bluuebunny/crossref_metadata_2025 huggingface dataset:
Microevolution between paired antral and paired antrum and corpus Helicobacter pylori isolates recovered from individual patients
Authors: Carroll Ian M. , Ahmed Niyaz , Beesley Sarah M. , Khan Aleem A. , Ghousunnissa Sheikh , Moráin Colm A.Ó , Habibullah C. M. , Smyth Cyril J.
Published: June 2004

Sequence variations located at the signal sequence and mid-region within the vacA gene, the 3′-end of the cagA gene, the indel motifs at the 3′-end of the cag pathogenicity island and the regions upstream of the vacA and ribA genes were determined by PCR in 19 paired antral or antrum and corpus Helicobacter pylori isolates obtained at the same endoscopic session, and three antral pairs taken sequentially. Random amplification of polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (FAFLP)-PCR fingerprinting were applied to these paired clinical isolates. The FAFLP-PCR profiles generated were phylogenetically analysed. For the 22 paired isolates there were no differences within pairs at five of the genetic loci studied. However, six pairs of isolates (27 %), of which four were antrum and corpus pairs, showed differences in the numbers of repeats located at the 3′-end of the cagA gene. RAPD-PCR fingerprinting showed that 16 (73 %) pairs, nine of which were antrum and corpus pairs, possessed identical profiles, while six (27 %) displayed distinctly different profiles, indicating mixed infections. Three of the six pairs showing differences at the 3′-end of the cagA gene yielded identical RAPD-PCR fingerprints. FAFLP-PCR fingerprinting and phylogenetic analysis revealed that all 16 pairs that displayed identical RAPD-PCR profiles had highly similar, but not identical, fingerprints, demonstrating that these pairs were ancestrally related but had undergone minor genomic alterations. Two antrum and corpus pairs of isolates, within the latter group, were isolates obtained from two siblings from the same family. This analysis demonstrated that each sibling was colonized by ancestrally related strains that exhibited differences in vacA genotype characteristics.
Aetiology of acute pharyngitis: the role of atypical bacteria
Authors: Esposito Susanna , Blasi Francesco , Bosis Samantha , Droghetti Roberta , Faelli Nadia , Lastrico Annalisa , Principi Nicola
Published: June 2004

In order to establish the role of atypical bacteria and compare characteristics of different infectious agents in acute pharyngitis, 127 patients with acute pharyngitis (66 males; median age, 5.33 years; range, 6 months to 14 years) and 130 healthy subjects of similar sex and age were studied. Serology with paired samples and PCR on nasopharyngeal aspirates and throat cultures were used to identify bacteria and viruses. Viruses were identified in 43 patients (33.8 %) and five controls (3.8 %;P< 0.0001), potential bacterial pathogens in 34 patients (26.8 %) and 26 controls (20 %;P= 0.256) and mixed viral/bacterial pathogens in 26 patients (20.5 %) and none of the controls (P< 0.0001). The main aetiological agents were adenovirus, respiratory syncytial virus (RSV),Mycoplasma pneumoniae,Streptococcus pyogenesandChlamydia pneumoniae.M. pneumoniaewas the agent found most frequently as a single pathogen. A history of recurrent pharyngitis, having older siblings and a negative outcome were significantly more common among patients with acuteM. pneumoniaeinfection than among those with infections due to other pathogens or healthy controls. This study demonstrates that: (i) adenovirus and RSV have a prominent role in acute pharyngitis; (ii)S. pyogenesis found frequently, but it is not possible to distinguish simple carriers from patients with a true infection; (iii)M. pneumoniaeappears to be able to cause acute pharyngitisper se; and (iv)C. pneumoniaeseems to be mainly a co-pathogen. To avoid the risk of an incorrect therapeutic approach, simple laboratory investigations that allow rapid identification ofM. pneumoniaeinfections are urgently needed.
Switches, cross-talk and memory in Escherichia coli adherence
Authors: Holden Nicola J. , Gally David L.
Published: June 2004

Escherichia coli is a successful commensal and pathogen. Its pathogenic diversity stems from the acquisition and expression of multiple virulence-associated loci. Many of the key virulence factors are surface structures involved in adherence and motility. These are important antigens and their expression is limited by phase-variable genetic switches that are considered to act randomly. This review considers the possibility that such stochastic expression within a bacterial population belies sequential or co-ordinate control at the level of the individual bacterium. Co-ordinated expression or cross-talk between virulence loci can lead to a programmed set of events within a bacterium analogous to a simple form of electronic memory that is of benefit during infection.
Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation
Authors: Stapper Andres Plata , Narasimhan Giri , Ohman Dennis E. , Barakat Johnny , Hentzer Morten , Molin Søren , Kharazmi Arsalan , Høiby Niels , Mathee Kalai
Published: June 2004

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced byPseudomonas aeruginosastrain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+PAOmucA22and Alg−PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.
Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation
Authors: McDowell Andrew , Mahenthiralingam Eshwar , Dunbar Kerstin E.A. , Moore John E. , Crowe Mary , Elborn J. Stuart
Published: June 2004

Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79 % of patients were infected with Burkholderia cenocepacia (genomovar III), 18 % with Burkholderia multivorans (genomovar II) and less than 5 % of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested.
Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients
Authors: Hierl Thomas , Reischl Udo , Lang Peter , Hebart Holger , Stark Maik , Kyme Pierre , Autenrieth Ingo B.
Published: June 2004

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify Toxoplasma gondii DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed T. gondii DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of T. gondii DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of T. gondii DNA in some samples.
Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay
Authors: Iijima Yoshio , Asako Nahoko T. , Aihara Masanori , Hayashi Kozaburo
Published: June 2004

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, includingSalmonella enterica,Vibrio parahaemolyticus,Campylobacter jejuniand Shiga toxin-producingEscherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40 %) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70 %) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18 %) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6 %) patients were found to be positive by PCR analysis.
Revised description and classification of atypical isolates of Pasteurella multocida from bovine lungs based on genotypic characterization to include variants previously classified as biovar 2 of Pasteurella canis and Pasteurella avium
Authors: Christensen Henrik , Angen Øystein , Olsen John Elmerdahl , Bisgaard Magne
Published: June 2004

Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship withPasteurella multocida. Twenty-two strains ofPasteurella aviumbiovar 2, including variants in indole, xylose and mannitol, 18 strains ofPasteurella canisbiovar 2 and variants of this taxon, five strains ofP. multocidasubsp.septicashowing variations in indole and ornithine decarboxylase, nine strains ofP. multocidasubsp.multocidashowing variation in ornithine decarboxylase and mannitol, and type strains of the subspecies ofP. multocidawere included. Ribotyping was used to examine the relationship of the strains, and 13 types, each containing between one and 20 isolates, were observed. Identical ribotypes were observed in some cases forP. aviumbiovar 2 and eitherP. canisbiovar 2 orP. multocidasubsp.septica. ITS (16S–23S rRNA internal transcribed spacer) fragment-length profiling showed identity of the majority of strains (47 of 52), representing all four taxa, with only five divergent strains. A 16S rRNA sequence comparison of 11 strains representing the main ribotype clusters showed 99·9 % similarity to the type strain ofP. multocidasubsp.multocida, but only 97·4 % similarity was obtained toP. canis(biovar 1) and 93·7 % toP. avium(biovar 1). A species-specific PCR test forP. multocidagave a positive result with biovar 2 variants ofP. aviumandP. canis. DNA–DNA hybridizations between strains ofP. multocida, biovar 2 variants ofP. aviumandP. canis, andP. multocidasubsp.septicaconfirmed similarity at the species level. It is proposed, on the basis of genotypic similarity, thatP. multocidabe reclassified to include the biovar 2 variants ofP. aviumandP. canisand that the existence of the biovar 2 variants ofP. aviumandP. canisis highly questionable. It is concluded that the redefinedP. multocidais genotypically homogeneous, although phenotypically diverse lineages exist with respect to ornithine decarboxylase, indole and mannitol, characters that have been regarded as essential for identification to the species level. A formal reclassification of the species is not possible, however, since too few strains have been found to vary in these key characters. Considering the phenotypic diversity ofP. multocida, identification will have to depend partly on genotypic methods and the source host also seems important for safe diagnosis.
Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms
Authors: Gomez-Gil Bruno , Soto-Rodríguez Sonia , García-Gasca Alejandra , Roque Ana , Vazquez-Juarez Ricardo , Thompson Fabiano L. , Swings Jean
Published: June 2004

Fifty strains belonging to Vibrio harveyi, Vibrio campbellii, and the recently described Vibrio rotiferianus, were analysed using phenotypic and genomic techniques with the aim of analysing the usefulness of the different techniques for the identification of V. harveyi-related species. The species V. harveyi and V. campbellii were phenotypically indistinguishable by more than 100 phenotypic features. Thirty-nine experimental strains were phenotypically identified as V. harveyi, but FAFLP, REP-PCR, IGS-PCR and DNA–DNA hybridization proved that they in fact belong to the species V. campbellii. Similar groupings were found among all fingerprinting methodologies (except IGS-PCR). Thirty-two experimental strains clustered with the V. campbellii type and one reference strain; seven strains clustered with the V. harveyi type and three reference strains; and the type and four reference strains of V. rotiferianus grouped together. The correlations between DNA–DNA hybridization and the genomic fingerprinting by FAFLP and (GTG)5-PCR were found to be above 0·68 and statistically significant, suggesting the value of the latter techniques for the reliable identification of V. harveyi-related species. The results presented indicate that strains phenotypically identified as V. harveyi are in fact V. campbellii; these findings position V. campbellii as an important species involved in diseases of reared aquatic organisms.
The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex
Authors: Pinto Rachel , Tang Quing Xui , Britton Warwick J. , Leyh Thomas S. , Triccas James A.
Published: June 2004

Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence ofMycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 5′-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of theM. tuberculosis cysDgene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates thatcysDandcysNCorthologues exist inM. tuberculosisand constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. ThecysDNCgenes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation byM. tuberculosisin response to toxic oxidants. To ensure that thecysDNCoperon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed inEscherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway.
Diversity and distribution of Microcystis (Cyanobacteria) oligopeptide chemotypes from natural communities studied by single-colony mass spectrometry
Authors: Welker Martin , Brunke Matthias , Preussel Karina , Lippert Indra , von Döhren Hans
Published: June 2004

Microcystis sp. has been recognized in recent years as a producer of a high number of secondary metabolites. Among these, peptides that are produced by the non-ribosomal peptide synthetase pathway often show bioactivity or are toxic to humans. The production of particular peptides is specific for individual Microcystis clones, allowing their characterization as chemotypes by analysing the peptidome. The authors studied the in situ diversity of peptides and chemotypes in Microcystis communities from lakes in and around Berlin, Germany, by direct analysis of individual colonies by MALDI-TOF mass spectrometry. From 165 colonies analysed a total of 46 individual peptides could be identified, 21 of which have not been described previously. For six of the new peptides the structures could be elucidated from fragment patterns, while for others only a preliminary classification could be achieved. In most colonies, two to ten individual peptides were detected. In 19 colonies, 16 of which were identified as M. wesenbergii, no peptide metabolites could be detected. The peptide data of 146 colonies were subjected to an ordination (principal components analysis). The principal components were clearly formed by the microcystin variants Mcyst-LR, -RR and -YR, anabaenopeptins B and E/F, a putative microviridin, and a new cyanopeptolin. In the resulting ordination plots most colonies were grouped into five distinct groups, while 40 colonies scattered widely outside these groups. In some cases colonies from different lakes clustered closely, indicating the presence of similar chemotypes in the respective samples. With respect to colony morphology no clear correlation between a chemotype and a morphospecies could be established, but M. aeruginosa, for example, was found to produce predominantly microcystins. In contrast, M. ichthyoblabe colonies were mostly negative for microcystins and instead produced anabaenopeptins. The number of peptides detected in a limited number of samples and the various combinations of peptides in individual Microcystis colonies highlights the immense metabolic potential and diversity of this genus.
Evolution of compatible replicons of the related IncQ-like plasmids, pTC-F14 and pTF-FC2
Authors: Gardner Murray N. , Rawlings Douglas E.
Published: June 2004

Two closely related but compatible plasmids of the IncQ-2αand IncQ-2βgroups, pTF-FC2 and pTC-F14, were discovered in two acidiphilic chemolithotrophic bacteria. Cross-complementation and cross-regulation experiments by the replication proteins were carried out to discover what changes were necessary when the plasmids evolved to produce two incompatibility groups. The requirement of a pTC-F14oriVfor a RepC DNA-binding protein was plasmid specific, whereas the requirement for the RepA helicase and RepB primase was less specific and could be complemented by the IncQ-2αplasmid pTC-FC2, and the IncQ-1βplasmid pIE1108. None of the IncQ-1αplasmid replication proteins could complement the pTC-F14oriV, and pTC-F14 and RSF1010 were incompatible. This incompatibility was associated with the RepC replication protein and was not due to iteron incompatibility. Replication of pTC-F14 took place from a 5·7 kb transcript that originated upstream of themobBgene located within the region required for mobilization. A pTC-F14mobB–lacZfusion was regulated by the pTC-F14repBgene product and was plasmid specific, as it was not regulated by the RepB proteins of pTF-FC2 or the IncQ-1αand IncQ-1βplasmids. Plasmid pTC-F14 appears to have evolved independently functioning iterons and a plasmid-specific RepC-binding protein; it also has a major replication transcript that is independently regulated from that of pTF-FC2. However, the RepA and RepB proteins have the ability to function with either replicon.
A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans
Authors: Shah Deepan S. H. , Russell Roy R. B.
Published: June 2004

Streptococcus mutansproduces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called ‘A’ repeats. TheS. mutansgenome sequence was searched for ORFs containing ‘A’ repeats, and one novel gene,gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three ‘A’ repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with aKDof 2–3 nM. Construction of truncated derivatives of GbpD confirmed that the ‘A’ repeat region was essential for binding. Furthermore, agbpDknockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to theα/βhydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site ‘lipase box’ and an ‘oxyanion hole’. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. ThegbpDmutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity ofS. mutansand another plaque bacterium,Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) ofS. sanguinis, but had no effect on LTA fromS. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.
Effects of high hydrostatic pressure on bacterial cytoskeleton FtsZ polymers in vivo and in vitro
Authors: Ishii Akihiro , Sato Takako , Wachi Masaaki , Nagai Kazuo , Kato Chiaki
Published: June 2004

Some rod-shaped bacteria, including Escherichia coli, exhibit cell filamentation without septum formation under high-hydrostatic-pressure conditions, indicating that the cell-division process is affected by hydrostatic pressure. The effects of elevated pressure on FtsZ-ring formation in E. coli cells were examined using indirect immunofluorescence microscopy. Elevated pressure of 40 MPa completely inhibited colony formation of E. coli cells under the cultivation conditions used, and the cells exhibited obviously filamentous shapes. In the elongated cells, normal cell-division processes appeared to be inhibited, because no FtsZ rings were observed by indirect immunofluorescent staining. In addition, it was observed that hydrostatic pressure dissociated the E. coli FtsZ polymers in vitro. These results suggest that high hydrostatic pressure directly affects cell survival and morphology through the dissociation of the cytoskeletal frameworks.
Phylogeny of Mycobacterium avium strains inferred from glycopeptidolipid biosynthesis pathway genes
Authors: Krzywinska Elzbieta , Krzywinski Jaroslaw , Schorey Jeffrey S.
Published: June 2004

TheMycobacterium aviumcomplex (MAC) encompasses two species,M. aviumandMycobacterium intracellulare, which are opportunistic pathogens of humans and animals. The standard method of MAC strain differentiation is serotyping based on a variation in the antigenic glycopeptidolipid (GPL) composition. To elucidate the relationships amongM. aviumserotypes a phylogenetic analysis of 13 reference and clinicalM. aviumstrains from 8 serotypes was performed using as markers two genomic regions (890 bp of thegtfBgene and 2150 bp spanning thertfA–mtfCgenes) which are associated with the strains' serological properties. Strains belonging to three other knownM. aviumserotypes were not included in the phylogeny inference due to apparent lack of the marker sequences in their genomes, as revealed by PCR and Southern blot analysis. These studies suggest that serotypes prevalent in AIDS patients have multiple origins. In trees inferred from both markers, serotype 1 strains, known to have the simplest and shortest GPLs among all other serotypes, were polyphyletic. Likewise, comparisons of the inferred phylogenies with the molecular typing results imply that the existing tools used in epidemiological studies may be poor estimators ofM. aviumstrain relatedness. Additionally, trees inferred from each marker had significantly incongruent topologies due to a well supported alternative placement of strain 2151, suggesting a complex evolutionary history of this genomic region.
DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases
Authors: Kwiatek Agnieszka , Kobes Monika , Olejnik Kamil , Piekarowicz Andrzej
Published: June 2004

The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli. The enzymes were purified to apparent homogeneity on Ni-NTA agarose columns, yielding proteins of 49±1 kDa and 39·6±1 kDa, respectively, under denaturing conditions. M.NmeDI recognizes the degenerate sequence 5′-RCCGGB-3′. It methylates the first 5′ cytosine residue on both strands within the core sequence CCGG. The enzyme shows higher affinity with the hemimethylated degenerate sequence than with the unmethylated degenerate sequence. Comparison of the amino acid sequence of the target-recognizing domain of M.NmeDI with the closest neighbours recognizing the sequence 5′-RCCGGY-3′ showed the presence of the homologous domain and an additional domain that may be responsible for recognizing the degenerate sequence. M.NmeAI recognizes the sequence 5′-CCGG-3′ and methylates the second 5′ cytosine residue on both DNA strands. In Neisseria gonorrhoeae strain FA1090 the homologues of these ORFs are truncated due to a variety of mutations.
Dosage-dependent functions of fatty acid desaturase Ole1p in growth and morphogenesis of Candida albicans
Authors: Krishnamurthy Shankarling , Plaine Armêl , Albert Juliane , Prasad Tulika , Prasad Rajendra , Ernst Joachim F.
Published: June 2004

Conditions in the infected human host trigger virulence attributes of the fungal pathogenCandida albicans. Specific inducers and elevated temperatures lead to hyphal development or regulate chlamydospore development. To explore if these processes are affected by membrane lipids, an investigation of the functions of the Ole1 fatty acid desaturase (stearoyl-CoA desaturase) inC. albicans, which synthesizes oleic acid, was undertaken. A conditional strain expressingOLE1from the regulatableMET3promoter was unable to grow in repressing conditions, indicating thatOLE1is an essential gene. In contrast, a mutant lacking both alleles ofOLE2, encoding a Ole1p homologue, was viable and had no apparent phenotypes. Partial repression ofMET3p–OLE1slightly lowered oleic acid levels and decreased membrane fluidity; these conditions permitted growth in the yeast form, but prevented hyphal development in aerobic conditions and blocked the formation of chlamydospores. In contrast, in hypoxic conditions, which trigger an alternative morphogenetic pathway, hyphal morphogenesis was unaffected. Because aerobic morphogenetic signalling and oleic acid biosynthesis require oxygen, it is proposed that oleic acid may function as a sensor activating specific morphogenetic pathways in normoxic conditions.
The RAM1 gene encoding a protein-farnesyltransferase β-subunit homologue is essential in Cryptococcus neoformans
Authors: Vallim Marcelo A. , Fernandes Larissa , Alspaugh J. Andrew
Published: June 2004

Many small G proteins require post-translational modification to allow functional association to the cell membrane. This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein. The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases. The human fungal pathogenCryptococcus neoformansrequires functional Ras and Rho proteins in order to undergo normal growth and differentiation. Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus. We cloned theRAM1gene encoding the single protein-farnesyltransferaseβ-chain homologue inC. neoformans. Using a gene-disruption strategy in a diploidC. neoformansstrain, we demonstrated that this gene encodes an essential function, in contrast to the case inSaccharomyces cerevisiae, in which the homologousRAM1gene is not essential for growth. Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis ofC. neoformans, as well as prevention of hyphal differentiation. Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth. Protein farnesylation is required for the growth and cellular differentiation ofC. neoformansand may provide novel targets for antifungal therapy.
Saccharomyces kluyveri FAD3 encodes an ω3 fatty acid desaturase
Authors: Oura Takahiro , Kajiwara Susumu
Published: June 2004

Fungi, like plants, are capable of producing the 18-carbon polyunsaturated fatty acids linoleic acid andα-linolenic acid. These fatty acids are synthesized by catalytic reactions of Δ12 andω3 fatty acid desaturases. This paper describes the first cloning and functional characterization of a yeastω3 fatty acid desaturase gene. The deduced protein encoded by theSaccharomyces kluyveri FAD3gene (Sk-FAD3) consists of 419 amino acids, and shows 30–60 % identity with Δ12 fatty acid desaturases of several eukaryotic organisms and 29–31 % identity withω3 fatty acid desaturases of animals and plants. DuringSk-FAD3expression inSaccharomyces cerevisiae,α-linolenic acid accumulated only when linoleic acid was added to the culture medium. The disruption ofSk-FAD3led to the disappearance ofα-linolenic acid inS. kluyveri. These findings suggest thatSk-FAD3is the onlyω3 fatty acid desaturase gene in this yeast. Furthermore, transcriptional expression ofSk-FAD3appears to be regulated by low-temperature stress in a manner different from the other fatty acid desaturase genes inS. kluyveri.
Design and analysis of pilot studies: recommendations for good practice
Authors: Lancaster Gillian A. , Dodd Susanna , Williamson Paula R.
Published: June 2004

Pilot studies play an important role in health research, but they can be misused, mistreated and misrepresented. In this paper we focus on pilot studies that are used specifically to plan a randomized controlled trial (RCT). Citing examples from the literature, we provide a methodological framework in which to work, and discuss reasons why a pilot study might be undertaken. A well‐conducted pilot study, giving a clear list of aims and objectives within a formal framework will encourage methodological rigour, ensure that the work is scientifically valid and publishable, and will lead to higher quality RCTs. It will also safeguard against pilot studies being conducted simply because of small numbers of available patients.
Latent Pattern Mixture Models for Informative Intermittent Missing Data in Longitudinal Studies
Authors: Lin Haiqun , McCulloch Charles E. , Rosenheck Robert A.
Published: June 2004

Summary.  A frequently encountered problem in longitudinal studies is data that are missing due to missed visits or dropouts. In the statistical literature, interest has primarily focused on monotone missing data (dropout) with much less work on intermittent missing data in which a subject may return after one or more missed visits. Intermittent missing data have broader applicability that can include the frequent situation in which subjects do not have common sets of visit times or they visit at nonprescheduled times. In this article, we propose a latent pattern mixture model (LPMM), where the mixture patterns are formed from latent classes that link the longitudinal response and the missingness process. This allows us to handle arbitrary patterns of missing data embodied by subjects' visit process, and avoids the need to specify the mixture patterns a priori. One assumption of our model is that the missingness process is assumed to be conditionally independent of the longitudinal outcomes given the latent classes. We propose a noniterative approach to assess this key assumption. The LPMM is illustrated with a data set from a health service research study in which homeless people with mental illness were randomized to three different service packages and measures of homelessness were recorded at multiple time points. Our model suggests the presence of four latent classes linking subject visit patterns to homeless outcomes.
Nonignorable Missingness in Matched Case–Control Data Analyses
Authors: Cho Paik Myunghee
Published: June 2004

Summary.  Matched case–control data analysis is often challenged by a missing covariate problem, the mishandling of which could cause bias or inefficiency. Satten and Carroll (2000, Biometrics56, 384–388) and other authors have proposed methods to handle missing covariates when the probability of missingness depends on the observed data, i.e., when data are missing at random. In this article, we propose a conditional likelihood method to handle the case when the probability of missingness depends on the unobserved covariate, i.e., when data are nonignorably missing. When the missing covariate is binary, the proposed method can be implemented using standard software. Using the Northern Manhattan Stroke Study data, we illustrate the method and discuss how sensitivity analysis can be conducted.
A Cautionary Note on the Robustness of Latent Class Models for Estimating Diagnostic Error without a Gold Standard
Authors: Albert Paul S. , Dodd Lori E.
Published: June 2004

Summary.  Modeling diagnostic error without a gold standard has been an active area of biostatistical research. In a majority of the approaches, model‐based estimates of sensitivity, specificity, and prevalence are derived from a latent class model in which the latent variable represents an individual's true unobserved disease status. For simplicity, initial approaches assumed that the diagnostic test results on the same subject were independent given the true disease status (i.e., the conditional independence assumption). More recently, various authors have proposed approaches for modeling the dependence structure between test results given true disease status. This note discusses a potential problem with these approaches. Namely, we show that when the conditional dependence between tests is misspecified, estimators of sensitivity, specificity, and prevalence can be biased. Importantly, we demonstrate that with small numbers of tests, likelihood comparisons and other model diagnostics may not be able to distinguish between models with different dependence structures. We present asymptotic results that show the generality of the problem. Further, data analysis and simulations demonstrate the practical implications of model misspecification. Finally, we present some guidelines about the use of these models for practitioners.
A Conditional Markov Model for Clustered Progressive Multistate Processes under Incomplete Observation
Authors: Cook Richard J. , Yi Grace Y. , Lee Ker‐Ai , Gladman Dafna D.
Published: June 2004

Summary.  Clustered progressive chronic disease processes arise when interest lies in modeling damage in paired organ systems (e.g., kidneys, eyes), in diseases manifest in different organ systems, or in systemic conditions for which damage may occur in several locations of the body. Multistate Markov models have considerable appeal for modeling damage in such settings, particularly when patients are only under intermittent observation. Generalizations are necessary, however, to deal with the fact that processes within subjects may not be independent. We describe a conditional Markov model in which the clustering in processes within subjects is addressed by the use of multiplicative random effects for each transition intensity. The random effects for the different transition intensities may be correlated within subjects, but are assumed to be independent for different subjects. We apply the mixed Markov model to a motivating data set of patients with psoriatic arthritis, and characterize the progressive course of damage in joints of the hand. A generalization to accommodate a subpopulation of “stayers” and extensions which facilitate regression are indicated and illustrated.
Assessing the Goodness‐of‐Fit of Hidden Markov Models
Authors: MacKay Altman Rachel
Published: June 2004

Summary.  In this article, we propose a graphical technique for assessing the goodness‐of‐fit of a stationary hidden Markov model (HMM). We show that plots of the estimated distribution against the empirical distribution detect lack of fit with high probability for large sample sizes. By considering plots of the univariate and multidimensional distributions, we are able to examine the fit of both the assumed marginal distribution and the correlation structure of the observed data. We provide general conditions for the convergence of the empirical distribution to the true distribution, and demonstrate that these conditions hold for a wide variety of time‐series models. Thus, our method allows us to compare not only the fit of different HMMs, but also that of other models as well. We illustrate our technique using a multiple sclerosis data set.
A Population Pharmacokinetic Model with Time‐Dependent Covariates Measured with Errors
Authors: Li Lang , Lin Xihong , Brown Morton B. , Gupta Suneel , Lee Kyung‐Hoon
Published: June 2004

Summary.  We propose a population pharmacokinetic (PK) model with time‐dependent covariates measured with errors. This model is used to model S‐oxybutynin's kinetics following an oral administration of Ditropan, and allows the distribution rate to depend on time‐dependent covariates blood pressure and heart rate, which are measured with errors. We propose two two‐step estimation methods: the second‐order two‐step method with numerical solutions of differential equations (2orderND), and the second‐order two‐step method with closed form approximate solutions of differential equations (2orderAD). The proposed methods are computationally easy and require fitting a linear mixed model at the first step and a nonlinear mixed model at the second step. We apply the proposed methods to the analysis of the Ditropan data, and evaluate their performance using a simulation study. Our results show that the 2orderND method performs well, while the 2orderAD method can yield PK parameter estimators that are subject to considerable biases.
Adaptive Two‐Stage Designs for Single‐Arm Phase IIA Cancer Clinical Trials
Authors: Lin Yong , Shih Weichung J.
Published: June 2004

Summary.  The main purpose of a phase IIA trial of a new anticancer therapy is to determine whether the therapy has sufficient promise against a specific type of tumor to warrant its further development. The therapy will be rejected for further investigation if the true response rate is less than some uninteresting level and the test of hypothesis is powered at a specific target response rate. Two‐stage designs are commonly used for this situation. However, in many situations investigators often express concern about uncertainty in targeting the alternative hypothesis to study power at the planning stage. In this article, motivated by a real example, we propose a strategy for adaptive two‐stage designs that will use the information at the first stage of the study to either reject the therapy or continue testing with either an optimistic or a skeptic target response rate, while the type I error rate is controlled. We also introduce new optimal criteria to reduce the expected total sample size.
Circular Distributions Based on Nonnegative Trigonometric Sums
Authors: Fernández‐Durán J. J.
Published: June 2004

Summary.  A new family of distributions for circular random variables is proposed. It is based on nonnegative trigonometric sums and can be used to model data sets which present skewness and/or multimodality. In this family of distributions, the trigonometric moments are easily expressed in terms of the parameters of the distribution. The proposed family is applied to two data sets, one related with the directions taken by ants and the other with the directions taken by turtles, to compare their goodness of fit versus common distributions used in the literature.
Biplots for Multifactorial Analysis of Distance
Authors: Krzanowski W. J.
Published: June 2004

Summary. Many data sets in practice fit a multivariate analysis of variance (MANOVA) structure, but do not accord with MANOVA assumptions for their analysis. One way forward is to calculate the matrix of dissimilarities or distances between every pair of individuals, and then to conduct an analysis of distance on the resulting data. Various metric scaling plots can be used to interpret the results of the analysis. However, developments to date of this approach have focused mainly on the individuals in the sample, and little attention has been paid to the assessment of influence of the original variables on the results. The present article attempts to rectify this omission. We discuss the inclusion of biplots on all forms of metric scaling representations in the analysis of distance. Exact biplots will often be nonlinear so we propose a simple linear approximation, and contrast it with other simple linear possibilities. An example from ecology illustrates the methodology.
General Ranked Set Sampling with Cost Considerations
Authors: Wang You‐Gan , Chen Zehua , Liu Jianbin
Published: June 2004

Summary.  Nahhas, Wolfe, and Chen (2002, Biometrics58, 964–971) considered optimal set size for ranked set sampling (RSS) with fixed operational costs. This framework can be very useful in practice to determine whether RSS is beneficial and to obtain the optimal set size that minimizes the variance of the population estimator for a fixed total cost. In this article, we propose a scheme of general RSS in which more than one observation can be taken from each ranked set. This is shown to be more cost‐effective in some cases when the cost of ranking is not so small. We demonstrate using the example in Nahhas, Wolfe, and Chen (2002, Biometrics58, 964–971), by taking two or more observations from one set even with the optimal set size from the RSS design can be more beneficial.
Confidence Interval for Rate Ratio in a 2 × 2 Table with Structural Zero: An Application in Assessing False‐Negative Rate Ratio When Combining Two Diagnostic Tests
Authors: Tang Man‐Lai , Tang Nian‐Sheng , Carey Vincent J.
Published: June 2004

Summary.  In this article, we consider problems with correlated data that can be summarized in a 2 × 2 table with structural zero in one of the off‐diagonal cells. Data of this kind sometimes appear in infectious disease studies and two‐step procedure studies. Lui (1998, Biometrics54, 706–711) considered confidence interval estimation of rate ratio based on Fieller‐type, Wald‐type, and logarithmic transformation statistics. We reexamine the same problem under the context of confidence interval construction on false‐negative rate ratio in diagnostic performance when combining two diagnostic tests. We propose a score statistic for testing the null hypothesis of nonunity false‐negative rate ratio. Score test–based confidence interval construction for false‐negative rate ratio will also be discussed. Simulation studies are conducted to compare the performance of the new derived score test statistic and existing statistics for small to moderate sample sizes. In terms of confidence interval construction, our asymptotic score test–based confidence interval estimator possesses significantly shorter expected width with coverage probability being close to the anticipated confidence level. In terms of hypothesis testing, our asymptotic score test procedure has actual type I error rate close to the pre‐assigned nominal level. We illustrate our methodologies with real examples from a clinical laboratory study and a cancer study.
The Treatment of Nonepileptic Seizures: Historical Perspectives and Future Directions
Authors: LaFrance W. Curt , Devinsky Orrin
Published: June 2004

Summary:  Nonepileptic seizures (NES) are neuropsychiatric disorders presenting with a combination of neurologic signs and underlying psychological conflicts. For more than a century, the medical community has accumulated data and insights about the phenomenology, epidemiology, risks, comorbidities, and prognosis of NES. However, we have not progressed much beyond anecdotal reports of treatments for NES, and no randomized, controlled trials of treatment for the disorder have been conducted. We review the diagnosis and treatment of NES and suggest directions for future research in these areas.
Recognition of the Various Expressions of Anxiety, Psychosis, and Aggression in Epilepsy
Authors: Kanner Andres M.
Published: June 2004

Summary: Anxiety, psychosis, and aggressive behavior are among the frequent comorbid psychiatric disorders identified in patients with epilepsy. Often the clinical manifestations of these disorders vary according to their temporal relation relative to seizure occurrence. Thus, postictal symptoms of anxiety or psychosis differ in severity, duration, and response to treatment with interictal symptomatology. Psychiatric symptomatology in epilepsy can appear concurrently with the seizure disorder and improve or remit on the abolition of epileptic activity. We refer to these as paraictal psychiatric phenomena. Such is the case of aggressive disturbances associated with gelastic seizures caused by hypothalamic hamartomas. In this article, three case studies are presented to illustrate the importance of distinguishing psychiatric symptoms of anxiety, psychosis, and aggression, with respect to their temporal relation with seizure occurrence.