bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenological shifts and the fate of mutualisms
Authors: Rafferty Nicole E. , CaraDonna Paul J. , Bronstein Judith L.
Published: September 2014

Climate change is altering the timing of life history events in a wide array of species, many of which are involved in mutualistic interactions. Because many mutualisms can form only if partner species are able to locate each other in time, differential phenological shifts are likely to influence their strength, duration and outcome. At the extreme, climate change‐driven shifts in phenology may result in phenological mismatch: the partial or complete loss of temporal overlap of mutualistic species. We have a growing understanding of how, when, and why phenological change can alter one type of mutualism–pollination. However, as we show here, there has been a surprising lack of attention to other types of mutualism. We generate a set of predictions about the characteristics that may predispose mutualisms in general to phenological mismatches. We focus not on the consequences of such mismatches but rather on the likelihood that mismatches will develop. We explore the influence of three key characteristics of mutualism: 1) intimacy, 2) seasonality and duration, and 3) obligacy and specificity. We predict that the following characteristics of mutualism may increase the likelihood of phenological mismatch: 1) a non‐symbiotic life history in which co‐dispersal is absent; 2) brief, seasonal interactions; and 3) facultative, generalized interactions. We then review the limited available data in light of our a priori predictions and point to mutualisms that are more and less likely to be at risk of becoming phenologically mismatched, emphasizing the need for research on mutualisms other than plant–pollinator interactions. Future studies should explicitly focus on mutualism characteristics to determine whether and how changing phenologies will affect mutualistic interactions.
Genetic use restriction technologies: a review
Authors: Lombardo Luca
Published: September 2014

Genetic use restriction technologies (GURTs), developed to secure return on investments through protection of plant varieties, are among the most controversial and opposed genetic engineering biotechnologies as they are perceived as a tool to force farmers to depend on multinational corporations' seed monopolies. In this work, the currently proposed strategies are described and compared with some of the principal techniques implemented for preventing transgene flow and/or seed saving, with a simultaneous analysis of the future perspectives of GURTs taking into account potential benefits, possible impacts on farmers and local plant genetic resources (PGR), hypothetical negative environmental issues and ethical concerns related to intellectual property that have led to the ban of this technology.
β‐catenin mutation in ovarian solid pseudopapillary neoplasm
Authors: Kominami Ayami , Fujino Masahiko , Murakami Hideki , Ito Masafumi
Published: September 2014

Primaly solid pseudopapillary neoplasm (SPN) of the ovary is a rare tumor; recently 6 cases have been reported. Its pathogenesis, however, remains largely unclear. We report an additional case of primary ovarian SPN of an 18‐year‐old girl. The aim of this study is to define the difference between pancreatic and ovarian SPN by histological and molecular examination. Microscopically the tumor predominantly showed a solid pattern and focally a pseudopapillary pattern. The tumor cells showed two patterns of abundant eosinophilic cytoplasm and intracytoplasmic vacuoles. Immunohistochemistry of the tumor was positive for β‐catenin (nuclear and cytoplasmic reactivity), α1‐antitrypsin, vimentin, CD56, synaptophysin (focal weak), CD10. Mutation analyses revealed a point mutation, c.110C >T, in exon 3 of the the β‐catenin gene (CTNNB1), which causes the replacement of serine with phenylalanine at codon 37. A Ser37 point mutation is known to be one of the oncogenic somatic mutations in pancreatic SPN and the major oncogenic β‐catenin mutation. Ovarian SPN of our case was similar to pancreatic SPN histologicaly and had the same genomic characteristics. We expected that both ovarian and pancreatic SPNs shared the same oncogenesis related to Wnt/β‐catenin pathway for tumorgenesis.
Embryonal tumor with abundant neuropil and true rosettes with only one structure suggestive of an ependymoblastic rosette
Authors: Nobusawa Sumihito , Orimo Kenta , Horiguchi Keishi , Ikota Hayato , Yokoo Hideaki , Hirato Junko , Nakazato Yoichi
Published: September 2014

Embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a very aggressive embryonal central nervous system (CNS) tumor, histologically featuring ependymoblastic rosettes and neuronal differentiation in a neuropil‐like background. 19q13.42 amplification was identified in ETANTR and epndymoblastoma, suggesting that these tumors constitute a single entity, called embryonal tumor with multilayered rosettes (ETMR). Here, we report a case involving a 2‐year‐old boy with a pontine embryonal tumor composed of clusters of poorly differentiated neuroepithelial cells, and smaller neuroblastic/neurocytic cells in a fibrillary and paucicellular neuropil‐like matrix, where clear ependymoblastic rosettes were not detected but only one structure suggestive of an ependymoblastic multilayered rosette was found. Fluorescence in situ hybridazation analysis revealed 19q13.42 amplification, supporting the diagnosis of ETANTR. This report indicates that rare ependymoblasic rosettes found in embryonal tumors, which are otherwise CNS primitive neuroectodermal tumors or medulloblastomas, are significant for considering the examination of 19q13.42 amplification to confirm the diagnosis of ETMR.
Characterization of Fusarium diseases on commercially grown orchids in Hawaii
Authors: Swett C. S. , Uchida J. Y.
Published: September 2014

A decline of unknown aetiology has become a major problem for commercial orchid production in Hawaii, one of the primary orchid‐producing states in the USA. The major symptoms of decline include root degradation, foliar blight, pseudobulb rot and sheath rot. It was unclear whether all these symptoms are caused by the same or different pathogens, but preliminary research indicated that Fusarium species may be involved. In this study, the incidence of Fusarium species was examined across 186 plants, from 29 orchid genera and intergeneric hybrids across three islands in the state of Hawaii. The main five species associated with diseased orchids were F. proliferatum (38% of samples), F. solani (16%), F. oxysporum (16%) and two previously undescribed species (8% for both species combined). The two undescribed species were similar in appearance to F. subglutinans, and were designated FS‐A and FS‐B. Pathogenicity tests established that both F. proliferatum and FS‐B caused foliar spots, foliar blight and pseudostem rot on Dendrobium orchids, and that F. proliferatum isolates from diseased tissue of several genera could also induce symptoms on Dendrobium orchids. Although orchids have increasing importance in floriculture, relatively little is known about orchid pathogens, and previous studies focused primarily on Cymbidium and Phalaenopsis. This study provides new information concerning Dendrobium orchid pathogens and suggests a much wider host range than previously recognized for the five Fusarium species recovered from tissue with symptoms. These findings can contribute to better management of Fusarium diseases, which represent a significant challenge to orchid production in Hawaii.
Phenotypic plasticity toward water regime: response of leaf growth and underlying candidate genes in Populus
Authors: Bizet François , Bogeat‐Triboulot Marie‐Béatrice , Montpied Pierre , Christophe Angélique , Ningre Nathalie , Cohen David , Hummel Irène
Published: September 2014

Phenotypic plasticity is considered as an important mechanism for plants to cope with environmental challenges. Leaf growth is one of the first macroscopic processes to be impacted by modification of soil water availability. In this study, we intended to analyze and compare plasticity at different scales. We examined the differential effect of water regime (optimal, moderate water deprivation and recovery) on growth and on the expression of candidate genes in leaves of different growth stages. Candidates were selected to assess components of growth response: abscisic acid signaling, water transport, cell wall modification and stomatal development signaling network. At the tree scale, the four studied poplar hybrids responded similarly to water regime. Meanwhile, leaf growth response was under genotype × environment interaction. Patterns of candidate gene expression enriched our knowledge about their functionality in poplars. For most candidates, transcript levels were strongly structured according to leaf growth performance while response to water regime was clearly dependent on genotype. The use of an index of plasticity revealed that the magnitude of the response was higher for gene expression than for macroscopic traits. In addition, the ranking of poplar genotypes for macroscopic traits well paralleled the one for gene expression.
Do you feel alright? Attenuated neural processing of aversive interoceptive stimuli in current stimulant users
Authors: Stewart Jennifer L. , Juavinett Ashley L. , May April C. , Davenport Paul W. , Paulus Martin P.
Published: September 2014

Inability to appropriately process afferent interoceptive stimuli may contribute to initiation and/or escalation of substance use. An aversive interoceptive stimulus probed neural processing in problem stimulant users (PSU; n = 19), 18 desisted stimulant users (DSU; n = 18), and healthy comparison subjects (CTL; n = 21). Participants completed a continuous performance task while they anticipated and experienced 40 cm H2O/L/sec inspiratory breathing loads during fMRI. PSU exhibited lower left dorsolateral prefrontal cortex and inferior frontal gyrus (IFG) activation than DSU and CTL across trials. Greater lifetime drug use due to stimulants was also linked to lower activation in these regions. In addition, PSU displayed lower right IFG and insula activation during breathing load than DSU and CTL. Findings suggest that transition to stimulant use disorders is marked by weakened attentional salience of aversive stimuli.
Egg stickiness in artificial reproduction of sturgeon: an overview
Authors: Siddique Mohammad A. Momin , Psenicka Martin , Cosson Jacky , Dzyuba Borys , Rodina Marek , Golpour Amin , Linhart Otomar
Published: September 2014

The egg structure of sturgeon is complex, with multiple micropyles and an envelope structure different from that of teleosts and other fish groups. In the sturgeon, an adhesive layer in the follicular epithelium is responsible for the egg adhesiveness. This is a problem for artificial reproduction of many fish species, including sturgeon, when a large number of eggs are incubated in hatchery conditions. Although several techniques for removing adhesiveness have been developed and successfully applied to eggs of common carp, tench, pikeperch and European catfish, de‐sticking methods for sturgeon are limited. Proteolytic enzymes have been successfully applied to common carp, tench and African catfish eggs, leading to a hatching rate over 80% following 2‐min treatment. In sturgeon, blue clay is the most commonly used de‐sticking substance. The chemical reactions determining egg adhesiveness in sturgeon are poorly studied, and optimum methods for removing stickiness remain to be developed.
Metaphysics, Intuitions and Physics
Authors: Tallant Jonathan
Published: September 2014

Ladyman and Ross (2007) do not think that contemporary metaphysics is in good standing. However, they do think that there is a version of metaphysics that can be made to work – provided we approach it using appropriate principles. My aim in this paper is to undermine some of their arguments against contemporary metaphysics as it is currently practiced.
Efficacy of azithromycin in the treatment of bronchiectasis
Authors: Lourdesamy Anthony Albert I. , Muthukumaru Umadevi
Published: September 2014

Background and objective

We evaluated the efficacy of a 12‐week oral treatment with azithromycin in adult patients with bronchiectasis. The objectives were to demonstrate that this treatment reduces sputum volume, improves quality of life and to assess the lengths of effects after cessation of therapy.

Methods

Seventy‐eight patients with bronchiectasis confirmed by high‐resolution computed tomography were included in this study. Subjects received oral azithromycin or placebo in a randomized manner for 12 weeks followed by placebo for another 12 weeks. Sputum volume, St George's Respiratory Questionnaire (SGRQ) score and spirometry were recorded at baseline, 12 weeks and 24 weeks, respectively. End‐point measurements were compared from baseline to the end of each study phase.

Results

Sixty‐eight subjects were included in the analysis. Mean 24‐h sputum volume significantly decreased (P < 0.01) during the active treatment phase and remained low during the control phase (P < 0.01). The mean SGRQ total score with azithromycin decreased (i.e. improved health status) from baseline by more than the 4 points at the end of 12 and 24 weeks. Lung functions remained stable during oral azithromycin therapy and the subsequent control phase.

Conclusions

Twelve weeks administration of azithromycin in bronchiectasis produces significant reductions in mean sputum volume, health status and stabilization of lung function values. Sputum volume reduction and the improvement of quality of life were sustained for 12 weeks after cessation of azithromycin. (Clinicaltrials.gov number NCT02107274).
Partially Supervised Spatiotemporal Clustering for Burglary Crime Series Identification
Authors: Reich Brian J. , Porter Michael D.
Published: September 2014

Statistical clustering of criminal events can be used by crime analysts to create lists of potential suspects for an unsolved crime, to identify groups of crimes that may have been committed by the same individuals or group of individuals, for offender profiling and for predicting future events. We propose a Bayesian model-based clustering approach for criminal events. Our approach is semisupervised, because the offender is known for a subset of the events, and utilizes spatiotemporal crime locations as well as crime features describing the offender's modus operandi. The hierarchical model naturally handles complex features that are often seen in crime data, including missing data, interval-censored event times and a mix of discrete and continuous variables. In addition, our Bayesian model produces posterior clustering probabilities which allow analysts to act on model output only as warranted. We illustrate the approach by using a large data set of burglaries in 2009–2010 in Baltimore County, Maryland.
Reshaping the Past: Gorky's Reminiscences of Korolenko
Authors: SCHERR BARRY P.
Published: September 2014

Although not as well‐known as his memoirs of Tolstoy, Chekhov, and Andreev, Gorky's two essays devoted to Korolenko are of no less interest. First, like some of Gorky's other works that draw on his life experiences, they show clear evidence of artistic reworking: he is not beyond playing with the chronology and introducing certain elements to reshape his memories into effective narratives. The existence of a third item on Korolenko, a speech that Gorky gave in 1918, provides comparative data that helps reveal the artistic embellishment in the two later works. Second, these two pieces straddle the boundary between memoir and autobiography, both in terms of content and within the context of Gorky's career. He composed them in 1922, just three years after he initially published his memoirs of Tolstoy and Andreev; like those works and others devoted to a single prominent figure, the writings on Korolenko seemingly belong to the category of memoir. However, they begin with the period that immediately follows the final events in the third part of his autobiographical trilogy, My Universities, which was written at about the same time, and so can also be regarded as a continuation of that work. Third, his relationship with Korolenko is of intrinsic interest. Their first meetings go back to Gorky's literary beginnings and so offer some insights into his early steps as a writer and Korolenko's role in those. In more subtle ways, the two essays also reflect aspects of an epistolary exchange between the writers toward the very end of Korolenko's life and contain a veiled critique of the social and political atmosphere of the post‐Revolutionary years.
The impact of electronic decision support and electronic remote blood issue on transfusion practice
Authors: Hibbs S. P. , Noel S. , Miles D. , Staves J. , Murphy M. F.
Published: September 2014

Objectives

To assess the impact on transfusion practice of a two‐stage electronic intervention: the introduction of a decision support system (DSS) followed by the addition of electronic remote blood issue (ERBI).

Background

With increasing evidence to show the benefit of restrictive transfusion policies, it is important to ascertain which interventions can increase clinician compliance with their implementation. A DSS provides patient‐specific recommendations to clinicians. ERBI reduces delays in acquiring blood and may alter the transfusion behaviour of clinicians.

Methods

All electronically requested blood transfusions administered outside of surgical theatres or recovery were identified in an orthopaedic hospital. These were divided into three time periods corresponding to pre‐intervention, the successive introduction of DSS alone and DSS with ERBI. Pre‐ and post‐transfusion haemoglobin (Hb) concentration levels, and the number of units ordered and transfused were recorded.

Results

A total of 204 transfusions for 92 patients were assessed; 38 of 85 (45%) transfusions in the first time period were compliant. This did not significantly change after introduction of the DSS, but with DSS and ERBI together significantly increased to 39 of 60 (65%) (P < 0·05). Mean pre‐transfusion Hb reduced from 8·24 g dl−1 in the first time period to 7·67 g dl−1 in the third (P < 0·0001). There was no significant change in overall blood usage, although ERBI significantly reduced the amount of unused blood orders from 70 to 25%.

Conclusion

Electronic DSS was not sufficient to change practice in the form implemented in this study. ERBI can contribute to significant improvements in blood usage as well as the efficiency of blood provision.
Evaluation of a transcutaneous blood gas monitoring system in critically ill dogs
Authors: Holowaychuk Marie K. , Fujita Hiroshi , Bersenas Alexa M. E.
Published: September 2014

Objectives

To describe the use of a transcutaneous blood gas monitoring system in critically ill dogs, determine if transcutaneous and arterial blood gas values have good agreement, and verify if clinical or laboratory variables are correlated with differences between transcutaneous and arterial blood gas measurements.

Design

Prospective observational study.

Setting

University teaching hospital ICU.

Animals

Twenty‐three client‐owned dogs.

Interventions

In critically ill dogs undergoing arterial blood gas monitoring, a transcutaneous blood gas monitor was used to measure transcutaneous partial pressure of carbon dioxide (PtcCO2) and transcutaneous partial pressure of oxygen (PtcO2) values 30 minutes after sensor placement, which were compared to PaCO2and PaO2values measured simultaneously. Clinical and laboratory variables were concurrently recorded to determine if they were correlated with the difference between transcutaneous and arterial blood gas measurements.

Measurements and Main Results

Bland‐Altman analysis revealed a mean bias of 4.6 ± 26.3 mm Hg (limits of agreement [LOA]: −46.9/+56.1 mm Hg) between PtcO2and PaO2and a mean bias of 9.3 ± 8.5 mm Hg (LOA: −7.5/+26.0 mm Hg) between PtcCO2and PaCO2. The difference between PtcCO2–PaCO2was strongly negatively correlated with HCO3−(r2= 0.52,P< 0.001) and PaCO2(r2= 0.58,P< 0.001) and weakly positively correlated with diastolic blood pressure (r2= 0.21,P= 0.044), whereas the difference between PtcCO2–PaCO2was moderately negatively correlated with diastolic blood pressure (r2= 0.33,P= 0.008).

Conclusions

Agreement between transcutaneous and arterial PO2and PCO2measurements in these critically ill dogs was inferior to that reported in similar adult and pediatric human studies. The transcutaneous monitor consistently over‐estimated PaO2and PaCO2and should not be used to replace arterial blood gas measurements in critically ill dogs requiring blood gas interpretation.
Absolutely continuous representing measures for R(X)
Authors: Brennan J. E.
Published: September 2014

Let be a compact nowhere dense subset of the complex plane , and let be the uniform closure of the rational functions having no poles on . We present a short proof of the fact that each non‐peak point for admits a representing measure absolutely continuous with respect area. If happens to have finite perimeter, then every non‐peak point admits a representing measure absolutely continuous with respect to arc length, provided the corresponding integral is taken in a principal value sense.
A refinement of the abc conjecture
Authors: Robert Olivier , Stewart Cameron L. , Tenenbaum Gérald
Published: September 2014

Based on recent work, by the first and third authors, on the distribution of the squarefree kernel of an integer, we present precise refinements of the famous conjecture. These rest on the sole heuristic assumption that, whenever and are coprime, then the kernels of , and are statistically independent.
A Population Pharmacokinetic Modeling Approach Shows that Serum Penicillin G Concentrations Are Below Inhibitory Concentrations by Two Weeks after Benzathine Penicillin G Injection in the Majority of Young Adults
Authors: Neely Michael , Kaplan Edward L. , Blumer Jeffrey L. , Faix Dennis J. , Broderick Michael P.
Published: September 2014

Serum penicillin G falls to low levels 2 weeks after injection as benzathine penicillin G (BPG) in young adults. Using Pmetrics and previously reported penicillin G pharmacokinetic data after 1.2 million units were given as BPG to 329 male military recruits, here we develop the first reported population pharmacokinetic model of penicillin G after BPG injection. We simulated time-concentration profiles over a broad range of pediatric and adult weights after alternative doses and dose frequencies to predict the probability of maintaining serum penicillin G concentrations of >0.02 mg/liter, a proposed protective threshold against group A
Streptococcus pyogenes
(GAS). The final population model included linear absorption into a central compartment, distribution to and from a peripheral compartment, and linear elimination from the central compartment, with allometrically scaled volumes and rate constants. With 1.2 million units of BPG given intramuscularly every 4 weeks in four total doses, only 23.2% of 5,000 simulated patients maintained serum penicillin G trough concentrations of >0.02 mg/liter 4 weeks after the last dose. When the doses were 1.8 million units and 2.4 million units, the percentages were 30.2% and 40.7%, respectively. With repeated dosing of 1.2 million units every 3 weeks and every 2 weeks for 4 doses, the percentages of simulated patients with a penicillin G trough concentration of >0.02 mg/liter were 37.8% and 65.2%, respectively. Our simulations support recommendations for more frequent rather than higher BPG doses to prevent recurrent rheumatic heart disease in areas of high GAS prevalence or during outbreaks.
Mutations in the
embC-embA
Intergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol
Authors: Cui Zhenling , Li Yuanyuan , Cheng Song , Yang Hua , Lu Junmei , Hu Zhongyi , Ge Baoxue
Published: September 2014

The rapid increase in
Mycobacterium tuberculosis
resistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in the
embC-embA
intergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767
M. tuberculosis
clinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and the
embC-embA
IGRs of these strains were sequenced. The transcriptional activity of the
embC-embA
IGR mutations was examined by reporter gene assays in recombinant
Mycobacterium smegmatis
strains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator of
embAB
, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that the
embC-embA
IGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level of
embA
and
embB
mRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed into
M. smegmatis
strains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of the
embC-embA
IGR enhance the binding of EmbR to the promoter region of
embAB
and increase the expression of
embAB
, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.
Comparative Pharmacodynamics of Posaconazole in Neutropenic Murine Models of Invasive Pulmonary Aspergillosis and Mucormycosis
Authors: Lewis Russell E. , Albert Nathaniel D. , Kontoyiannis Dimitrios P.
Published: September 2014

We used two established neutropenic murine models of pulmonary aspergillosis and mucormycosis to explore the association between the posaconazole area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC) and treatment outcome. Posaconazole serum pharmacokinetics were verified in infected mice to ensure that the studied doses reflected human exposures with the oral suspension, delayed-release tablet, and intravenous formulations of posaconazole. Sinopulmonary infections were then induced in groups of neutropenic mice with
Aspergillus fumigatus
strain 293 (posaconazole MIC, 0.5 mg/liter) or
Rhizopus oryzae
strain 969 (posaconazole MIC, 2 mg/liter) and treated with escalating daily dosages of oral posaconazole, which was designed to achieve AUCs ranging from 1.10 to 392 mg · h/liter. After 5 days of treatment, lung fungal burden was analyzed by quantitative real-time PCR. The relationships of the total drug AUC/MIC and the treatment response were similar in both models, with 90% effective concentrations (EC
90
s) corresponding to an AUC/MIC threshold of 76 (95% confidence interval [CI], 46 to 102) for strain 293 versus 87 (95% CI, 66 to 101) for strain 969. Using a provisional AUC/MIC target of >100, these exposures correlated with minimum serum posaconazole concentrations (
C
min
s) of 1.25 mg/liter for strain 293 and 4.0 mg/liter for strain 969. The addition of deferasirox, but not liposomal amphotericin or caspofungin, improved the activity of a suboptimal posaconazole regimen (AUC/MIC, 33) in animals with pulmonary mucormycosis. However, no combination was as effective as the high-dose posaconazole monotherapy regimen (AUC/MIC, 184). Our analysis suggests that posaconazole pharmacodynamics are similar for
A. fumigatus
and
R. oryzae
when indexed to pathogen MICs.
Quantification of Rifapentine, a Potent Antituberculosis Drug, from Dried Blood Spot Samples Using Liquid Chromatographic-Tandem Mass Spectrometric Analysis
Authors: Parsons Teresa L. , Marzinke Mark A. , Hoang Thuy , Bliven-Sizemore Erin , Weiner Marc , Mac Kenzie William R. , Dorman Susan E. , Dooley Kelly E.
Published: September 2014

The quantification of antituberculosis drug concentrations in multinational trials currently requires the collection of modest blood volumes, centrifugation, aliquoting of plasma, freezing, and keeping samples frozen during shipping. We prospectively enrolled healthy individuals into the Tuberculosis Trials Consortium Study 29B, a phase I dose escalation study of rifapentine, a rifamycin under evaluation in tuberculosis treatment trials. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying rifapentine in whole blood on dried blood spots (DBS) to facilitate pharmacokinetic/pharmacodynamic analyses in clinical trials. Paired plasma and whole-blood samples were collected by venipuncture, and whole blood was spotted on Whatman protein saver 903 cards. The methods were optimized for plasma and then validated for DBS. The analytical measuring range for quantification of rifapentine and its metabolite was 50 to 80,000 ng/ml in whole-blood DBS. The analyte was stable on the cards for 11 weeks with a desiccant at room temperature and protected from light. The method concordance for paired plasma and whole-blood DBS samples was determined after correcting for participant hematocrit or population-based estimates of bias from Bland-Altman plots. The application of either correction factor resulted in acceptable correlation between plasma and whole-blood DBS (Passing-Bablok regression corrected for hematocrit;
y
= 0.98
x
+ 356). Concentrations of rifapentine may be determined from whole-blood DBS collected via venipuncture after normalization in order to account for the dilutional effects of red blood cells. Additional studies are focused on the application of this methodology to capillary blood collected by finger stick. The simplicity of processing, storage, shipping, and low blood volume makes whole-blood DBS attractive for rifapentine pharmacokinetic evaluations, especially in international and pediatric trials.
Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs
Authors: Sun Ren , Eriksson Staffan , Wang Liya
Published: September 2014

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK.In organellostudies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs.
Nitrotriazole- and Imidazole-Based Amides and Sulfonamides as Antitubercular Agents
Authors: Papadopoulou Maria V. , Bloomer William D. , Rosenzweig Howard S. , Arena Alexander , Arrieta Francisco , Rebolledo Joseph C. J. , Smith Diane K.
Published: September 2014

Twenty-three 3-nitrotriazole-based and 2-nitroimidazole-based amides and sulfonamides were screened for antitubercular (anti-TB) activity in aerobic
Mycobacterium tuberculosis
H37Rv by using the BacTiter-Glo (BTG) microbial cell viability assay. In general, 3-nitrotriazole-based sulfonamides demonstrated anti-TB activity, whereas 3-nitrotriazole-based amides and 2-nitroimidazole-based amides and sulfonamides were inactive. Three 3-nitrotriazole-based sulfonamides (compounds 4, 2, and 7) demonstrated 50% inhibitory concentration (IC
50
), IC
90
, and MIC values of 0.38, 0.43, and 1.56 μM (compound 4), 0.57, 0.98, and 3.13 μM (compound 2), and 0.79, 0.87, and 3.13 μM (compound 7), respectively. For 3-nitrotriazole-based sulfonamides, anti-TB activity increased with lipophilicity, whereas the one-electron reduction potential (E
1/2
) did not play a role. 2-Nitroimidazole-based analogs, which were inactive in the BTG assay, were significantly more active in the low-oxygen assay and more active than the 3-nitrotriazoles. All active nitrotriazoles in the BTG assay were similarly active or more potent (lower MIC values) against resistant strains, with the exception of compounds 2, 3, 4, and 8, which demonstrated greater MIC values against isoniazid-resistant strains. Five 3-nitrotriazole-based sulfonamides demonstrated activity in infected murine J774 macrophages, causing log reductions similar to those seen with rifampin. However, some compounds caused toxicity in uninfected macrophages. In conclusion, the classes of 3-nitrotriazole-based amides and sulfonamides merit further investigation as potential antitubercular agents.
Bacterial Lysis Liberates the Neutrophil Migration Suppressor YbcL from the Periplasm of Uropathogenic Escherichia coli
Authors: Lau Megan E. , Danka Elizabeth S. , Tiemann Kristin M. , Hunstad David A.
Published: September 2014

Uropathogenic
Escherichia coli
(UPEC) modulates aspects of the innate immune response during urinary tract infection to facilitate bacterial invasion of the bladder epithelium, a requirement for the propagation of infection. For example, UPEC-encoded YbcL suppresses the traversal of bladder epithelia by neutrophils in both an
in vitro
model and an
in vivo
murine cystitis model. The suppressive activity of YbcL requires liberation from the bacterial periplasm, though the mechanism of release is undefined. Here we present findings on the site of action of YbcL and demonstrate a novel mode of secretion for a UPEC exoprotein. Suppression of neutrophil migration by purified YbcL
UTI
, encoded by cystitis isolate UTI89, required the presence of a uroepithelial layer; YbcL
UTI
did not inhibit neutrophil chemotaxis directly. YbcL
UTI
was released to a greater extent during UPEC infection of uroepithelial cells than during that of neutrophils. Release of YbcL
UTI
was maximal when UPEC and bladder epithelial cells were in close proximity. Established modes of secretion, including outer membrane vesicles, the type II secretion system, and the type IV pilus, were dispensable for YbcL
UTI
release from UPEC. Instead, YbcL
UTI
was liberated during bacterial death, which was augmented upon exposure to bladder epithelial cells, as confirmed by detection of bacterial cytoplasmic proteins and DNA in the supernatant and enumeration of bacteria with compromised membranes. As YbcL
UTI
acts on the uroepithelium to attenuate neutrophil migration, this mode of release may represent a type of altruistic cooperation within a UPEC population during colonization of the urinary tract.
Functional Characterization of Anopheles Matrix Metalloprotease 1 Reveals Its Agonistic Role during Sporogonic Development of Malaria Parasites
Authors: Goulielmaki Evi , Sidén-Kiamos I. , Loukeris Thanasis G.
Published: September 2014

The ability to invade tissues is a unique characteristic of the malaria stages that develop/differentiate within the mosquitoes (ookinetes and sporozoites). On the other hand, tissue invasion by many pathogens has often been associated with increased matrix metalloprotease (MMP) activity in the invaded tissues. By employing cell biology and reverse genetics, we studied the expression and explored putative functions of one of the three MMPs encoded in the genome of the malaria vector
Anopheles gambiae
, namely, the
Anopheles gambiae
MMP1
(
AgMMP1
) gene, during the processes of blood digestion, midgut epithelium invasion by
Plasmodium
ookinetes, and oocyst development. We show that AgMMP1 exists in two alternative isoforms resulting from alternative splicing; one secreted (S-MMP1) and associated with hemocytes, and one membrane type (MT-MMP1) enriched in the cell attachment sites of the midgut epithelium. MT-MMP1 showed a remarkable response to ookinete midgut invasion manifested by increased expression, enhanced zymogen maturation, and subcellular redistribution, all indicative of an implication in the midgut epithelial healing that accompanies ookinete invasion. Importantly, RNA interference (RNAi)-mediated silencing of the
AgMMP1
gene revealed a postinvasion protective function of AgMMP1 during oocyst development. The combined results link for the first time an MMP with vector competence and mosquito-
Plasmodium
interactions.
Recognition of Streptococcus pneumoniae and Muramyl Dipeptide by NOD2 Results in Potent Induction of MMP-9, Which Can Be Controlled by Lipopolysaccharide Stimulation
Authors: Vissers Marloes , Hartman Yvonne , Groh Laszlo , de Jong Dirk J. , de Jonge Marien I. , Ferwerda Gerben
Published: September 2014

Matrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis duringStreptococcus pneumoniaeinfection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction byStreptococcus pneumoniaeand MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show thatStreptococcus pneumoniaeis able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved inStreptococcus pneumoniaepathogenesis.
DAP1, a Negative Regulator of Autophagy, Controls SubAB-Mediated Apoptosis and Autophagy
Authors: Yahiro Kinnosuke , Tsutsuki Hiroyasu , Ogura Kohei , Nagasawa Sayaka , Moss Joel , Noda Masatoshi
Published: September 2014

Autophagy and apoptosis play critical roles in cellular homeostasis and survival. Subtilase cytotoxin (SubAB), produced by non-O157 type Shiga-toxigenic
Escherichia coli
(STEC), is an important virulence factor in disease. SubAB, a protease, cleaves a specific site on the endoplasmic reticulum (ER) chaperone protein BiP/GRP78, leading to ER stress, and induces apoptosis. Here we report that in HeLa cells, activation of a PERK (RNA-dependent protein kinase [PKR]-like ER kinase)-eIF2α (α subunit of eukaryotic initiation factor 2)-dependent pathway by SubAB-mediated BiP cleavage negatively regulates autophagy and induces apoptosis through death-associated protein 1 (DAP1). We found that SubAB treatment decreased the amounts of autophagy markers LC3-II and p62 as well as those of mTOR (mammalian target of rapamycin) signaling proteins ULK1 and S6K. These proteins showed increased expression levels in PERK knockdown or DAP1 knockdown cells. In addition, depletion of DAP1 in HeLa cells dramatically inhibited the SubAB-stimulated apoptotic pathway: SubAB-induced Bax/Bak conformational changes, Bax/Bak oligomerization, cytochrome
c
release, activation of caspases, and poly(ADP-ribose) polymerase (PARP) cleavage. These results show that DAP1 is a key regulator, through PERK-eIF2α-dependent pathways, of the induction of apoptosis and reduction of autophagy by SubAB.
cbb
3
-Type Cytochrome
c
Oxidases, Aerobic Respiratory Enzymes, Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1
Authors: Hamada Masakaze , Toyofuku Masanori , Miyano Tomoki , Nomura Nobuhiko
Published: September 2014

For bacteria, many studies have focused on the role of respiratory enzymes in energy conservation; however, their effect on cell behavior is poorly understood.
Pseudomonas aeruginosa
can perform both aerobic respiration and denitrification. Previous studies demonstrated that
cbb
3
-type cytochrome
c
oxidases that support aerobic respiration are more highly expressed in
P. aeruginosa
under anoxic conditions than are other aerobic respiratory enzymes. However, little is known about their role under such conditions. In this study, it was shown that
cbb
3
oxidases of
P. aeruginosa
PAO1 alter anaerobic growth, the denitrification process, and cell morphology under anoxic conditions. Furthermore, biofilm formation was promoted by the
cbb
3
oxidases under anoxic conditions.
cbb
3
oxidases led to the accumulation of nitric oxide (NO), which is produced during denitrification. Cell elongation induced by NO accumulation was reported to be required for robust biofilm formation of
P. aeruginosa
PAO1 under anoxic conditions. Our data show that
cbb
3
oxidases promote cell elongation by inducing NO accumulation during the denitrification process, which further leads to robust biofilms. Our findings show that
cbb
3
oxidases, which have been well studied as aerobic respiratory enzymes, are also involved in denitrification and influence the lifestyle of
P. aeruginosa
PAO1 under anoxic conditions.
Structural and Functional Characterization of Pseudomonas aeruginosa Global Regulator AmpR
Authors: Caille Olivier , Zincke Diansy , Merighi Massimo , Balasubramanian Deepak , Kumari Hansi , Kong Kok-Fai , Silva-Herzog Eugenia , Narasimhan Giri , Schneper Lisa , Lory Stephen , Mathee Kalai
Published: September 2014

Pseudomonas aeruginosais a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC β-lactamase is a common mechanism of β-lactam resistance among clinical isolates. The inducible expression ofampCis controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important forampCinduction. Specifically, theampC(PampC) andampR(PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5′ rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ54and σ70consensus sequences were identified at PampRand PampC, respectively. Sigma factor RpoN was found to negatively regulateampRexpression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampCextends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampCin vitrowhereasin vivobinding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role inP. aeruginosaAmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, anin vivocross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulatesampCexpression by direct binding.
Redox-Responsive Repressor Rex Modulates Alcohol Production and Oxidative Stress Tolerance in Clostridium acetobutylicum
Authors: Zhang Lei , Nie Xiaoqun , Ravcheev Dmitry A. , Rodionov Dmitry A. , Sheng Jia , Gu Yang , Yang Sheng , Jiang Weihong , Yang Chen
Published: September 2014

Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD
+
ratio, has recently been found to play a role in the solventogenic shift of
Clostridium acetobutylicum
. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in
C. acetobutylicum
. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO
2
/CO fixation. The predicted Rex-binding sites in the genomes of
Clostridium
spp. were verified by
in vitro
binding assays with purified Rex protein. Novel members of the
C. acetobutylicum
Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and
rex
-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H
2
O
2
on intracellular NADH and NAD
+
concentrations, expression of Rex regulon genes, and physiology of the wild type and
rex
-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD
+
ratio
in vivo
to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in
C. acetobutylicum
. It is suggested that Rex plays an important role in maintaining NADH/NAD
+
homeostasis in clostridia.
Chemotaxis of Escherichia coli to Norepinephrine (NE) Requires Conversion of NE to 3,4-Dihydroxymandelic Acid
Authors: Pasupuleti Sasikiran , Sule Nitesh , Cohn William B. , MacKenzie Duncan S. , Jayaraman Arul , Manson Michael D.
Published: September 2014

Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic
Escherichia coli
(EHEC). Here we show that nonpathogenic
E. coli
K-12 grown in the presence of 2 μM NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for
E. coli
. Only strains of
E. coli
K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is ≤5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 μM. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit
E. coli
and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection.