance suppression occurred at an ELF AUC/MIC ratio of 110.6. For combination therapy, the near-maximal effect was reached at 60 mg/kg/day and 50 mg/kg/day of meropenem and tobramycin, which produced a 35.25% time > MIC in ELF and an ELF AUC/MIC ratio of 80.1. The interaction was additive. All combination regimens suppressed resistance. Combination therapy produced additive drug interaction and suppressed all resistance amplification. It is likely that optimal therapy for
Pseudomonas aeruginosa
pneumonia will involve a combination of agents.
ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding
Authors: Greninger Alexander L. , Knudsen Giselle M. , Betegon Miguel , Burlingame Alma L. , DeRisi Joseph L.
Published: April 2013

Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism.

IMPORTANCE
Multiple viruses use the same Golgi protein (ACBD3) to recruit the lipid kinase phosphatidylinositol 4-kinase class III beta (PI4KB) in order to replicate. We identify a new binding partner of ACBD3 in the evolutionarily conserved Rab GTPase-activating proteins (RabGAPs) TBC1D22A and -B. Interestingly, TBC1D22A directly competes with PI4KB for binding to the same location of ACBD3 by utilizing a similar binding domain. Different viruses are able to influence this interaction through distinct mechanisms to promote the association of PI4KB with ACBD3. This work informs our knowledge of both the physical interactions of the proteins that help maintain metazoan Golgi structure and how viruses subvert these evolutionarily conserved interactions for their own purposes.