Gene-gene interaction between FGF20 and MAOB in Parkinson disease.

The fibroblast growth factor 20 (FGF20) and monoamine oxidase B (MAOB) genes are associated with Parkinson Disease (PD) risk and both are in the dopamine bio-pathway. Therefore, we investigated the joint effect between polymorphisms in the FGF20 and MAOB genes for evidence of interaction contributing to PD risk. Fourteen polymorphisms (eight for FGF20, six for MAOB) were genotyped in 736 families and analyzed using conditional logistic regression (CLR). Significant two-locus interactions were found in females between the polymorphisms rs1721100 of FGF20 and rs1799836 of MAOB, and between the polymorphisms rs1721082 of FGF20 and rs1799836 of MAOB. The risk alleles for each SNP identified from CLR, rs1721100 C, rs1721082 T and rs1799836 A, are consistent with previous reports. Using indicator variables for the SNP genotypes, rs1721100 GC with rs1799836 AA showed significant interaction (P = 0.021), compared with the reference group rs1721100 GG with rs1799836 GG. Using an allele-dose model for the risk alleles, rs1721100 and rs1799836 showed significant interaction (P = 0.019). We found similar interaction results between rs1721082 and rs1799836. In conclusion, variants in FGF20 and MAOB show evidence of statistical interactions, which emphasizes the importance of considering them jointly in genetic analysis of PD and illustrates potential patterns of biological interaction contributing to PD risk.

Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.

Pathogens that infect and/or are transmitted by mosquitoes typically are exposed to the body cavity, and to haemocytes circulating therein, during development or dissemination. Aedes aegypti haemocytes produce a range of immune response-related gene products, and an endpoint response of phagocytosis and/or melanization that is temporally and structurally distinct for the invading pathogen. Expressed sequence tags were generated from haemocyte libraries and then used to design oligonucleotide microarrays. Arrays were screened with haemocyte material collected 1-, 8- and 24-h post-inoculation with Escherichia coli or Micrococcus luteus bacteria. Data from these studies support the discovery of novel immune response-activated genes, provide an expanded understanding of antimicrobial peptide biology and highlight the coordination of immune factors that leads to an endpoint response.

Investigation of the PARK10 gene in Parkinson disease.

Two recent association mapping studies in Parkinson disease (PD) reported three candidate genes for the PARK10 locus: EIF2B3 as a modifier of age-at-onset of PD (min P= 0.0004) and HIVEP3 as a PD risk gene (P < or = 0.006) (Oliveira et al. 2005); and LOC200008 (CDCP2) identified by the whole genome association (WGA) study of PD of Maraganore et al. (2005). However, evaluation of the on-line PD WGA results revealed two significant SNPs in HIVEP3 in the two datasets, Tier 1 and Tier 2, used by Maraganore et al. (P < or = 0.008 for Tier 1 and P=0.03 for Tier 2 dataset). Here, we revisited both the HIVEP3 and CDCP2 loci by examining 47 SNPs, mostly tagging, in an expanded PD family dataset (293 multiplex and 467 singleton families). A discordant sibpair (DSP) dataset (one DSP per family), with similar data structure as the WGA Tier 1 dataset, was also tested. We confirmed our and other previous negative findings for CDCP2. However, five significant SNPs in HIVEP3 (min P=0.004) were observed, although the two significant HIVEP3 SNPs from the PD WGA study were not significant in our datasets. Even though the sets of significant HIVEP3 markers differ between studies, these findings strongly support HIVEP3 as a candidate for PARK10. Further testing of HIVEP3 by other groups is encouraged.

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli.

We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

Diffusion of mepivacaine between adjacent synovial structures in the horse. Part 1: forelimb foot and carpus.

This paper tests the hypothesis that the local analgesic agent mepivacaine diffuses between adjacent equine synovial structures in the forelimb and with greater frequency than latex, gelatine dye or contrast media. We report the incidence of diffusion of mepivacaine between the distal interphalangeal joint (DIPJ) and navicular bursa (NB) of the forelimbs and between the intercarpal (IC) and radiocarpal (RC) joints of 31 fresh equine cadavers. The DIPJ of one forelimb and the NB of the contra lateral forelimb and the RC joint of one forelimb and the IC joint of the contra lateral forelimb were injected with mepivacaine. After flexion and extension of the joints, synovial fluid was obtained from the synovial structures adjacent to the injected synovial structures. The concentration of mepivacaine in these samples was determined using an enzyme linked immunosorbent assay. For samples obtained by dilution of synovial fluid, the concentration of mepivacaine was determined by comparing the concentrations of urea in the diluted synovial fluid and the concentration of serum urea. Mepivacaine diffused from the DIPJ to the NB or from the NB to the DIPJ in 25/25 (100%) limbs. Mepivacaine diffused from the IC to RC joints in 24/25 (96%) limbs and from the RC to IC joints in 21/25 (84%) limbs. It was detected at concentrations >0.3 mg/l in 9/25 (36%) of IC joints after RC joint injection and in 25/25 (100%) of the NB after DIPJ injection; at concentrations >100 mg/l in 2/25 (8%) of IC and RC joints and 12/25 (48%) of NB following DIPJ injection; and at concentrations >300 mg/l in 1/25 (4%) in the IC joints following RC joint injection and in 11/25 (44%) of DIPJ following NB injection. The results show greater diffusion of mepivacaine between adjacent synovial structures than assumed from previous anatomical, latex injection and contrast arthrographic studies. This study showed that commonly performed intrasynovial analgesic techniques in the forelimb of the horse are not as specific as previously reported.

Urea as a measure of dilution of equine synovial fluid.

This paper tests the hypothesis that serum and synovial urea concentrations are similar and that urea concentration can be used as an accurate marker for synovial fluid dilution in normal equine joints. Serum and synovial fluid urea concentrations were compared in 42 horses and were equivalent for individual horses (P<0.0001). Mean +/- s.e. serum concentration was 6.1+/-0.552 mmol/l and synovial concentration 6.0+/-0.459 mmol/l. The normal range for synovial urea concentration was determined as 2.5-7.7 mmol/l. The synovial urea concentration from different synovial structures in individual horses were compared and were equivalent (P = 0.002). Known dilutions of synovial fluid with saline were made. The actual and expected synovial urea concentrations were compared and were equivalent (P<0.001). An accurate method of calculating synovial fluid dilution has been determined.

Diffusion of mepivacaine between adjacent synovial structures in the horse. Part 2: tarsus and stifle.

This paper tests the hypothesis that the local analgesic agent mepivacaine diffuses between adjacent equine synovial structures in the hindlimb and with greater frequency than latex, gelatine dye or contrast media. We report the incidence of diffusion of mepivacaine between the tarsometatarsal, centrodistal and tarsocrural joints, and the 3 synovial compartments of the stifle in 33 fresh equine cadavers. The tarsometatarsal joint and one synovial compartment of the stifle in the left limb and the centrodistal joint and a different synovial compartment of the stifle in the right limbs were injected with mepivacaine. Following flexion and extension of the limb, synovial fluid was aspirated from the noninjected centrodistal and tarsometatarsal joints and the tarsocrural joints of the hock and the noninjected compartments of the stifle. Concentrations of mepivacaine in these samples were assayed using an enzyme linked immunosorbent assay. For samples obtained by dilution of synovial fluid the concentration of mepivacaine was determined by comparing the concentration of urea in the diluted synovial fluid and the concentrations of the serum urea. Mepivacaine was detected in 25/25 (100%) adjacent tarsometatarsal and centrodistal joints after diffusion in both directions, in 23/25 (92%) of tarsocrural joints after diffusion from tarsometatarsal joints and in 22/25 (88%) tarsocrural joints after diffusion from centrodistal joints in the hocks. Diffusion from the femoropatellar to medial and lateral femorotibial joints and between the medial and lateral femorotibial joints in both directions were 20/20 (100%). Diffusion from the lateral femorotibial to the femoropatellar joint was 18/20 (90%) and from the medial femorotibial to femoropatellar joints 17/20 (85%). Mepivacaine was detected at concentrations >0.3 mg/l in a proportion of samples ranging from 15/25 (60%) in the tarsocrural joint following tarsometatarsal joint injection to 18/20 (90%) in the lateral femorotibial joint after femoropatellar joint injection. At mepivacaine concentrations >100 mg/l, detection ranged from 3/20 (15%) in the lateral femorotibial joint from the medial femorotibial joint to 19/25 (76%) in the centrodistal joint from the tarsometatarsal joint. At mepivacaine concentrations >300 mg/l, detection ranged from 1/25 (4%) in the tarsocrural joint from the tarsometatarsal joint to 16/25 (64%) in the from centrodistal joint the tarsometatarsal joint. The results show greater diffusion of mepivacaine between these adjacent synovial structures than assumed from previous anatomical, latex injection and contrast arthrographic studies. Therefore, commonly performed intrasynovial local analgesic techniques in the hindlimb of the horse are not as specific as first thought.

Transmissibility of adenovirus-induced adiposity in a chicken model.

BACKGROUND: We previously reported that human adenovirus Ad-36 induces adiposity and paradoxically lower levels of serum cholesterol (CHOL) and triglycerides (TG) in animals. OBJECTIVE: To evaluate the transmissibility of Ad-36 and Ad-36 induced adiposity using a chicken model. DESIGN: Experiment 1--four chickens were housed (two per cage) and one from each cage was inoculated with Ad-36. Duration of presence of Ad-36 DNA in the blood of all chickens was monitored. Experiment 2--two groups of chickens were intranasally inoculated with Ad-36 (infected donors, I-D) or media (control donors, C-D). Blood drawn 36 h later from I-D and C-D groups was inoculated into wing veins of recipient chickens (infected receivers, I-R, and control receivers, C-R, respectively). On sacrifice, 5 weeks post-inoculation, blood was drawn, body weight noted and visceral fat was separated and weighed. RESULTS: Experiment 1--Ad-36 DNA appeared in the blood of the inoculated chickens and that of uninoculated chickens (cage mates) within 12 h of inoculation and the viral DNA persisted up to 25 days in the blood. Experiment 2--compared with C-D, visceral and total body fat were significantly greater and CHOL significantly lower for the I-D and I-R. TG were significantly lower for the I-D. Ad-36 was isolated from 12 out of 16 blood samples of the I-D that were used for inoculating I-R chickens. Ad-36 DNA was present in the blood and the adipose tissue of the I-D and I-R but not in the skeletal muscles of animals selected randomly for testing. CONCLUSION: As seen in experiment 1, Ad-36 infection can be transmitted horizontally from an infected chicken to another chicken sharing the cage. Additionally, experiment 2 demonstrated blood-borne transmission of Ad-36-induced adiposity in chickens. Transmissibility of Ad-36-induced adiposity in chicken model raises serious concerns about such a possibility in humans that needs further investigation.

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Increased adiposity in animals due to a human virus.

BACKGROUND: Four animal models of virus-induced obesity including adiposity induced by an avian adenovirus have been described previously. This is the first report of adiposity induced in animals by a human virus. OBJECTIVE: We investigated the adiposity promoting effect of a human adenovirus (Ad-36) in two different animal models. DESIGN: Due to the novel nature of the findings we replicated the experiments using a chicken model three times and a mammal model once. In four separate experiments, chickens and mice were inoculated with human adenovirus Ad-36. Weight matched groups inoculated with tissue culture media were used as non-infected controls in each experiment. Ad-36 inoculated and uninfected control groups were housed in separate rooms under biosafety level 2 or better containment. The first experiment included an additional weight matched group of chickens that was inoculated with CELO (chick embryo lethal orphan virus), an avian adenovirus. Food intakes and body weights were measured weekly. At the time of sacrifice blood was drawn and visceral fat was carefully separated and weighed. Total body fat was determined by chemical extraction of carcass fat. RESULTS: Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides. This syndrome was not seen in chickens inoculated with CELO virus. Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes. Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation. CONCLUSIONS: Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.

Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restriction sites.

A transposon-based method of introducing unique restriction sites was used for subdivision of the Escherichia coli genome into a contiguous series of large non-overlapping segments spanning 2.5Mb. The segments, sizes ranging from 150 to 250kb, were isolated from the chromosome using the inserted restriction sites and shotgun cloned into an M13 vector for DNA sequencing. These shotgun sizes proved easily manageable, allowing the genomic sequence of E. coli to be completed more efficiently and rapidly than was possible by previously available methods. The 9bp duplication generated during transposition was used as a tag for accurate splicing of the segments; no further sequence redundancy at the junction sites was needed. The system is applicable to larger genomes even if they are not already well-characterized. We present the technology for segment sequencing, results of applying this method to E. coli, and the sequences of the transposon cassettes.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis.

BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

Complete DNA sequence and detailed analysis of the Yersinia pestis KIM5 plasmid encoding murine toxin and capsular antigen.

Yersinia pestis, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the Y. pestis-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of Y. pestis KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in Y. pestis. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the parABS bacteriophage P1 and P7 system. Y. pestis pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.

Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7.

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic Escherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic E. coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E. coli strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.

The complete genome sequence of Escherichia coli K-12.

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

Familial resemblances in disgust sensitivity and animal phobias.

This paper reports the results of two studies, both of which suggest there are close intrafamilial resemblances between parental disgust sensitivity and various measures of offspring animal phobia. In Study 1, a multiple regression analysis identified parental disgust sensitivity as a primary predictor of offspring animal fear in general (as measured by the animal phobia sub-scale of the Fear Survey Schedule), and offspring spider fear in particular. Study 2 indicated that parental disgust sensitivity was only related to offsprings' fear of animals known to be associated with the disgust reaction. These intrafamilial resemblances are discussed in relation to genetic and cultural transmission of the disgust reaction.

Incidental teaching of mentally retarded students within a token system.

Six mentally retarded students were taught to name sight words during the token-exchange periods of a token-reinforcement system. Words appeared on 25% of the tokens, and a student was given two opportunities to name a word written on a token before the token could be exchanged. Sequential teaching of new sets of sight words via a multiple-baseline design was used to evaluate the procedure. Five of the 6 students acquired sight-word vocabularies. The data support the contention that token-exchange periods may be used for educational purposes.

Social reinforcement and the naturally occurring social responses of severely and profoundly retarded adolescents.

This study was designed to determine if the deficits in social behavior of retarded persons might be due, in part, to the failure of their environment to maintain that behavior. Using an ABAB design, we systematically ignored severely and profoundly retarded, institutionalized adolescents and gave them social reinforcement for appropriate social behavior. Social behavior decreased during nonreinforcement conditions and increased during reinforcement conditions. The data suggest that deficits in social behavior of retarded persons may be due to failure of their environment to maintain such behavior. Implications of these findings for staff behavior and the utility of the present procedure for increasing social behavior of institutionalized retarded persons were discussed.