bluuebunny/crossref_metadata_2025 huggingface dataset:
Macroautophagy in Immunity and Tolerance
Authors: Gannagé Monique , Münz Christian
Published: January 2009

Autophagy and proteasomal degradation constitute the two main catabolic pathways in cells. While the proteasome degrades primarily short‐lived soluble proteins, macroautophagy, the main constitutive autophagic pathway, delivers cell organelles and protein aggregates for lysosomal degradation. Both the proteasome and macroautophagy are attractive effector mechanisms for the immune system because they can be used to degrade foreign substances, including pathogenic proteins, within cells. Therefore, both innate and adaptive immune responses use these pathways for intracellular clearance of pathogens as well as for presentation of pathogen fragments to the adaptive immune system. Because, however, the same mechanisms are used for the steady‐state turnover of cellular self‐components, the immune system has to be desensitized not to recognize these. Therefore, proteasomal degradation and macroautophagy are also involved in tolerizing the immune system prior to pathogen encounter. We will discuss recent advances in our understanding how macroautophagy selects self‐structures in the steady state, how presentation of these on major histocompatibility complex class II molecules leads to tolerance and how macroautophagy assists both innate and adaptive immunity. This new knowledge on the specialized functions of the metabolic process macroautophagy in higher eukaryotes should allow us to target it for therapy development against immunopathologies and to improve vaccinations.
Control of Granule Mobility and Exocytosis by Ca2+‐Dependent Formation of F‐Actin in Pancreatic Duct Epithelial Cells
Authors: Jung Seung‐Ryoung , Kim Mean‐Hwan , Hille Bertil , Koh Duk‐Su
Published: January 2009

Elevation of intracellular Ca2+ concentration ([Ca2+]i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca2+]i increase, kinetically coincident with formation of filamentous actin (F‐actin) in the whole cytoplasm. At high resolution, the new F‐actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F‐actin with latrunculin B blocked both the F‐actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F‐actin meshwork inhibited exocytosis at small Ca2+ rises <1 μm. Larger [Ca2+]i rises increased exocytosis because of the co‐ordinate translocation of granules and fusion to the membrane. We propose that the Ca2+‐dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.
Basal Body Assembly in Ciliates: The Power of Numbers
Authors: Pearson Chad G. , Winey Mark
Published: January 2009

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium, historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.
Calsyntenins Mediate TGN Exit of APP in a Kinesin‐1‐Dependent Manner
Authors: Ludwig Alexander , Blume Jessica , Diep Tu‐My , Yuan Ju , Mateos José María , Leuthäuser Kerstin , Steuble Martin , Streit Peter , Sonderegger Peter
Published: January 2009

Kinesin motors are required for the export of membranous cargo from the trans‐Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin‐1 docking protein calsyntenin‐1 localizes to the TGN in vivo and directly and specifically recruits Kinesin‐1 to Golgi/TGN membranes as well as to dynamic post‐Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin‐1‐decorated tubular structures which may serve as carriers for calsyntenin‐1‐mediated post‐TGN transport of APP. Abrogation of this pathway via virus‐mediated knockdown of calsyntenin‐1 expression in primary cultured neurons caused a marked elevation of APP C‐terminal fragments. Together, these results indicate a role for calsyntenin‐1 in Kinesin‐1‐dependent TGN exit and post‐Golgi transport of APP‐containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.
Analyses of the Recycling Receptor, FcRn, in Live Cells Reveal Novel Pathways for Lysosomal Delivery
Authors: Gan Zhuo , Ram Sripad , Vaccaro Carlos , Ober Raimund J. , Ward E. Sally
Published: January 2009

Lysosomes play a central role in the degradation of proteins and other macromolecules. The mechanisms by which receptors are transferred to lysosomes for constitutive degradation are poorly understood. We have analyzed the processes that lead to the lysosomal delivery of the Fc receptor, FcRn. These studies provide support for a novel pathway for receptor delivery. Specifically, unlike other receptors that enter intraluminal vesicles in late endosomes, FcRn is transferred from the limiting membrane of such endosomes to lysosomes, and is rapidly internalized into the lysosomal lumen. By contrast, LAMP‐1 persists on the limiting membrane. Receptor transfer is mediated by tubular extensions from late endosomes to lysosomes, or by interactions of the two participating organelles in kiss‐and‐linger‐like processes, whereas full fusion is rarely observed. The persistence of FcRn on the late endosomal limiting membrane, together with selective transfer to lysosomes, allows this receptor to undergo recycling or degradation. Consequently, late endosomes have functional plasticity, consistent with the presence of the Rab5 GTPase in discrete domains on these compartments.
Working profiles of dental hygienists in public and private practice in Finland and Norway
Authors: Tseveenjav B , Virtanen JI , Wang NJ , Widström E
Published: January 2009

Abstract:  Aim:  The aim was to compare the working profiles of Finnish and Norwegian dental hygienists in public and private practice. To this end, we compared the procedures performed, the type of patients and the time devoted to different tasks.

Subjects and methods:  A questionnaire survey was originally conducted among a representative sample of dental hygienists in Finland (n = 595) and all authorized dental hygienists in Norway (n = 1 138) in 2004. The questionnaires collected data on the dental hygienists’ age, gender, year of graduation, working experience, work sector (private or public), working time spent on different activities and patient groups. The questionnaire also assessed how frequently the dental hygienists performed 25 different treatment measures.

Results:  The Norwegian dental hygienists spent 45.4% of their clinical time on check‐ups, whereas the Finns spent 49.9% of their time scaling. Dental hygienists in Finland and Norway working in the public sector spent 42.9% and 74.6% of their working time dealing with children and youth respectively.

Conclusions:  The working profiles of dental hygienists in Finland and Norway were quite similar, although differences in distribution by activities, type of patients and treatment measures do exist. The main activity of the dental hygienists was clinical work. The most commonly practised clinical activity among Finnish dental hygienists was scaling, and among Norwegians, check‐ups. Public dental hygienists in both countries dealt mainly with children and youths. Oral hygiene instruction was the most commonly reported treatment measure among both Finns and Norwegians.
A discourse on the nature of dental hygiene knowledge and knowing
Authors: Cobban SJ , Edgington EM , Myrick F , Keenan L
Published: January 2009

Abstract:  Objective:  Historically, dental hygiene has adopted theory and research from other health disciplines, without adequately modifying these concepts to reflect the unique dental hygiene practice context, leaving dental hygiene’s research and theory base underdeveloped. Dental hygiene has yet to articulate its epistemological assumptions – the nature, scope and object of dental hygiene knowledge – or to fully describe the patterns of knowing that are brought to practice.

Methods:  This paper uses a method of inquiry from philosophy to begin the discourse about dental hygiene ways of knowing. In nursing, Carper identified four fundamental patterns of knowing: empirics or the science of nursing; aesthetics or the art of nursing; personal knowledge and ethical or moral knowledge. These patterns were used to explore this concept within dental hygiene.

Results:  There is more to the nature of dental hygiene knowledge and knowing than rote application of technique‐related or research‐based information in practice, including judgements about when and how to use different types of information that are used. Currently, empirical forms of knowledge seem to be disproportionately valued, yet evidence was found for all of Carper’s four patterns of knowing.

Conclusions:  Carper’s work on patterns of knowing in nursing provided a useful framework to initiate the discourse on ways of knowing in dental hygiene. These results are submitted for others to challenge, refine and extend, for continuing the discussion. Dental hygiene leaders and scholars need to engage in discourse about extending the epistemological assumptions to reflect reality.
The WHO’s action plan for oral health
Authors: Monajem S
Published: January 2009

Abstract:  The oral health action plan, recommended for adoption to the Sixtieth World Health Assembly of the World Health Organization in January 2007, included many of the necessary components. Had fissure sealants been added to the list of prevention methods and the roles of dental educational institutions and hygiene community better clarified, the action plan would have made a more viable and realistic package for the ministries and their directors of national programmes receiving the support of the World Health Organization and partners. Sealants remain under‐utilized, few dental hygienists are integrated in the primary oral health team and fewer dental graduates have had service‐learning experiences – all contradictory to the evidence in the literature. Translating research findings into public health action programmes is one of the recommendations made in the action plan and one way we can begin this is by implementing sealant programmes in ‘Health Promoting Schools’ that are WHO’s brain child and borne of the wisdom of using schools as ‘platforms’ for the promotion and delivery of health care to the community.
Older peoples’ perceptions of oral health: ‘it’s just not that simple’
Authors: McKenzie‐Green B , Giddings LS , Buttle L , Tahana K
Published: January 2009

Abstract:  Objectives:  Little is known about older persons’ perceptions of oral health and oral health care. The purpose of this study was to explore the viewpoint of older adults’ regarding their oral health care practices.

Methods:  A qualitative interpretive methodology was employed comprising three analytic levels: coding of data into concepts, analysis of concepts into themes, followed by an in‐depth analysis of relationships within concepts and between themes. In‐depth individual interviews were conducted with 19 participants aged 65 to 87 years.

Results:  Older people’s decision to access oral health care involves complex and personally meaningful strategies. A dental visit surfaces hopes and fears based on past and present experiences. Mouth and teeth are not merely objects of dental care; they represent a person’s social and relational self. Age‐related changes challenge the relational self as represented in societal ideal images of youth and perfection (the perfect smile). This study highlights older peoples’ resilience and determination when faced with the dilemmas in accessing oral health care – it costs, personally as well as financially. Contrary to the assumption that older peoples’ oral health status is related to neglect, rather for many, it is the result of the intersection of their history with technological advances.

Conclusions:  These findings challenge oral health care practitioners to be sensitive to the contexts affecting their older client’s oral health care status. They do not ‘just go’ to the dentist; they bring with them their past dental experiences and their hopes for the future. It matters how one is treated at this vulnerable time.
Periodontitis from the patient’s perspective, a qualitative study
Authors: Karlsson E , Lymer U‐B , Hakeberg M
Published: January 2009

Abstract:  Periodontitis is an infectious disease that impacts people’s oral health and can lead to the loss of teeth. There are several factors that explain why some people develop this disease. Purpose: The aim of this study was to describe patients’ perceptions of living with periodontitis. Method: A qualitative method inspired by phenomenography was chosen, because the focal point was the patients’ perceptions of this phenomenon. A total of 10 patients, all of whom were being treated at a specialist clinic for periodontology in the south of Sweden, were interviewed. Results: Two main categories were identified: perceptions of disease and perceptions of having the disease under control, together with subcategories. The first category highlights the patients’ perception of what causes periodontitis, being given the diagnosis, after the diagnosis and the consequences of periodontitis. The other main category highlights the patients’ perception of their own responsibility and the professionals’ responsibility. The patients’ oral health‐related quality of life had been influenced by periodontitis. There is no guarantee that the patient has understood what is actually required of him/her during treatment, despite information about the diagnosis. It can often take some time for patients to understand the nature of their disease. Conclusion: Acceptance and control of the disease result in less inconvenience in the patients’ daily lives. It creates an optimistic future perspective, despite feelings of anxiety about facing consequences such as tooth loss.
Tobacco use in relation to socioeconomic factors and dental care habits among Swedish individuals 15–70 years of age, 1983–2003
Authors: Hellqvist L , Rolandsson M , Birkhed D , Hugoson A
Published: January 2009

Abstract:  Objectives:  The aim of this study was to describe the use of tobacco and changes in its use over time among individuals living in Jönköping, Sweden, and to analyse tobacco habits in relation to socioeconomic conditions, personality aspects and dental care habits.

Methods:  This study comprised three epidemiological cross‐sectional studies, involving a random selection of individuals aged between 15 and 70 years, and was conducted in 1983, 1993 and 2003. The participants were asked to complete a questionnaire.

Results:  The results revealed a statistically significant reduction from 34% tobacco users in 1983 to 27% in 1993 and 28% in 2003. The main decrease was seen among smokers. At the same time, the number of users of snuff increased in all the age groups between 20 and 60 years of age. The use of tobacco was therefore largely unchanged in 1993 and 2003. In 2003, there was a statistically significant difference between users and non‐tobacco users when it came to the frequency of dental visits; more tobacco users than non‐tobacco users did not visit a dentist at all or did not visit a dentist regularly. In 1993, non‐tobacco users brushed their teeth more frequently than tobacco users and this difference was statistically significant.

Conclusions:  During the 20‐year study, there was a reduction in the number of smokers and an increase in the number of snuff users. There was a difference between tobacco users and non‐tobacco users when it came to the frequency of dental visits and oral hygiene habits.
Smoking and caries experience in subjects with various form of periodontal diseases from a teaching hospital clinic
Authors: Al‐Habashneh R , Al‐Omari MAO , Taani DQ
Published: January 2009

Abstract:  Objective:  The aim of this study was to assess the relationships between aggressive periodontitis (AgP), caries and smoking.

Method and materials:  A cross‐sectional study was conducted among patients who were specifically referred to the Dental Teaching Clinic in Irbid, Jordan for periodontal treatment. Self‐administered questionnaire related to socio‐demographic data and smoking habits was completed. The oral hygiene, gingival status, periodontal health and dental status of the participants was determined by using the plaque index of Silness and Loe [Acta Odontol Scand, 22 (1964), 121], the gingival index of Loe and Silness [Acta Odontol Scand, 21 (1963), 233], clinical attachment level (CAL) and decayed, missing and filled teeth (DMFT) index respectively.

Result:  The prevalence of smoking was greater in chronic periodontitis (CP) group (44.2%) than in either chronic gingivitis (CG) (27.4%) or AgP (29.9%) group. Self‐reported perio‐diseases in the close family was more prevalent (77%) among subjects diagnosed with AgP. The mean plaque scores were significantly higher for smoker than non‐smoker in AgP group only (P = 0.04), with significantly greater plaque and gingival scores in CG and CP groups than AgP group (P = 0.012, 0.004). A significantly greater mean gingival scores were noted among CG and CP groups than AgP group (P = 0.004). The mean CAL was higher in smokers than in non‐smokers in the three groups, with statistically significant differences in CP and AgP groups (P = 0.04, 0.01 respectively).The mean number of DMFT was significantly higher in smoker than in non‐smoker of all age groups (P = 0.016, 0.043 and 0.01). However, mean DMFT was significantly greater in CP and CG than AgP groups.

Conclusion:  It was concluded that (i) higher plaque and gingival index among smokers in all groups; (ii) significant difference in the CAL between smoker and non‐smoke in CP and AgP groups; (iii) significant increase in caries risk among smokers in all groups; (iv) smokers and non‐smokers of AgP group had significantly lower mean DMFT scores than those of CG or CP groups.
Tobacco use cessation provided by dental hygienists
Authors: Ramseier CA , Fundak A
Published: January 2009

Abstract:  Second to regular mechanical plaque control, tobacco use cessation has become the most important measure for the treatment of periodontal diseases. In contrast to general medical professionals, dental hygienists are seeing their patients regularly and are therefore available for supporting their patients to quit tobacco use. Tobacco use disease consists of both a physical addiction and a psychological dependence. Therefore, the combination of behaviour change support with pharmacotherapy is recommended for tobacco use cessation counselling. The use of brief motivational interviewing for tobacco use short interventions in the dental practice appears to be suitable. In addition to behavioural support, the use for nicotine replacement therapy is the treatment of choice for the dental practice. Following a critical review of the literature on this topic, a step by step approach for tobacco use cessation is presented for the dental hygienists to be implemented in their daily practice routine.
Organization of ventricular fibrillation in the human heart: experiments and models
Authors: Ten Tusscher K. H. W. J. , Mourad A. , Nash M. P. , Clayton R. H. , Bradley C. P. , Paterson D. J. , Hren R. , Hayward M. , Panfilov A. V. , Taggart P.
Published: January 2009

Sudden cardiac death is a major health problem in the industrialized world. The lethal event is typically ventricular fibrillation (VF), during which the co‐ordinated regular contraction of the heart is overthrown by a state of mechanical and electrical anarchy. Understanding the excitation patterns that sustain VF is important in order to identify potential therapeutic targets. In this paper, we studied the organization of human VF by combining clinical recordings of electrical excitation patterns on the epicardial surface during in vivo human VF with simulations of VF in an anatomically and electrophysiologically detailed computational model of the human ventricles. We find both in the computational studies and in the clinical recordings that epicardial surface excitation patterns during VF contain around six rotors. Based on results from the simulated three‐dimensional excitation patterns during VF, which show that the total number of electrical sources is 1.4 ± 0.12 times greater than the number of epicardial rotors, we estimate that the total number of sources present during clinically recorded VF is 9.0 ± 2.6. This number is approximately fivefold fewer compared with that observed during VF in dog and pig hearts, which are of comparable size to human hearts. We explain this difference by considering differences in action potential duration dynamics across these species. The simpler spatial organization of human VF has important implications for treatment and prevention of this dangerous arrhythmia. Moreover, our findings underline the need for integrated research, in which human‐based clinical and computational studies complement animal research.
Dietary pectin up‐regulates monocaboxylate transporter 1 in the rat gastrointestinal tract
Authors: Kirat Doaa , Kondo Koji , Shimada Ritsu , Kato Seiyu
Published: January 2009

This work was undertaken to study the effect of pectin feeding on the expression level, cellular localization and functional activity of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of rats. The results indicated that MCT1 protein level was significantly increased along the entire length of the gastrointestinal tract of pectin‐fed rats in comparison with control animals. Immunohistochemical analysis revealed an increase in MCT1 in the stratified squamous epithelia of the forestomach as well as in the basolateral membranes of the cells lining the gastric pit of the glandular stomach of pectin‐fed rats when compared with control animals. The parietal cells, which showed barely any or no detectable MCT1 in the control group, exhibited a strong intensity of MCT1 on the basolateral membranes in pectin‐fed rats. In the small intestine of pectin‐fed rats, strong immunopositivity for MCT1 was detected in the brush border and basolateral membranes of the absorptive enterocytes lining the entire villi, while in control rats, weak reactivity was detected on the brush border membrane in a few absorptive enterocytes in the villus tip. In the large intestine of control animals, MCT1 was detected on the basolateral membranes of the epithelia lining the caecum and colon. This staining intensity was markedly increased in pectin‐fed rats, along with the appearance of strong reactivity for MCT1 on the apical membranes of the surface and crypt epithelia of caecum and colon. Our results also showed that MCT1 co‐localizes with its chaperone, basigin (CD147), in the rat gastrointestinal tract, and that the pectin feeding increased the expression of CD147. In vivo functional studies revealed an enhanced acetate absorption in the colon of pectin‐fed in comparison with control animals. We conclude that MCT1 is up‐regulated along the gastrointestinal tract of pectin‐fed rats, which might represent an adaptive response to the increased availability of its substrates.
Sudden Death of Entanglement
Authors: Yu Ting , Eberly J. H.
Published: January 2009

A new development in the dynamical behavior of elementary quantum systems is the surprising discovery that correlation between two quantum units of information called qubits can be degraded by environmental noise in a way not seen previously in studies of dissipation. This new route for dissipation attacks quantum entanglement, the essential resource for quantum information as well as the central feature in the Einstein-Podolsky-Rosen so-called paradox and in discussions of the fate of Schrödinger's cat. The effect has been labeled ESD, which stands for early-stage disentanglement or, more frequently, entanglement sudden death. We review recent progress in studies focused on this phenomenon.
Studies of the Production of Fungal Polyketides inAspergillus nidulansby Using Systems Biology Tools
Authors: Panagiotou Gianni , Andersen Mikael R. , Grotkjaer Thomas , Regueira Torsten B. , Nielsen Jens , Olsson Lisbeth
Published: January 2009

Many filamentous fungi produce polyketide molecules with great significance as human pharmaceuticals; these molecules include the cholesterol-lowering compound lovastatin, which was originally isolated fromAspergillus terreus. The chemical diversity and potential uses of these compounds are virtually unlimited, and it is thus of great interest to develop a well-described microbial production platform for polyketides. Using genetic engineering tools available for the model organismAspergillus nidulans, we constructed two recombinant strains, one expressing thePenicillium griseofulvum6-methylsalicylic acid (6-MSA) synthase gene and one expressing the 6-MSA synthase gene and overexpressing the native xylulose-5-phosphate phosphoketolase gene (xpkA) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and that of a reference wild-type strain were characterized on glucose, xylose, glycerol, and ethanol media in controlled bioreactors. Glucose was found to be the preferred carbon source for 6-MSA production, and 6-MSA concentrations up to 455 mg/liter were obtained for the recombinant strain harboring the 6-MSA gene. Our findings indicate that overexpression ofxpkAdoes not directly improve 6-MSA production on glucose, but it is possible, if the metabolic flux through the lower part of glycolysis is reduced, to obtain quite high yields for conversion of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA-producing strains grown on glucose and xylose in the presence and absence ofxpkAoverexpression, combined with flux and physiology data, enabled us to propose anxpkA-msaSinteraction model describing the competition between biomass formation and 6-MSA production for the available acetyl coenzyme A.
Changes in Barotolerance, Thermotolerance, and Cellular Morphology throughout the Life Cycle of
Listeria monocytogenes
Authors: Wen Jia , Anantheswaran Ramaswamy C. , Knabel Stephen J.
Published: January 2009

Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of
Listeria monocytogenes
were investigated. For part 1 of this analysis,
L. monocytogenes
ATCC 19115 was grown to log, stationary, death, and long-term-survival phases at 35°C in tryptic soy broth with yeast extract (TSBYE). Cells were diluted in whole milk that had been subjected to ultrahigh temperatures (UHT whole milk) and then high-pressure processed (HPP) at 400 MPa for 180 s or thermally processed at 62.8°C for 30 s. As cells transitioned from the log to the long-term-survival phase, the D
400 MPa
and D
62.8°C
values increased 10- and 19-fold, respectively. Cells decreased in size as they transitioned from the log to the long-term-survival phase. Rod-shaped cells transitioned to cocci as they entered the late-death and long-term-survival phases.
L. monocytogenes
strains F5069 and Scott A showed similar results. For part 2 of the analysis, cells in long-term-survival phase were centrifuged, suspended in fresh TSBYE, and incubated at 35°C. As cells transitioned from the long-term-survival phase to log and the stationary phase, they increased in size and log reductions increased following HPP or heat treatment. In part 3 of this analysis, cells in long-term-survival phase were centrifuged, suspended in UHT whole milk, and incubated at 4°C. After HPP or heat treatment, similar results were observed as for part 2. We hypothesize that cells of
L. monocytogenes
enter a dormant, long-term-survival phase and become more barotolerant and thermotolerant due to cytoplasmic condensation when they transition from rods to cocci. Further research is needed to test this hypothesis and to determine the practical significance of these findings.
Longitudinal Study of Shiga Toxin-Producing
Escherichia coli
Shedding in Sheep Feces: Persistence of Specific Clones in Sheep Flocks
Authors: Sánchez Sergio , Martínez Remigio , García Alfredo , Blanco Jorge , Blanco Jesús E. , Blanco Miguel , Dahbi Ghizlane , López Cecilia , Mora Azucena , Rey Joaquín , Alonso Juan M.
Published: January 2009

To provide information on the persistence and maintenance of colonization with Shiga toxin-producing
Escherichia coli
(STEC) in sheep, pulsed-field gel electrophoresis analysis of STEC isolates (
n
= 145) belonging to serogroups O5, O91, and O146 from 39 healthy animals was performed in a 12-month longitudinal study carried out with four sheep flocks. At the flock level as well as the individual-animal level, the same clones were obtained on sampling occasions separated by as much as 11 months.
Reconfiguring the Quorum-Sensing Regulator SdiA ofEscherichia coliTo Control Biofilm Formation via Indole andN-Acylhomoserine Lactones
Authors: Lee Jintae , Maeda Toshinari , Hong Seok Hoon , Wood Thomas K.
Published: January 2009

SdiA is a homolog of quorum-sensing regulators that detectsN-acylhomoserine lactone (AHL) signals from other bacteria.Escherichia coliuses SdiA to reduce its biofilm formation in the presence of both AHLs and its own signal indole. Here we reconfigured SdiA (240 amino acids) to control biofilm formation using protein engineering. Four SdiA variants were obtained with altered biofilm formation, including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and K153X) and SdiA14C3 (W9R, P49T, N87T, frameshift at N96, and L123X), which reduced biofilm formation by 5- to 20-fold compared to wild-type SdiA in the presence of endogenous indole. Whole-transcriptome profiling revealed that wild-type SdiA reduced biofilm formation by repressing genes related to indole synthesis and curli synthesis compared to when no SdiA was expressed, while variant SdiA1E11 induced genes related to indole synthesis in comparison to wild-type SdiA. These results suggested altered indole metabolism, and corroborating the DNA microarray results in regard to indole synthesis, variant SdiA1E11 produced ninefold more indole, which led to reduced swimming motility and cell density. Also, wild-type SdiA decreased curli production andtnaAtranscription, while SdiA1E11 increasedtnaAtranscription (tnaAencodes tryptophanase, which forms indole) compared to wild-type SdiA. Hence, wild-type SdiA decreased biofilm formation by reducing curli production and motility, and SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive variant (SdiA2D10, having four mutations at E31G, Y42F, R116H, and L165Q) increased biofilm formation sevenfold in the presence ofN-octanoyl-dl-homoserine lactone andN-(3-oxododecatanoyl)-l-homoserine lactone. Therefore, SdiA can be evolved to increase or decrease biofilm formation, and biofilm formation may be controlled by altering sensors rather than signals.
Real-Time PCR Assay for Detection of a New Simulant for Poxvirus Biothreat Agents
Authors: Garnier Laurence , Gaudin Jean-Christophe , Bensadoun Paul , Rebillat Isabelle , Morel Yannick
Published: January 2009

Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant “simulant” microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the
Cydia pomonella
granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per μl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows
Cydia pomonella
granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies.
Development of a Prophage Typing System and Analysis of Prophage Carriage in
Streptococcus pneumoniae
Authors: Romero Patricia , García Ernesto , Mitchell Tim J.
Published: January 2009

The frequency of prophage carriage was tested in a collection of 108 clinical isolates of
Streptococcus pneumoniae
. A PCR-based assay was developed to allow classification of the prophage into the three groups recently identified according to genome comparisons (P. Romero, N. Croucher, N. L. Hiller, F. Z. Hu, G. D. Ehrlich, S. D. Bentley, E. García, and T. J. Mitchell, submitted for publication). Use of the assay showed that more than half of the isolates studied were lysogenic with prophage belonging to group 1 being the most abundant (56%), followed by those belonging to group 2 (26%) and those belonging to group 3 (11%). Four polylysogenic strains harboring a group 1 and a group 2 prophage were identified. Interestingly, lysogenic strains were found in 8 out of the 12 internationally distributed, relevant clones of
S. pneumoniae
contained in our strain collection. The high percentage of clinical pneumococcal isolates harboring prophage strongly suggests an important contribution to the diversification of the genome architecture in this species as well as a role for bacteriophage in the virulence/and or fitness of
S. pneumoniae
, although further studies using a significant number of isolates belonging to the most relevant pneumococcal clones are needed.
Nickel Promotes Biofilm Formation by
Escherichia coli
K-12 Strains That Produce Curli
Authors: Perrin Claire , Briandet Romain , Jubelin Gregory , Lejeune Philippe , Mandrand-Berthelot Marie-Andrée , Rodrigue Agnès , Dorel Corinne
Published: January 2009

The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads
Escherichia coli
bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in
E. coli
is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.
Tetracycline Resistome of the Organic Pig Gut
Authors: Kazimierczak Katarzyna A. , Scott Karen P. , Kelly Denise , Aminov Rustam I.
Published: January 2009

The occurrence of genes conferring resistance to tetracyclines in the organic pig gut was assessed through the metagenomic approach. Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known
tet
(C),
tet
(W), and
tet
(40) genes, as well as novel genes encoding resistance to the tetracyclines minocycline and doxycycline. The latter are different from the known
tet
genes and are homologous to genes encoding UDP-glucose 4-epimerases, with the domain structure characteristic for these enzymes. The majority of the resistance genes were associated with putative mobile genetic elements. The sequence of a novel 9.7-kb plasmid carrying
tet
(W) and
tet
(40) was also identified. Conserved flanking regions identified around the
tet
(W) and
tet
(40) genes in our metagenomic library may play a role in genetic exchange of these genes. This is the first report describing the occurrence of
tet
(40) outside the human intestine. The maintenance of antibiotic resistance genes in apparently antibiotic-free animals is probably due to their presence on mobile genetic elements, the fitness cost of which for the cell is ameliorated during the previous antibiotic selection.
Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract
Authors: Turroni Francesca , Foroni Elena , Pizzetti Paola , Giubellini Vanessa , Ribbera Angela , Merusi Paolo , Cagnasso Patrizio , Bizzarri Barbara , de'Angelis Gian Luigi , Shanahan Fergus , van Sinderen Douwe , Ventura Marco
Published: January 2009

Although the health-promoting roles of bifidobacteria are widely accepted, the diversity of bifidobacteria among the human intestinal microbiota is still poorly understood. We performed a census of bifidobacterial populations from human intestinal mucosal and fecal samples by plating them on selective medium, coupled with molecular analysis of selected rRNA gene sequences (16S rRNA gene and internally transcribed spacer [ITS] 16S-23S spacer sequences) of isolated colonies. A total of 900 isolates were collected, of which 704 were shown to belong to bifidobacteria. Analyses showed that the culturable bifidobacterial population from intestinal and fecal samples include six main phylogenetic taxa, i.e.,
Bifidobacterium longum
,
Bifidobacterium pseudocatenulatum
,
Bifidobacterium adolescentis
,
Bifidobacterium pseudolongum
,
Bifidobacterium breve
, and
Bifidobacterium bifidum
, and two species mostly detected in fecal samples, i.e.,
Bifidobacterium dentium
and
Bifidobacterium animalis
subp.
lactis
. Analysis of bifidobacterial distribution based on age of the subject revealed that certain identified bifidobacterial species were exclusively present in the adult human gut microbiota whereas others were found to be widely distributed. We encountered significant intersubject variability and composition differences between fecal and mucosa-adherent bifidobacterial communities. In contrast, a modest diversification of bifidobacterial populations was noticed between different intestinal regions within the same individual (intrasubject variability). Notably, a small number of bifidobacterial isolates were shown to display a wide ecological distribution, thus suggesting that they possess a broad colonization capacity.
Comparative Analysis of Attachment of Shiga-Toxigenic
Escherichia coli
and
Salmonella
Strains to Cultured HT-29 and Caco-2 Cell Lines
Authors: Mellor Glen E. , Goulter Rebecca M. , Chia T. W. Raymond , Dykes Gary A.
Published: January 2009

The ability of
Escherichia coli
and
Salmonella
isolates to attach to Caco-2 and HT-29 cell monolayers was measured. All isolates displayed a greater ability to attach to Caco-2 cells than HT-29 cells, and overall
E. coli
isolates attached better to both cell lines than
Salmonella
isolates. Bacteria that were considered to be pathogenic displayed no greater ability to attach to cell lines than those that were not considered to be pathogenic. Additionally, no correlation was found between cell line attachment and previously determined hydrophobicity results.
Phage-Mediated Shiga Toxin 2 Gene Transfer in Food and Water
Authors: Imamovic Lejla , Jofre Juan , Schmidt Herbert , Serra-Moreno Ruth , Muniesa Maite
Published: January 2009

Shiga toxin (
stx
) transduction in various food matrices has been evaluated with lysogens of Stx phages.
stx
transduction events were observed for many phages under appropriate conditions. Transduction did not occur at low pH and low temperatures. A total of 10
3
to 10
4
CFU ml
−1
was the minimal amount of donor and recipient strains necessary to generate transductants.
Diffusion of Macromolecules in Model Oral Biofilms
Authors: Takenaka Shoji , Pitts Betsey , Trivedi Harsh M. , Stewart Philip S.
Published: January 2009

The diffusive penetration of fluorescently tagged macromolecular solutes into model oral biofilms was visualized by time-lapse microscopy. All of the solutes tested, including dextrans, proteases, green fluorescent protein, and immunoglobulin G, accessed the interior of cell clusters 100 to 200 μm in diameter within 3 min or less.
C
29
Olefinic Hydrocarbons Biosynthesized by
Arthrobacter
Species
Authors: Frias Janice A. , Richman Jack E. , Wackett Lawrence P.
Published: January 2009

Arthrobacter aurescens
TC1,
Arthrobacter chlorophenolicus
A6,
Arthrobacter crystallopoietes
, and
Arthrobacter oxydans
produce long-chain monoalkenes, predominantly
cis
-3,25-dimethyl-13-heptacosene. Four other
Arthrobacter
strains did not form alkenes. The level of
cis
-3,25-dimethyl-13-heptacosene in
Arthrobacter chlorophenolicus
A6 remained proportional to cell mass during growth.
cis
-3,25-Dimethyl-13-heptacosene did not support growth of
A. chlorophenolicus
A6.
AgtA, the Dicarboxylic Amino Acid Transporter of
Aspergillus nidulans
, Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis
Authors: Apostolaki Angeliki , Erpapazoglou Zoi , Harispe Laura , Billini Maria , Kafasla Panagiota , Kizis Dimosthenis , Peñalva Miguel Angel , Scazzocchio Claudio , Sophianopoulou Vicky
Published: January 2009

We identified
agtA
, a gene that encodes the specific dicarboxylic amino acid transporter of
Aspergillus nidulans
. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.
Towards New Antifolates Targeting Eukaryotic Opportunistic Infections
Authors: Liu Jieying , Bolstad David B. , Bolstad Erin S. D. , Wright Dennis L. , Anderson Amy C.
Published: January 2009

Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa
Cryptosporidium
and
Toxoplasma
as well as the fungus
Candida glabrata
. A comparison of the structures of DHFR from the fungal species
Candida glabrata
and
Pneumocystis
suggests that the compounds may also potently inhibit
Pneumocystis
DHFR.
Role of CpSUB1, a Subtilisin-Like Protease, in
Cryptosporidium parvum
Infection In Vitro
Authors: Wanyiri Jane W. , Techasintana Patsharaporn , O'Connor Roberta M. , Blackman Michael J. , Kim Kami , Ward Honorine D.
Published: January 2009

The apicomplexan parasite
Cryptosporidium
is a significant cause of diarrheal disease worldwide. Previously, we reported that a
Cryptosporidium parvum
subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in
C. parvum
lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the
C. parvum
genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from
C. parvum
-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, for
C. parvum
lysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by
C. parvum
lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in
C. parvum
and for processing of gp40/15. Importantly, the recombinant prodomain inhibited
C. parvum
infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.