Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Temporal and spatial responses of Chironomidae (Diptera) and other benthic invertebrates to urban stormwater runoff

In a longitudinal study of two streams whose lower reaches received unattenuated urban stormwater runoff, physical disturbance by stormflow was less important than the persistant unidentified chemical impacts of urban stormwater in limiting the distribution of Chironomidae, and Ephemeroptera, Trichoptera and Plecoptera (EPT). A hierarchical spatial analysis showed that chironomid density did not decrease from rural to urban stream reaches. Instead, the taxonomic composition of chironomid assemblages was significantly altered in urban versus rural reaches; chironomid assemblages in urban reaches exhibited higher average pollution tolerance scores. In contrast, the density of EPT was significantly lower in urban reaches. Despite higher values of stormflow tractive force in urban reaches, streambed stability tended to be greater in urban reaches. Modeling of temporal variation in chironomid density showed similar patterns in both rural and urban reaches: chironomid density had a unimodal relationship to rainfall index (RI), with highest densities at intermediate values of RI. Models of EPT density over time in rural reaches showed no significant relation to RI, and temporal variation in EPT density in urban reaches was not predictable. The abundance of fine particulate organic matter, including periphyton (FPOM), on cobbles was greater in urban reaches and showed a much greater degree of temporal variation than in rural reaches. In urban reaches, a negative relation between FPOM and RI indicated the importance of stormflow abrasion. Handling editor: K. Martens     

Comparative genomics of emerging human ehrlichiosis agents

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.     

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.