bluuebunny/crossref_metadata_2025 huggingface dataset:
Mobility of Protozoa through Narrow Channels
Authors: Wang Wei , Shor Leslie M. , LeBoeuf Eugene J. , Wikswo John P. , Kosson David S.
Published: August 2005

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 μm s
−1
(or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 μm s
−1
. Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.
Critical Factors Influencing the Occurrence of
Vibrio cholerae
in the Environment of Bangladesh
Authors: Huq Anwar , Sack R. Bradley , Nizam Azhar , Longini Ira M. , Nair G. Balakrish , Ali Afsar , Morris J. Glenn , Khan M. N. Huda , Siddique A. Kasem , Yunus Mohammed , Albert M. John , Sack David A. , Colwell Rita R.
Published: August 2005

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype
Vibrio cholerae
O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic
Vibrio cholerae
serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a “reemerging” disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis.
187:
96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably
V. cholerae
). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of
V. cholerae
is proving useful in developing environmental models for the prediction of cholera epidemics.
Molecular Characterization of Korean
Bacillus anthracis
Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis
Authors: Ryu Chunsun , Lee Kyunghee , Hawng Han-Jun , Yoo Cheon-Kwon , Seong Won-Keun , Oh Hee-Bok
Published: August 2005

We analyzed the genetic relationships and molecular characteristics of 34
Bacillus anthracis
isolates from soil and clinical samples in various regions of Korea and 17 related
Bacillus
species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of
B. anthracis
isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate
B. anthracis
strains from other
Bacillus
species. The
B. cereus
group was separated from other
Bacillus
species at a level of similarity of 0.68. Among them, some
B. cereus
strains showed genetic interspersion with
B. thuringiensis
strains. The evolutionary pattern of nucleotide differences among
B. anthracis
strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic
B. anthracis
isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.
Expression of 17 Genes in
Clostridium thermocellum
ATCC 27405 during Fermentation of Cellulose or Cellobiose in Continuous Culture
Authors: Stevenson David M. , Weimer Paul J.
Published: August 2005

Clostridium thermocellum
is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in
C. thermocellum
ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a ∼10-fold range of dilution rates (0.013 to 0.16 h
−1
). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (
recA
and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type:
sdbA
(cellulosomal scaffoldin-dockerin binding protein),
cdp
(cellodextrin phosphorylase),
cbp
(cellobiose phosphorylase),
hydA
(hydrogenase),
ldh
(lactate dehydrogenase),
ack
(acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h
−1
:
cipA
(cellulosomal scaffolding protein),
celS
(exoglucanase),
manA
(mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.
Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis
Authors: Ben-Amor Kaouther , Heilig Hans , Smidt Hauke , Vaughan Elaine E. , Abee Tjakko , de Vos Willem M.
Published: August 2005

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to
Clostridium coccoides
,
Clostridium leptum
, and
Bacteroides
. We found that some of the butyrate-producing related bacteria, such as
Eubacterium rectale
and
Eubacterium hallii
, were obviously viable at the time of sampling. However, amplicons affiliated with
Bacteroides
and
Ruminococcus obeum
- and
Eubacterium biforme
-like bacteria, as well as
Butyrivibrio crossotus
, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the
Clostridium leptum
cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to
Bifidobacterium adolescentis
,
Bifidobacterium longum
,
Bifidobacterium infantis
,
Bifidobacterium pseudocatenulatum
, and
Bifidobacterium bifidum
. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations;
B. longum
and
B. infantis
were retrieved from all sorted fractions, while
B. adolescentis
was recovered mostly from the sorted dead fraction.
Study of Spanish Grape Mycobiota and Ochratoxin A Production by Isolates of
Aspergillus tubingensis
and Other Members of
Aspergillus
Section
Nigri
Authors: Medina Angel , Mateo Rufino , López-Ocaña Laura , Valle-Algarra Francisco Manuel , Jiménez Misericordia
Published: August 2005

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were
Alternaria
,
Cladosporium
, and
Aspergillus
. The isolation frequency of
Aspergillus
spp. section
Nigri
in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of
Aspergillus carbonarius
and 14.3% of
Aspergillus tubingensis
isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No
Aspergillus niger
isolate had the ability to produce this toxin under the conditions assayed. Identification of the
A. niger
aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of
A. niger
CECT 2807 and
A. tubingensis
CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of
A. tubingensis
matched 99 to 100% with the nucleotide sequence of strain
A. tubingensis
CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of
A. tubingensis
to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.
Proteins Encoded bySphingomonas elodeaATCC 31461rmlAandugpGGenes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities
Authors: Silva Elisabete , Marques Ana Rita , Fialho Arsénio Mendes , Granja Ana Teresa , Sá-Correia Isabel
Published: August 2005

The commercial gelling agent gellan is a heteropolysaccharide produced bySphingomonas elodeaATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of therml4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gelcluster). Based on sequence homology, the putativermloperon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared fromEscherichia coliIPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Kmof 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Kmof 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. TheS. elodeaATCC 31461 UgpG protein, encoded by theugpGgene which maps outside thegelcluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing ofugpGgene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).
Engineering of Phenylacetaldehyde Reductase for Efficient Substrate Conversion in Concentrated 2-Propanol
Authors: Makino Yoshihide , Inoue Kousuke , Dairi Tohru , Itoh Nobuya
Published: August 2005

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.
Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates
Authors: Green Stefan J. , Minz Dror
Published: August 2005

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from
Streptomyces
spp., and selective restriction of the
Streptomyces
16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.
Biological Control of Hog Waste Odor through Stimulated Microbial Fe(III) Reduction
Authors: Coates John D. , Cole Kimberly A. , Michaelidou Urania , Patrick Jennifer , McInerney Michael J. , Achenbach Laurie A.
Published: August 2005

Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family
Geobacteraceae
as the dominant species. These organisms were present at concentrations as high as 2 × 10
5
cells g
−1
. Several pure cultures of Fe(III) reducers, including
Geobacter metallireducens
,
Geobacter humireducens
,
Geobacter sulfurreducens
,
Geobacter grbiciae
,
Geothrix fermentans
, and
Geovibrio ferrireducens
, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast,
Shewanella algae
did not degrade any of these compounds. We isolated an Fe(III) reducer,
Geobacter
strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.
Effect of pH on an In Vitro Model of Gastric Microbiota in Enteral Nutrition Patients
Authors: O'May Graeme A. , Reynolds Nigel , Macfarlane George T.
Published: August 2005

Patients with dysphagia due to oropharyngeal disease or cerebrovascular accident require long-term nutritional support via enteral feeding, which often results in microbial overgrowth in the upper gastrointestinal (GI) tract. Gastric acid is the primary innate defense mechanism in the stomach and has been assumed to provide an effective barrier to microbial colonization at pH values of <4. To evaluate the efficacy of gastric acid as a barrier to overgrowth, the microbiota of gastric and duodenal aspirates was assessed by culturing methods. Additionally, a fermentor-based model incorporating enteral nutrition tubing of the gastric microbiota of enteral nutrition (EN) patients was constructed to assess the effect of pH on the microbiota. Results showed that gastric acidity had a relatively small effect on the numbers of microorganisms recovered from intestinal aspirates but did influence microbiota composition. Similarly, at pH 3 in the fermentor, a complex microbiota developed in the planktonic phase and in biofilms. The effect of pH on microbiota composition was similar in aspirates and in the fermentors. Candidas and lactobacilli were aciduric, while recoveries of
Escherichia coli
and
Klebsiella pneumoniae
decreased as pH was reduced, although both were still present in significant numbers at pH 3. Only
Staphylococcus aureus
and
Bifidobacterium adolescentis
persisted at higher pH values both in vitro and in vivo. Lactate and acetate were the main organic acids detected in both aspirates and fermentors. These data show that the simulator used in this investigation was capable of modeling the effects of environmental influences on the upper GI microbiota of EN patients and that gastric pH of <4 is not sufficient to prevent microbial overgrowth in these individuals.
Composition and Structure of Microbial Communities from Stromatolites of Hamelin Pool in Shark Bay, Western Australia
Authors: Papineau Dominic , Walker Jeffrey J. , Mojzsis Stephen J. , Pace Norman R.
Published: August 2005

Stromatolites, organosedimentary structures formed by microbial activity, are found throughout the geological record and are important markers of biological history. More conspicuous in the past, stromatolites occur today in a few shallow marine environments, including Hamelin Pool in Shark Bay, Western Australia. Hamelin Pool stromatolites often have been considered contemporary analogs to ancient stromatolites, yet little is known about the microbial communities that build them. We used DNA-based molecular phylogenetic methods that do not require cultivation to study the microbial diversity of an irregular stromatolite and of the surface and interior of a domal stromatolite. To identify the constituents of the stromatolite communities, small subunit rRNA genes were amplified by PCR from community genomic DNA with universal primers, cloned, sequenced, and compared to known rRNA genes. The communities were highly diverse and novel. The average sequence identity of Hamelin Pool sequences compared to the >200,000 known rRNA sequences was only ∼92%. Clone libraries were ∼90% bacterial and ∼10% archaeal, and eucaryotic rRNA genes were not detected in the libraries. The most abundant sequences were representative of novel proteobacteria (∼28%), planctomycetes (∼17%), and actinobacteria (∼14%). Sequences representative of cyanobacteria, long considered to dominate these communities, comprised <5% of clones. Approximately 10% of the sequences were most closely related to those of α-proteobacterial anoxygenic phototrophs. These results provide a framework for understanding the kinds of organisms that build contemporary stromatolites, their ecology, and their relevance to stromatolites preserved in the geological record.
Aphid-Symbiotic Bacteria Cultured in Insect Cell Lines
Authors: Darby A. C. , Chandler S. M. , Welburn S. C. , Douglas A. E.
Published: August 2005

The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “
Candidatus
Consessoris aphidicola” for T type and “
Candidatus
Adiaceo aphidicola” for U type.
Escherichia coli
K1's Capsule Is a Barrier to Bacteriophage T7
Authors: Scholl Dean , Adhya Sankar , Merril Carl
Published: August 2005

Escherichia coli
strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in
E. coli
physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.
Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA
Authors: Suenaga Hikaru , Liu Rui , Shiramasa Yuko , Kanagawa Takahiro
Published: August 2005

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium,
Sphaerotilus natans
, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.
Correlation of
Campylobacter
Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca
Authors: Atterbury R. J. , Dillon E. , Swift C. , Connerton P. L. , Frost J. A. , Dodd C. E. R. , Rees C. E. D. , Connerton I. F.
Published: August 2005

Campylobacter jejuni
and
Campylobacter
-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (
n
= 205).
C. jejuni
counts in the presence of bacteriophage (mean log
10
5.1 CFU/g) were associated with a significant (
P
< 0.001) reduction compared to samples with
Campylobacter
alone (mean log
10
6.9 CFU/g).
Insertional Inactivation of Determinants for Mg
2+
and Co
2+
Transport as a Tool for Screening Recombinant
Lactococcus
Species Clones
Authors: Mills Susan , Coffey Aidan , Hill Colin , Fitzgerald Gerald F. , McAuliffe Olivia , Ross R. Paul
Published: August 2005

Insertional inactivation of the plasmid-encoded determinants for Mg
2+
and Co
2+
transport,
orf18
/
corA
, provides a tool for screening recombinant clones in
Lactococcus
, based on the observation that overexpression of
orf18
/
corA
results in cell growth inhibition on certain concentrations of CoCl
2
. The lacticin 3147 immunity gene,
ltnI
, was used to insertionally inactivate
orf18
/
corA
. The resulting clones were capable of growth on concentrations of CoCl
2
that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor
corA
, the system has potential as a screen for selecting recombinant lactococcal clones.
Habits of Magnetosome Crystals in Coccoid Magnetotactic Bacteria
Authors: Lins Ulysses , McCartney Martha R. , Farina Marcos , Frankel Richard B. , Buseck Peter R.
Published: August 2005

High-resolution transmission electron microscopy and electron holography were used to study the habits of exceptionally large magnetite crystals in coccoid magnetotactic bacteria. In addition to the crystal habits, the crystallographic positioning of successive crystals in the magnetosome chain appears to be under strict biological control.
Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of
Escherichia coli
O45 and O55 Serogroups
Authors: DebRoy Chitrita , Fratamico Pina M. , Roberts Elisabeth , Davis Michael A. , Liu Yanhong
Published: August 2005

The
Escherichia coli
O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the
wzx
(O-antigen flippase) and
wzy
(O-antigen polymerase) genes. In addition, PCR assays targeting the
E. coli
O55
wzx
and
wzy
genes were designed based on previously published sequences. PCR assays targeting
E. coli
O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for
E. coli
O55, 97/102 strains serotyped as
E. coli
O55 were positive for
wzx
and 98/102 for
wzy
. Multiplex PCR assays targeting the
E. coli
O45 and the
E. coli
O55
wzx
and
wzy
genes were used to detect the organisms in fecal samples spiked at levels of 10
6
and 10
8
CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify
E. coli
serogroups O45 and O55.
Specificity of a
Bacteroides thetaiotaomicron
Marker for Human Feces
Authors: Carson C. Andrew , Christiansen Jessica M. , Yampara-Iquise Helen , Benson Verel W. , Baffaut Claire , Davis Jerri V. , Broz Robert R. , Kurtz William B. , Rogers Wendi M. , Fales William H.
Published: August 2005

A bacterial primer set, known to produce a 542-bp amplicon specific for
Bacteroides thetaiotaomicron
, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.
Patterns of CD8 T-Cell Epitopes within the Human Papillomavirus Type 16 (HPV 16) E6 Protein among Young Women Whose HPV 16 Infection Has Become Undetectable
Authors: Nakagawa Mayumi , Kim Kevin H. , Moscicki Anna-Barbara
Published: August 2005

The patterns of CD8 T-cell epitopes recognized within the E6 protein in women who had cleared their human papillomavirus 16 infection were examined. T-cell lines were established using autologous dendritic cells infected with a recombinant vaccinia virus. Evidence of potential antigenic epitopes was shown in 8 of 23 (34.8%) women.
Critical Evaluation of Urine-Based PCR Assay for Diagnosis of Lyme Borreliosis
Authors: Rauter Carolin , Mueller Markus , Diterich Isabel , Zeller Sabine , Hassler Dieter , Meergans Thomas , Hartung Thomas
Published: August 2005

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole
Borrelia
or
Borrelia
DNA was established. The development of a nested real-time PCR targeting
ospA
enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at −20°C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 ×
g
for 30 min resulted in a concentration of both spikes, i.e., whole
Borrelia
and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.
Modification of Rapid Human Immunodeficiency Virus (HIV) Antibody Assay Protocols for Detecting Recent HIV Seroconversion
Authors: Soroka Stephen D. , Granade Timothy C. , Candal Debra , Parekh Bharat S.
Published: August 2005

Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score = 0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics = 0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A11-LS SOD values (
R
2
> 0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.
Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay
Authors: Ko Young-Joon , Choi Kang-Seuk , Nah Jin-Ju , Paton David J. , Oem Jae-Ku , Wilsden Ginette , Kang Shien-Young , Jo Nam-In , Lee Joo-Ho , Kim Jae-Hong , Lee Hee-Woo , Park Jong-Myeong
Published: August 2005

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (
n
= 1,041). When tested using sera (
n
= 186) collected periodically from pigs (
n
= 19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.
Naturally Acquired Antibodies against Four
Enterococcus faecalis
Capsular Polysaccharides in Healthy Human Sera
Authors: Hufnagel Markus , Kropec Andrea , Theilacker Christian , Huebner Johannes
Published: August 2005

Healthy human sera (HHS) contain naturally acquired enterococcal antibodies which promote neutrophil-mediated killing. The target antigens remain unknown. The present study used a capsular polysaccharide (CPS)-enzyme-linked immunosorbent assay (ELISA) to investigate whether the HHS antibodies of 12 healthy donors bound to the CPS of four
E. faecalis
serotypes (CPS-A to CPS-D) and then employed an opsonic-killing assay to determine if these antibodies mediated phagocyte-dependent killing. All HHS contained immunoglobulin G (IgG) and IgM antibodies directed against capsular polysaccharides of the four serotypes. Absorption of the sera with homologous and heterologous strains showed a majority of antibodies to be cross-reactive among the prototype strains. The susceptibility of the four prototype strains to opsonic killing varied. Opsonic killing of CPS-A and CPS-B strains was significantly higher than killing of CPS-C and CPS-D strains. Absorption studies revealed that the opsonic killing of HHS was only partially type specific, with cross-reactivity between CPS-A and CPS-B strains and between CPS-C and CPS-D strains. These data indicate that healthy individuals possess opsonic antibodies specific for CPS-A and CPS-B but only low titers of opsonic antibodies against CPS-C and CPS-D. Titers of opsonic antibodies did not correlate with antibody titers measured by ELISA. Whether this lack of correlation is due to the low frequency of opsonic antibodies or to increased resistance to the opsonophagocytic killing of some serotypes remains to be determined.
Borrelia burgdorferi
BmpA, BmpB, and BmpD Proteins Are Expressed in Human Infection and Contribute to P39 Immunoblot Reactivity in Patients with Lyme Disease
Authors: Bryksin Anton V. , Godfrey Henry P. , Carbonaro Carol A. , Wormser Gary P. , Aguero-Rosenfeld Maria E. , Cabello Felipe C.
Published: August 2005

The Bmp proteins are a paralogous family of chromosomally encoded
Borrelia burgdorferi
lipoproteins. They have similar predicted immunogenicities and similar electrophoretic mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P39 reactivity against
Borrelia burgdorferi
lysate in immunoblots of Lyme disease patients has long been identified with reactivity to BmpA, but responses to other Bmp proteins have not been examined. To determine if patients with Lyme disease developed such responses, immunoglobulin G (IgG) anti-Bmp reactivity in patient and control sera was studied by using soluble recombinant Bmp (rBmp) proteins expressed in
Escherichia coli
. Although some patient sera contained IgG immunoblot and immunodot reactivities against all four Bmp proteins, analysis of IgG anti-Bmp fine specificity by a competitive enzyme-linked immunosorbent assay with graded doses of soluble homologous and heterologous rBmp proteins showed that only the responses to BmpA, BmpB, and BmpD were specific. This suggests that at least three of the four Bmp proteins are expressed by
B. burgdorferi
in infected patients and that specific antibodies to them are likely to be present in the P39 band in some patients.
Seroreactivity to and Avidity for Recombinant Antigens in Toxoplasmosis
Authors: Pfrepper Klaus-Ingmar , Enders Gisela , Gohl Marion , Krczal Doris , Hlobil Harald , Wassenberg Doris , Soutschek Erwin
Published: August 2005

To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinantToxoplasma gondiiantigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned inEscherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points.
Diagnosis and management of chronic pancreatitis
Authors: Gupta V , Toskes P P
Published: August 2005

Chronic pancreatitis represents a condition that is challenging for clinicians secondary to the difficulty in making an accurate diagnosis and the less than satisfactory means of managing chronic pain. This review emphasises the various manifestations that patients with chronic pancreatitis may have and describes recent advances in medical and surgical therapy. It is probable that many patients with chronic abdominal pain are suffering from chronic pancreatitis that is not appreciated. As the pathophysiology of this disorder is better understood it is probable that the treatment will be more successful.
Acanthosis nigricans: relation with type 2 diabetes mellitus, anthropometric variables, and body mass in Indians
Authors: Grandhe N P , Bhansali A , Dogra S , Kumar B
Published: August 2005

Objective

To determine the prevalence of acanthosis nigricans (AN) in type 2 diabetes mellitus (T2DM) and its correlation with various anthropometric measurements in Indians.

Methods

One hundred and fifty consecutive subjects with T2DM attending the diabetes clinic at a tertiary referral centre in North India were considered as cases and 150 age and sex matched healthy attendants of non-diabetic subjects as controls. All the cases and controls were screened for the presence of AN and its severity. Anthropometric measurements of all of them were measured in standard method. Regression analysis was done to determine the association of AN with T2DM and various anthropometric measurements.

Results

The prevalence of AN in subjects with diabetes and healthy controls was 62.6% and 40% respectively, and this difference was significant (p<0.05). Body mass index (BMI) between cases and controls was comparable by chance. There was a statistically significant correlation of increasing severity of AN with increasing BMI, waist circumference, hip circumference, waist-hip ratio, skinfold thickness, and body fat percentage in diabetic patients. However, in regression analysis after considering all the confounding factors there was a significant correlation of AN, only with diabetes mellitus and BMI.

Conclusions

Indians have high prevalence of AN and it is an independent cutaneous marker of both T2DM and BMI.
The heart and the oesophagus: intimate relations
Authors: Heatley M , Rose K , Weston C
Published: August 2005

The close anatomical relations of the heart and oesophagus, and the similarity of symptoms attributable to disorders of either organ, often lead to diagnostic difficulty in patients with chest pain. A definitive diagnosis of non-cardiac chest pain attributable to oesophageal reflux or spasm is hampered, both by the need for prolonged ambulatory monitoring of pH, manometry, and endoscopy, and by the common occurrence of asymptomatic reflux and spasm, and the corresponding difficulty in linking an episode of reflux or spasm with an episode of pain. Moreover, some patients with non-cardiac chest pain and normal tests of oesophageal structure and function have centrally mediated hypersensitivity, both within and without the oesophagus. Rather than proceed with investigations, in the absence of symptoms to suggest structural disease of the oesophagus, it would be reasonable to attempt symptomatic treatment with a proton pump inhibitor or an antidepressant.
The approach to patients with “non-epileptic seizures”
Authors: Mellers J D C
Published: August 2005

Up to one fifth of patients who present to specialist clinics with seizures do not have epilepsy. The majority of such patients suffer from psychologically mediated episodes; dissociative seizures, often referred to as “non-epileptic seizures”. This paper describes the diagnostic evaluation of seizure disorders, including clinical assessment and the role of special investigations. The organic and psychiatric imitators of epilepsy are outlined and findings on psychiatric assessment are reviewed. This group of patients often proves difficult to engage in appropriate treatment and an approach to explaining the diagnosis is described. As yet there are no controlled trials of treatment in this disorder but preliminary evidence suggests cognitive behavioural therapy is both a rational and promising way forward.
Mechanisms underlying recovery of motor function after stroke
Authors: Ward N S
Published: August 2005

Neurological damage, and stroke in particular, is the leading cause of long term disability worldwide. There is growing interest in the part that central nervous system reorganisation plays in recovery of function. Techniques such as functional magnetic resonance imaging and transcranial magnetic stimulation permit the non-invasive study of the working human brain, and suggest that functionally relevant adaptive changes occur in the human brain after focal damage. An understanding of how these changes are related to recovery will facilitate the development of novel therapeutic techniques that are based on neurobiological principles and that are designed to minimise impairment in appropriately targeted patients suffering from stroke.
Polymorphisms of CD14 gene and TLR4 gene are not associated with ulcerative colitis in Chinese patients
Authors: Guo Q S , Xia B , Jiang Y , Morré S A , Cheng L , Li J , Crusius J B A , Peña A S
Published: August 2005

Background and aims

Toll-like receptor 4 and CD14 are the components of the lipopolysaccharide receptor complex. The aim of this study was to investigate the associations between polymorphisms TLR4 Asp299Gly and CD14 C-260T and Chinese patients with ulcerative colitis (UC).

Methods

Using a polymerase chain reaction based restriction fragment length polymorphism, the study genotyped polymorphisms TLR4 Asp299Gly and CD14 C-260T in 114 patients with UC and 160 healthy controls in the Chinese Han population. Moreover a comparison was made with 170 healthy Dutch white subjects.

Results

No TLR4 Asp299Gly mutation was detected in any patients or healthy controls in the Chinese Han population, which was similar to Japanese subjects, but the mutation occurred in 10% of the Dutch white subjects. There were no significant differences of CD14 genotypes between healthy controls and the patients with UC.
Well leg compartment syndrome after pelvic and perineal surgery in the lithotomy position
Authors: Simms M S , Terry T R
Published: August 2005

Objective

Lower limb compartment syndrome after prolonged surgical procedures performed in the lithotomy position is a rare but potentially devastating complication. It is recognised after urological, colorectal, and gynaecological procedures. Sixteen cases of compartment syndrome after urological surgery have been reported. The objective of this study was to estimate the incidence of this complication in urological practice and identify risk factors for its development.

Design

A postal survey of UK consultant urologists was conducted.

Results

Replies were received from 261 consultants. In total there were 65 cases of compartment syndrome. Compartment syndrome occurred after radical cystectomy and urinary diversion in 51 cases and was rare in procedures lasting less than four hours. The incidence of compartment syndrome after cystectomy was estimated at around 1 in 500 cases. Risk factors for its development included perioperative blood loss, peripheral vascular disease, and obesity.

Conclusions

Compartment syndrome after use of the lithotomy position may be more common than is generally appreciated and has been underreported in the past. All staff should be aware of this serious complication and adopt strategies for its avoidance.
A NOTE ON THE LOCAL NILPOTENCE OF 4-ENGEL GROUPS
Authors: TRAUSTASON GUNNAR
Published: August 2005

Recently Havas and Vaughan-Lee proved that 4-Engel groups are locally nilpotent. Their proof relies on the fact that a certain 4-Engel group T is nilpotent and this they prove using a computer and the Knuth–Bendix algorithm. In this paper we give a short handproof of the nilpotency of T.