bluuebunny/crossref_metadata_2025 huggingface dataset:
Small Craft Combat Survivability Enhancement Through the Use of Smoke
Authors: KENNEDY JOHN R. , RINGWALD MICHAEL G. , SCHONBERGER JAMES R. , LOHKAMP CARL W.
Published: March 2009

Survivability is dependent on two basic factors, susceptibility, which is the probability of being hit by a damage‐causing mechanism, and vulnerability, which is the probability of being killed once hit. Visually aimed weapons present a major threat to the survivability of small waterborne craft in combat operations. Since this type of vessel is inherently quite vulnerable to virtually any modern weapons system, survivability is best enhanced by decreasing the susceptibility. Although combat experience in previous wars has proved that smoke is invaluable in protecting ships from visually aimed weapons, the ability to produce screening smoke on modern U.S. Navy vessels is very limited. In an effort to evaluate the effectiveness of using screening smoke to protect small craft under modern conditions, e.g., from enemy helicopters, small craft tests were conducted using a variety of smoke sources. Smoke sources tested included the U.S. Army M3A3E3 pulse‐jet smoke generator, pyrotechnic smoke blocks, and a turbine type generator. This paper discusses the results of these tests and draws conclusions regarding the feasibility of using smoke to help protect boats from visually aimed weapons.
CLEER: An AI System Developed to Assist Equipment Arrangements on Warships
Authors: ZHOU H. HARRY , SILVERMAN BARRY G. , SIMKOL JOEL
Published: March 2009

This paper describes a modularized AI system being built to help improve electromagnetic compatibility (EMC) among shipboard topside equipment and their associated systems. CLEER is intended to act as an easy to use integrator of existing expert knowledge and pre‐existing data bases and large scale analytical models. Due to these interfaces; to the need for portability of the software; and to artificial intelligence related design requirements (such as the need for spatial reasoning, expert data base management, model base management, track‐based reasoning, and analogical (similar ship) reasoning) it was realized that traditional expert system shells would be inappropriate, although relatively off‐the‐shelf AI technology could be incorporated. In the same vein, the rapid prototyping approach to expert system design and knowledge engineering was not pursued in favor of a rigorous systems engineering methodology. The critical design decisions affecting CLEER's development are summarized in this paper along with lessons learned to date all in terms of “how,” “why,” and “when” specific features are being developed.
Comparative Assessment of U.S. and Foreign Buoy Tenders
Authors: KEHOE JAMES W. , BROWER KENNETH S. , TUTTLE JOHN G.
Published: March 2009

The paper reports the results of a comparative study of seven modern foreign buoy tenders and two older U.S. buoy tenders that was conducted by the U.S. Coast Guard as part of its current project to replace its older medium size (WLM) and large seagoing (WLB) buoy tenders. As part of the assessment, the authors visited the foreign ships to observe them conducting buoy tending operations, to review engineering design data, and to discuss buoy tender design, technology and operations with the cognizant operating authorities. The buoy tenders included the Canadian 4,800‐ton Type 1100 and 2,800‐ton Type 1050, the British 2,900‐ton Mermaid, the Swedish 1,200‐ton Baltica, the Norwegian 1,100‐ton Skomvaer, the Dutch 700‐ton Rotterdam, and the Italian 600‐ton MIF. The U.S. buoy tenders included the Coast Guard 470‐ton, 157‐foot WLM and 1,000‐ton, 180‐foot WLB (SLEP), which were also visited and included in the study for comparative purposes.

The study's objectives were to identify design practices and technology incorporated in the foreign buoy tenders that the U.S. Coast Guard could adapt in designing future buoy tenders and to determine if any foreign buoy tender design meets the Coast Guard requirements for WLB and WLM replacements.

The design characteristics, technology, and unique features of each buoy tender are described and compared. Lessons learned from the study which could be beneficial in the design of future Coast Guard buoy tenders are identified. Finally, the capabilities of the foreign buoy tenders are assessed in relation to current Coast Guard requirements.
The Quantification of Interoperability
Authors: MENSH DENNIS R. , KITE ROBERT S. , DARBY PAUL H.
Published: March 2009

JCS Pub 1 defines interoperability as “The ability of systems, units or forces to provide services to and accept services from other systems, units or forces and to use the services so exchanged to enable them to operate effectively together.” With JCS Pub 1 as a foundation, interoperability of systems, units or forces can be factored into a set of components that can quantify interoperability. These components are: media, languages, standards, requirements, environment, procedures, and human factors. The concept described in this paper uses these components as an analysis tool to enable specific detailed analyses of the interoperability of BFC3 systems, units, or forces for the purpose of uncovering and resolving interoperability issues and problems in the U.S. Navy, Joint, and Allied arenas. Also, as a management tool, the components can help determine potential interoperability characteristics of future U.S. Navy BFC3 systems for compliance with battle force systems architectures.

The approach selected for the quantification of interoperability was the development of a set of measures of performance (MOPs) and measures of effectiveness (MOEs). The MOPs/MOEs were integrated with a candidate set of components, which were used to partition the totality of interoperability into measurable entities.

The methodology described employs basic truth table theory in conjunction with logic equations to evaluate the interoperability components in terms of MOPs that were aggregated to MOEs. It is believed that this concept, although elementary and based on fundamental principles, represents an operationally significant approach rather than a theoretical approach to the quantification of interoperability.

The vehicle used as a means to measure the MOPs and MOEs was the Research Evaluation and Systems Analysis (RESA) computer modeling and simulation capability at the Naval Ocean Systems Center (NOSC), San Diego, Calif. Data for the measurements were collected during a Tactical Information Management Exercise (TIMEX) in August 1987. The data consisted of printouts from computer data bases plus data collected by human observers.

It is hypothesized that subsequent repeated exercises will yield a sufficient number of meaningful measurements. These measurements may lead to the development of frequency distribution curves representing the degree to which each interoperability component was achieved. These curves will in turn be used to derive some probability function [P(I)] that empirically describes the degree of interoperability that could be achieved. The benefit of this approach is that complex interoperability problems can be partitioned into a smaller subset of problems that can be more easily managed and/or resolved. Also it provides the potential means to obtain a probabilistic measure of how often, operationally, a desired level of interoperability could be achieved.
An Application of Total Quality Management at an Intermediate Maintenance Activity
Authors: NEEDHAM CDR. WILLIAM D.
Published: March 2009

The current budget and resource constrained fiscal environment mandates positive measures to improve productivity. The management practices of the last four decades are no longer valid. The Department of Defense (DoD) has issued broad guidelines for a restructuring of all subordinate commands and agencies called total quality management (TQM). The precepts of TQM are not new. They are the very same precepts prescribed by numerous management and quality control experts for the past decade to reverse the downward trends in the productivity of American industry. The program elements of TQM are broad in scope, as they must be for an organization as large and diverse as DoD. The implementation of TQM at the local command level is left to the local commander. This is no mean task, as TQM is a radical departure from traditional management practice in every aspect of an organization. This article reports on the implementation of TQM at a local command, the USS Hunley, a naval auxiliary support ship whose primary mission is the repair and maintenance of a squadron of nuclear‐powered submarines in Norfolk, Virginia.
Preliminary Design of a Convertible Container Roll‐On/Roll‐Off (CCR) Ship
Authors: MCKENNEY TIMOTHY , INGLES EDWARD , KOWALICK DAVID
Published: March 2009

This paper develops an innovative yet practical solution to the present sealift deficiency in unit equipment capacity. A U.S. built and operated active merchant fleet that can be quickly converted to sealift use is the ideal solution to the sealift deficiencies facing the United States. The concept behind the convertible container roll‐on roll‐off (CCR) ship is simple: Design a ship which meets the needs of the commercial fleet as a fully cellular container ship that can be quickly converted into a roll‐on roll‐off (Ro‐Ro) ship to carry unit equipment. The result is a sealift solution that supports the Army at the theater of operations in less than two weeks at a fraction of the lifecycle cost of a dedicated sealift fleet The CCR concept also provides commercially viable containerships for U.S. flag operators at reduced capital cost and fosters an enhanced U.S. shipbuilding base.
On the Development of Seakeeping Criteria
Authors: GRAHAM ROSS , BAITIS A. ERICH , MEYERS WILLIAM G.
Published: March 2009

In order to determine the effects of ship motion on operational performance, quantitative seakeeping criteria are required. In this paper, we propose that seakeeping criteria should be presented in terms of limits on the incidence of whatever degrading event is determined to curtail the operation of interest, rather than limits on the underlying ship motions. For personnel operating on deck, the degrading events of most interest are the onset of loss‐of‐balance incidents, termed motion‐induced interruptions, due to tipping or sliding. For unsecured helicopter operations, the most important degrading event is the helicopter sliding.

We present a frequency‐domain method of predicting the incidence of personnel or of equipment sliding which includes the forces due to roll, pitch, longitudinal, lateral and vertical accelerations, and also the effects of non‐zero mean heel angles. We then present a method for predicting the incidence of personnel experiencing motion‐induced interruptions due to loss of balance which includes the above forces and also the effects of steady and unsteady winds. In order to work in the frequency‐domain, all of the forces are linearized and we consider only the linear turbulent wind force, and neglect the nonlinear term.

Two main applications of the method are anticipated. The first application is to design. The seakeeping criteria presented here are closely linked with the physical phenomena that are causing performance to be degraded, and are equally applicable to all ship types. The second application is to active operational guidance systems which provide real time information to the operators of naval vessels on the effects of changes in speed or heading on operations. Developing criteria in terms of the degrading events of most interest to the operator should result in an immediate improvement in the quality of the guidance.
The Testing of a New Ship Class—The USS Arleigh Burke
Authors: FOLEY J. KEVIN
Published: March 2009

The USS Arleigh Burke, being the first of a new class of ships, presented many challenges to both the production and test crews that worked on her. This paper discusses the unique conditions that the Aegis Test Team faced in the completion of the test program. A historical perspective of why the Arleigh Burke class was chosen as the battle force combatant, differences between the Ticonderoga class design and how they forced a re‐thinking of the classic test plan, time proven versus innovative testing techniques, and unique problems encountered and their solutions are discussed. The paper highlights the fundamental differences between the planned ship design, construction and test plan caused by the fluctuations of the dynamic shipyard environment.
Best Value Contractìng
Authors: ZACKS MICHAEL R. , DEMAS THOMAS A.
Published: March 2009

The best value concept is based on making decisions on an offeror's technical competence, proven past performance, management capability, life‐cycle costs, and product quality. The evaluation of these factors should be structured to ensure consideration is given to determine the overall benefit associated with the offered price. This paper advocates using the best value concept as the method for developing and rating proposal evaluation factors for the procurement of new ships. It discusses methods for establishing evaluation factors, developing standards to evaluate, associated documentation, and weighting and scoring the factors. The information for this paper was obtained from personal interviews, hands‐on experience developing and evaluating best value proposals, and documentation research. If properly executed, the best value concept will enable the Navy to improve ships while reducing operating costs.
Laser Cladding Refurbishment of Catapult Trough Covers
Authors: DENNEY PAUL E. , LOWRY ROBERT W.
Published: March 2009

A laser cladding process has been developed and is now in production for the cladding of new aircraft carrier catapult components. The Service Life Extension Program (SLEP) Office of Naval Sea Systems Command (NavSea) suggested that this process be utilized for the refurbishment of catapult tracks. Early experiments were conducted by the Naval Surface Warfare Center in conjunction with the IIT Research Institute. The program was then transitioned to the Applied Research Laboratory, Pennsylvania State University (ARL Penn State) under the sponsorship of the Naval Air Systems Command (NavAir)/Navy MANTECH program, thus providing an effective solution to a NavSea/NavAir interface hardware problem.

ARL Penn State was responsible for the production and evaluation of six laser clad aircraft carrier catapult tracks as part of a program sponsored and funded by NavAir Code 5512.

Six catapult tracks that had been taken out of service because of excessive wear were laser clad at the Westinghouse Electric Research and Development Center (Now “Science and Technology Center”) for ARL Penn State. The composition of the clads was Inconel 625, Stellite 6/Stainless Steel 304, and Ferrelium 255. The tracks were machined and installed on the USS Constellation. Wear measurements were taken for the laser clad tracks and standard tracks ahead and behind the clad tracks.

After 7,161 launches, the tracks were removed and returned to ARL Penn State for evaluation. The evaluation indicated that impact damage was observed on one track. No additional defects related to operations were found. Wear data indicated that the wear rate for it was 25%‐50% less than non‐clad. The results indicated that rejectable tracks (and one piece trough covers) could be successfully laser clad for extended operating life at acceptable costs.

With emphasis for the future on a more affordable Navy, the need for viable refurbishment processes will be necessary for extending life and performance for the 21st century. Laser cladding and laser processing offer a real potential for affordable extended life systems.
Advanced Waterjet Systems
Authors: Stricker John G. , Becnel Alan J. , Purnell John G.
Published: March 2009

ABSTRACT  Several advanced waterjet systems have recently been developed for Marine Corps applications. In developing propulsor technology for the Advanced Amphibious Assault Vehicle (AAAV) program, a compact system was designed to meet specified thrust, speed, and envelope requirements of a baseline planing vehicle configuration. A second propulsor design was completed to meet requirements of a revised vehicle. Concurrently, design studies of an outboard motor‐fitted pumpjet concept were undertaken for the MARS system development program, and modified components were designed and manufactured as part of an effort to improve performance of the commercially‐developed pumpjet units. Optimization procedures, detailed designs, and available performance data are discussed for each of these applications. In addition, new waterjet design concepts aiming toward improved thrust and efficiency for planing craft applications are discussed.
Identification and Resolution of Propulsion Plant Machinery Problems
Authors: BROWN RUSSELL W.
Published: March 2009

This paper presents propulsion plant problems caused by both manufacturing and design faults. These problems have been selected from the many that developed because they required challenging engineering in both their definition and resolution.

The problems described cover main propulsion shafting and bearings; propulsion turbines; ship service turbine generators; set turbines; main reduction gears; main condensers; steam distilling plants, valves and high pressure air systems. The topic discussed in that system is the identification of auto ignition in the first 4500 psig high pressure air system and its corrective action.

Each problem is described both in writing and with a sketch. In all, fourteen problems are discussed.
A Gas Turbine Condition‐Monitoring System
Authors: Hardin James R. , Howell Ivan L. , Mirilovich J. Richard , Hartranft John J. , Schreder David L.
Published: March 2009

ABSTRACT  This paper describes a program to develop a condition‐based maintenance system for the Navy's LM2500 main propulsion engine. The effort began with research and evaluation of the engine failure modes and effects. Using a well‐defined reliability‐centered maintenance (RCM) methodology, we identified the most appropriate maintenance approach for each subsystem and failure mode.

Expert rules were developed for the equipment and failures that will benefit from a condition‐based maintenance approach. The expert system is based on the Navy's Integrated Condition Assessment System (ICAS). The proposed system applies new and innovative expert system logic and instrumentation using an upgraded capable ICAS to identify degradation in the marine gas turbine (MGT) engine and its subsystems.
Influence of Human Engineering on Manning Levels and Human Performance on Ships
Authors: Anderson D.E. , Oberman F.R. , Malone T.B. , Baker C.C.
Published: March 2009

ABSTRACT  The objectives of Human Engineering (HE) are generally viewed as increasing human performance, reducing human error, enhancing personnel and equipment safety, and reducing training and related personnel costs. There are other benefits that are thoroughly consistent with the direction of the Navy of the future, chief among these is reduction of required numbers of personnel to operate and maintain Navy ships. The Naval Research Advisory Committee (NRAC) report on Man‐Machine Technology in the Navy estimated that one of the benefits from increased application of man‐machine technology to Navy ship design is personnel reduction as well as improving system availability, effectiveness, and safety. The objective of this paper is to discuss aspects of the human engineering design of ships and systems that affect manning requirements, and impact human performance and safety. The paper will also discuss how the application of human engineering leads to improved performance, and crew safety, and reduced workload, all of which influence manning levels. Finally, the paper presents a discussion of tools and case studies of good human engineering design practices which reduce manning.
A simplified dental examination for detection of cavitated carious lesions in school setting
Authors: Haleem Abdul , Khan Ayyaz Ali , Siddiqui Muhammad Irfanullah
Published: March 2009

Abstract – 

Objective:  The objective of the study was to compare a simplified dental examination involving the use of a wooden spatula and a toothpick (TS examination) with the conventional dental mirror and probe examination (MP examination) for detection of cavitated carious lesions in schoolchildren.

Methods:  The study involved three groups of caries‐positive schoolchildren aged 4–5, 9–10 and 13–14 years with 179, 188 and 202 children, respectively. All children were examined by a trained and calibrated examiner using a wooden spatula and a toothpick under natural light. After a week the same examiner examined the three groups of children by a dental mirror and a Community Periodontal Index (CPI) probe. ‘Teeth’ were considered as units of measurement for data analysis. The WHO recommended criteria (2) for decayed, missing and filled teeth were followed for recording dental caries on a specially designed recording form. The sensitivity, specificity, positive predictive value, negative predictive value, false‐positive rate and false‐negative rate of TS examination were calculated using MP examination as the standard method.

Results:  The specificity of TS examination was well above 95% in three types of dentition. Although the sensitivity of this type of examination in deciduous and mixed dentitions was almost comparable with that of MP examination, it was the lowest for permanent dentition but still within the acceptable limits.

Conclusion:  The TS examination can provide an alternative to traditional MP examination to undertake regular check‐ups of schoolchildren for dental caries.
Burning mouth syndrome: the role of contact hypersensitivity
Authors: Marino R , Capaccio P , Pignataro L , Spadari F
Published: March 2009

Background:  Burning mouth syndrome is a burning sensation or stinging disorder affecting the oral mucosa in the absence of any clinical signs or mucosal lesions. Some studies have suggested that burning mouth syndrome could be caused by the metals used in dental prostheses, as well as by acrylate monomers, additives and flavouring agents, although others have not found any aetiologic role for hypersensitivity to dental materials.

Objective:  To evaluate the extent and severity of adverse reactions to dental materials in a group of patients with burning mouth syndrome, and investigate the possible role of contact allergy in its pathogenesis.

Materials and methods:  We prospectively studied 124 consecutive patients with burning mouth syndrome (108 males; mean age 57 years, range 41–83), all of whom underwent allergen patch testing between 2004 and 2007.

Results:  Sixteen patients (13%) showed positive patch test reactions and were classified as having burning mouth syndrome type 3 or secondary burning mouth syndrome (Lamey’s and Scala’s classifications).

Conclusion:  Although we did not find any significant association between the patients and positive patch test reactions, it would be advisable to include hypersensitivity to dental components when evaluating patients experiencing intermittent oral burning without any clinical signs.
ASH Position Paper: Hypertension in Pregnancy
Authors: Lindheimer Marshall D. , Taler Sandra J. , Cunningham F. Gary
Published: March 2009

The American Society of Hypertension is publishing a series of Position Papers in their official journals throughout the 2008 ‐ 2009 years. The following Position Paper originally appeared: JASH. 2008;2(6):484–494.

Hypertension complicates 5% to 7% of all pregnancies. A subset of preeclampsia, characterized by new‐onset hypertension, proteinuria, and multisystem involvement, is responsible for substantial maternal and fetal morbidity and is a marker for future cardiac and metabolic disease. This American Society of Hypertension, Inc (ASH) position paper summarizes the clinical spectrum of hypertension in pregnancy, focusing on preeclampsia. Recent research breakthroughs relating to etiology are briefly reviewed. Topics include classification of the different forms of hypertension during pregnancy, status of the tests available to predict preeclampsia, and strategies to prevent preeclampsia and to manage this serious disease. The use of antihypertensive drugs in pregnancy, and the prevention and treatment of the convulsive phase of preeclampsia, eclampsia, with intravenous magnesium sulfate is also highlighted. Of special note, this guideline article, specifically requested, reviewed, and accepted by ASH, includes solicited review advice from the American College of Obstetricians and Gynecologists.
Estimating Nitrogen Loading to Ground Water and Assessing Vulnerability to Nitrate Contamination in a Large Karstic Springs Basin, Florida1
Authors: Katz Brian G. , Sepulveda A. Alejandro , Verdi Richard J.
Published: March 2009

Abstract: A nitrogen (N) mass‐balance budget was developed to assess the sources of N affecting increasing ground‐water nitrate concentrations in the 960‐km2karstic Ichetucknee Springs basin. This budget included direct measurements of N species in rainfall, ground water, and spring waters, along with estimates of N loading from fertilizers, septic tanks, animal wastes, and the land application of treated municipal wastewater and residual solids. Based on a range of N leaching estimates, N loads to ground water ranged from 262,000 to 1.3 million kg/year; and were similar to N export from the basin in spring waters (266,000 kg/year) when 80‐90% N losses were assumed. Fertilizers applied to cropland, lawns, and pine stands contributed about 51% of the estimated total annual N load to ground water in the basin. Other sources contributed the following percentages of total N load to ground water: animal wastes, 27%; septic tanks, 12%; atmospheric deposition, 8%; and the land application of treated wastewater and biosolids, 2%. Due to below normal rainfall (97.3 cm) during the 12‐month rainfall collection period, N inputs from rainfall likely were about 30% lower than estimates for normal annual rainfall (136 cm). Low N‐isotope values for six spring waters (δ15N‐NO3 = 3.3 to 6.3‰) and elevated potassium concentrations in ground water and spring waters were consistent with the large N contribution from fertilizers. Given ground‐water residence times on the order of decades for spring waters, possible sinks for excess N inputs to the basin include N storage in the unsaturated zone and parts of the aquifer with relatively sluggish ground‐water movement and denitrification. A geographical‐based model of spatial loading from fertilizers indicated that areas most vulnerable to nitrate contamination were located in closed depressions containing sinkholes and other dissolution features in the southern half of the basin.
How Far Is Georgia?: New Jersey's Teachers of Italian Evaluate Their Preparation
Authors: Antenos‐Conforti Enza
Published: March 2009

Abstract: This study examined the professional preparation of Italian teachers in New Jersey through the lens of Cooper's (2004) investigation in Georgia, which determined how K‐12 foreign language teachers perceived and evaluated the effectiveness of their professional preparation. Unfortunately, however, that report did not have an Italian representation. In view of the undeniable presence of Italian in New Jersey, this replicate study attempts to ascertain the degree to which the perceptions and evaluations of teachers of Italian in New Jersey were similar to those of foreign language teachers in Georgia and to determine if there were specific findings that could be considered distinctive to Italian programs. The present study had 37 participants, all current teachers of Italian, 75% of whom had received certification in New Jersey. The survey they completed was based on 40 of the 42 questions asked to foreign language teachers in Georgia, and included several additional items such as level taught, second field certification, and place of certification. The quantitative findings for Italian teachers in New Jersey and foreign language teachers in Georgia were consistent in terms of participants' evaluation of teacher education programs and their teaching competencies. Additionally, in their responses to the open‐ended questions, participants echoed many of the observations about foreign language teacher education programs outlined by Cooper (2004). However, Italian teachers in New Jersey also expressed concern about the following specific aspects of teacher preparation: (1) observe Italian classes, (2) work with mentors who help “fill‐the‐gaps” left by the programs, (3) participate in intensive language programs and methodology workshops (offered by some out‐of‐state programs), (4) participate in and network at conferences and in‐service workshops, and (5) become members of relevant associations.
Giant-Stroke, Superelastic Carbon Nanotube Aerogel Muscles
Authors: Aliev Ali E. , Oh Jiyoung , Kozlov Mikhail E. , Kuznetsov Alexander A. , Fang Shaoli , Fonseca Alexandre F. , Ovalle Raquel , Lima Márcio D. , Haque Mohammad H. , Gartstein Yuri N. , Zhang Mei , Zakhidov Anvar A. , Baughman Ray H.
Published: March 2009

Improved electrically powered artificial muscles are needed for generating force, moving objects, and accomplishing work. Carbon nanotube aerogel sheets are the sole component of new artificial muscles that provide giant elongations and elongation rates of 220% and (3.7 × 10
4
)% per second, respectively, at operating temperatures from 80 to 1900 kelvin. These solid-state–fabricated sheets are enthalpic rubbers having gaslike density and specific strength in one direction higher than those of steel plate. Actuation decreases nanotube aerogel density and can be permanently frozen for such device applications as transparent electrodes. Poisson's ratios reach 15, a factor of 30 higher than for conventional rubbers. These giant Poisson's ratios explain the observed opposite sign of width and length actuation and result in rare properties: negative linear compressibility and stretch densification.
Comparison of Planktonic and Biofilm Cultures of
Pseudomonas fluorescens
DSM 8341 Cells Grown on Fluoroacetate
Authors: Heffernan Barry , Murphy Cormac D. , Casey Eoin
Published: March 2009

Comparisons between the physiological properties of
Pseudomonas fluorescens
biofilm cells grown in a tubular biofilm reactor and planktonic cells grown in a chemostat were performed. Fluoroacetate was the sole carbon source for all experiments. The performance of cells was assessed using cell cycle kinetics and by determining specific fluoroacetate utilization rates. Cell cycle kinetics were studied by flow cytometry in conjunction with the fluorescent stain propidium iodide. Determination of the DNA content of planktonic and biofilm cultures showed little difference between the two modes of growth. Cultures with comparable specific glycolate utilization rates had similar percentages of cells in the B phase of the cell cycle, indicating similar growth rates. Specific fluoroacetate utilization rates showed the performance of planktonic cells to be superior to that of biofilm cells, with more fluoroacetate utilized per cell at similar specific fluoroacetate loading rates. A consequence of this decreased biofilm performance was the accumulation of glycolate in the effluent of biofilm cultures. This accumulation of glycolate was not observed in the effluent of planktonic cultures. Spatial stratification of oxygen within the biofilm was identified as a possible explanation for the overflow metabolism of glycolate and the decreased performance of the biofilm cells.
Osmoadaptation among
Vibrio
Species and Unique Genomic Features and Physiological Responses of
Vibrio parahaemolyticus
Authors: Naughton Lynn M. , Blumerman Seth L. , Carlberg Megan , Boyd E. Fidelma
Published: March 2009

Vibrio parahaemolyticus
is a moderately halophilic bacterium found in estuarine and marine coastal ecosystems worldwide. Although the ability of
V. parahaemolyticus
to grow and proliferate in fluctuating saline environments is well known, the underlying molecular mechanisms of osmoadaptation are unknown. We performed an in silico analysis of
V. parahaemolyticus
strain RIMD2210633 for genes homologous to osmotic stress response genes in other bacteria. We uncovered two putative compatible solute synthesis systems (encoded by
ectABC
and
betABI
) and six putative compatible solute transporters (encoded by four
bcct
loci and two
proVWX
loci). An ectoine synthesis system clustered with a betaine/carnitine/choline transporter and a ProU transporter (encoded by homologues of
proVWX
from
Escherichia coli
), and a betaine synthesis system clustered with a ProU transporter (encoded by homologues of
proVXW
from
Pseudomonas syringae
). This is at least double the number present in
V. cholerae
,
V. fischeri
, or
V. vulnificus
. Six additional
Vibrio
species contain both
ectABC
and
betABI
, i.e.,
V. alginolyticus
12G01,
V. angustum
,
V. harveyi
BAA-1116,
V. splendidus
LGP32,
Vibrio
sp. strain MED222, and
Vibrio
sp. strain Ex25.
V. harveyi
HY01 and
V. splendidus
12B01 only encoded the betaine system. In addition,
V. alginolyticus
had a compendium of systems identical to that found in
V. parahaemolyticus
. Comparative physiological analysis of RIMD2210633 with
V. vulnificus
YJ016,
V. cholerae
N16961, and
V. fischeri
ES114 grown at different salinities and temperatures demonstrated that
V. parahaemolyticus
had a growth advantage under all of the conditions examined. We demonstrate, by one-dimensional nuclear magnetic resonance analysis, that
V. parahaemolyticus
is capable of de novo synthesis of ectoine at high salinity whereas a Δ
ectB
knockout strain is not. We constructed a single-knockout mutation in
proU1
, but no growth defect was noted, indicating transporter system redundancy. We complemented
E. coli
MKH13, a compatible solute transporter-negative strain, with
bcct2
and demonstrated uptake of betaine at high salt concentrations.
Global Distribution and Evolution of a Toxinogenic
Burkholderia-Rhizopus
Symbiosis
Authors: Lackner Gerald , Möbius Nadine , Scherlach Kirstin , Partida-Martinez Laila P. , Winkler Robert , Schmitt Imke , Hertweck Christian
Published: March 2009

Toxinogenic endobacteria were isolated from a collection of
Rhizopus
spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the
Burkholderia rhizoxinica
complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.
Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of
Shewanella oneidensis
MR-1
Authors: Lower Brian H. , Yongsunthon Ruchirej , Shi Liang , Wildling Linda , Gruber Hermann J. , Wigginton Nicholas S. , Reardon Catherine L. , Pinchuk Grigoriy E. , Droubay Timothy C. , Boily Jean-François , Lower Steven K.
Published: March 2009

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living
Shewanella oneidensis
MR-1 cells when Fe(III), including solid-phase hematite (Fe
2
O
3
), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.
Infection ofMelanoplus sanguinipesGrasshoppers following Ingestion of Rangeland Plant Species Harboring Vesicular Stomatitis Virus
Authors: Drolet Barbara S. , Stuart Melissa A. , Derner Justin D.
Published: March 2009

Knowledge of the many mechanisms of vesicular stomatitis virus (VSV) transmission is critical for understanding of the epidemiology of sporadic disease outbreaks in the western United States. Migratory grasshoppers [Melanoplus sanguinipes(Fabricius)] have been implicated as reservoirs and mechanical vectors of VSV. The grasshopper-cattle-grasshopper transmission cycle is based on the assumptions that (i) virus shed from clinically infected animals would contaminate pasture plants and remain infectious on plant surfaces and (ii) grasshoppers would become infected by eating the virus-contaminated plants. Our objectives were to determine the stability of VSV on common plant species of U.S. Northern Plains rangelands and to assess the potential of these plant species as a source of virus for grasshoppers. Fourteen plant species were exposed to VSV and assayed for infectious virus over time (0 to 24 h). The frequency of viable virus recovery at 24 h postexposure was as high as 73%. The two most common plant species in Northern Plains rangelands (western wheatgrass [Pascopyrum smithii] and needle and thread [Hesperostipa comata]) were fed to groups of grasshoppers. At 3 weeks postfeeding, the grasshopper infection rate was 44 to 50%. Exposure of VSV to a commonly used grasshopper pesticide resulted in complete viral inactivation. This is the first report demonstrating the stability of VSV on rangeland plant surfaces, and it suggests that a significant window of opportunity exists for grasshoppers to ingest VSV from contaminated plants. The use of grasshopper pesticides on pastures would decrease the incidence of a virus-amplifying mechanical vector and might also decontaminate pastures, thereby decreasing the inter- and intraherd spread of VSV.
Molecular Epidemiology and Characterization of
Campylobacter
spp. Isolated from Wild Bird Populations in Northern England
Authors: Hughes Laura A. , Bennett Malcolm , Coffey Peter , Elliott John , Jones Trevor R. , Jones Richard C. , Lahuerta-Marin Angela , Leatherbarrow A. Howard , McNiffe Kenny , Norman David , Williams Nicola J. , Chantrey Julian
Published: March 2009

Campylobacter
infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of
Campylobacter
spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of
Campylobacter
spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales.
Campylobacter jejuni
,
Campylobacter lari
, and
Campylobacter coli
were detected by PCR, and the prevalences of different
Campylobacter
spp. in different avian families ranged from 0% to 33%. Characterization of 36
C. jejuni
isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of
C. jejuni
. However, the apparent absence of unique wild bird strains of
C. jejuni
in livestock suggests that the direction of infection is predominantly from livestock to wild birds.
C. lari
was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen
C. lari
isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of
C. lari
in Eurasian oystercatchers (
Haematopus ostralegus
) and clonal populations in magpies (
Pica pica
).
Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Responses of Bifidobacteria in Breast- and Formula-Fed Infants
Authors: Klaassens Eline S. , Boesten Rolf J. , Haarman Monique , Knol Jan , Schuren Frank H. , Vaughan Elaine E. , de Vos Willem M.
Published: March 2009

Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.
High Rate of N
2
Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum
Authors: Hara Shintaro , Hashidoko Yasuyuki , Desyatkin Roman V. , Hatano Ryusuke , Tahara Satoshi
Published: March 2009

For evaluating N
2
fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N
2
-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N
2
fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N
2
-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N
2
fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as
Pseudomonas fluorescens
and
Burkholderia xenovorans
, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including
Luteibacter
sp.,
Janthinobacterium
sp.,
Paenibacillus
sp., and
Arthrobacter
sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast,
Pseudomonas
spp. and
Burkholderia
spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.
De Novo Biosynthesis of Vanillin in Fission Yeast (
Schizosaccharomyces pombe
) and Baker's Yeast (
Saccharomyces cerevisiae
)
Authors: Hansen Esben H. , Møller Birger Lindberg , Kock Gertrud R. , Bünner Camilla M. , Kristensen Charlotte , Jensen Ole R. , Okkels Finn T. , Olsen Carl E. , Motawia Mohammed S. , Hansen Jørgen
Published: March 2009

Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (
Vanilla planifolia
), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in
Schizosaccharomyces pombe
, also known as fission yeast or African beer yeast, as well as in baker's yeast,
Saccharomyces cerevisiae
. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold
Podospora pauciseta
, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the
Nocardia
genus, and an
O
-methyltransferase from
Homo sapiens
. In
S. cerevisiae
, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a
Corynebacterium glutamicum
phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase
ADH6
. In
S. pombe
, the biosynthesis was further improved by introduction of an
Arabidopsis thaliana
family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-
d
-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose.
S. pombe
yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.
Fungal Functional Genomics: Tunable Knockout-Knock-in Expression and Tagging Strategies
Authors: Larrondo Luis F. , Colot Hildur V. , Baker Christopher L. , Loros Jennifer J. , Dunlap Jay C.
Published: March 2009

Strategies for promoting high-efficiency homologous gene replacement have been developed and adopted for many filamentous fungal species. The next generation of analysis requires the ability to manipulate gene expression and to tag genes expressed from their endogenous loci. Here we present a suite of molecular tools that provide versatile solutions for fungal high-throughput functional genomics studies based on locus-specific modification of any target gene. Additionally, case studies illustrate caveats to presumed overexpression constructs. A tunable expression system and different tagging strategies can provide valuable phenotypic information for uncharacterized genes and facilitate the analysis of essential loci, an emerging problem in systematic deletion studies of haploid organisms.