A simulated heat wave-but not herbicide exposure-alters resource investment strategy in an insect.

Animals are increasingly exposed to potential stressors related to environmental change, and multiple stressors may alter the dynamics by which animals acquire resources and invest those resources into important life-history traits. Stress may lead to the prioritization of current reproduction to maximize lifetime reproduction (i.e., terminal investment [TI]) or, in contrast, prioritize somatic investment over current reproduction to facilitate future reproductive opportunities (i.e., reproductive restraint [RR]). Tests of the TI and RR hypotheses typically use immune challenges as stressors, and have not been explicitly tested in the context of environmental change even though warming influences resource allocation patterns across taxa. Further, the multiple-stressor framework has been a useful construct to clarify the costs of complex environmental shifts to animals, but it has not been leveraged to understand such effects on investment strategy. Thus, we tested the TI and RR hypotheses by manipulating widespread features of environmental change-glyphosate-based herbicide (GBH; Roundup®) exposure and a simulated heat wave-in the variable field cricket (Gryllus lineaticeps). A simulated heat wave affected the life-history tradeoff between investment into reproduction and soma. Specifically, heat wave prioritized investment into ovary mass over non-reproductive tissue, even after accounting for food consumption, in support of the TI hypothesis. In contrast, GBH exposure did not affect any measured trait, and crickets did not discriminate between tap water and GBH solution during drinking. Therefore, some-but not all-aspects of environmental change may alter resource investment strategies in animals. We encourage continued integration of the multiple-stressor framework and life-history theory to better understand how animals respond to their rapidly changing environments.

Pesticides in a warmer world: Effects of glyphosate and warming across insect life stages.

Glyphosate (GLY) is a broad-spectrum herbicide that is the most commonly applied pesticide in terrestrial ecosystems in the U.S. and, potentially, worldwide. However, the combined effects of warming associated with climate change and exposure to GLY and GLY-based formulations (GBFs) on terrestrial animals are poorly understood. Animals progress through several life stages (e.g., embryonic, larval, and juvenile stages) that may exhibit different sensitivities to stressors. Therefore, we factorially manipulated temperature and GLY/GBF exposure in the variable field cricket (Gryllus lineaticeps) during two life stages-nymphal development and adulthood-and examined key animal traits, such as developmental rate, body size, food consumption, reproductive investment, and lifespan. A thermal environment simulating future climate warming obligated several costs to fitness-related traits. For example, warming experienced during nymphal development reduced survival, adult body mass and size, and investment into flight capacity and reproduction. Warming experienced by adults reduced lifespan and growth rate. Alternatively, the effects of GBF exposure were more subtle, often context-dependent (e.g., effects were only detected in one sex or temperature regime), and were stronger during adult exposure relative to exposure during development. There was evidence of additive costs of warming and GBF exposure to rates of feeding and growth in adults. Yet, the negative effect of GBF exposure to adult lifespan did not occur in warming conditions, suggesting that ongoing climate change may obscure some of the costs of GBFs to non-target organisms. The effects of GLY alone (i.e., in the absence of proprietary surfactants found in commercial formulations) were non-existent. Animals will be increasingly exposed to warming and GBFs, and our results indicate that GBF exposure and warming can entail additive costs for an animal taxon (insects) that plays critical roles in terrestrial ecosystems.

Technical innovation changes standard radiographic protocols in veterinary medicine: is it necessary to obtain two dorsoproximal-palmarodistal oblique views of the equine foot when using computerised radiography systems?

Since the 1950s, veterinary practitioners have included two separate dorsoproximal-palmarodistal oblique (DPr-PaDiO) radiographs as part of a standard series of the equine foot. One image is obtained to visualise the distal phalanx and the other to visualise the navicular bone. However, rapid development of computed radiography and digital radiography and their post-processing capabilities could mean that this practice is no longer required. The aim of this study was to determine differences in perceived image quality between DPr-PaDiO radiographs that were acquired with a computerised radiography system with exposures, centring and collimation recommended for the navicular bone versus images acquired for the distal phalanx but were subsequently manipulated post-acquisition to highlight the navicular bone. Thirty images were presented to four clinicians for quality assessment and graded using a 1-3 scale (1=textbook quality, 2=diagnostic quality, 3=non-diagnostic image). No significant difference in diagnostic quality was found between the original navicular bone images and the manipulated distal phalanx images. This finding suggests that a single DPr-PaDiO image of the distal phalanx is sufficient for an equine foot radiographic series, with appropriate post-processing and manipulation. This change in protocol will result in reduced radiographic study time and decreased patient/personnel radiation exposure.

Analysis of a band 7/MEC-2 family gene of Porphyromonas gingivalis.

In vivo-induced antigen technology has previously been used to identify 115 genes induced in Porphyromonas gingivalis W83 during human infection. The aim of this study was to determine if one of these genes, PG1334, was important for the virulence of P. gingivalis. Analysis of plaque samples from persons with periodontitis revealed that PG1334 was expressed in 88.0% of diseased sites, compared with 42.1% of healthy sites, even though P. gingivalis was detected in equal numbers from both sites. A mutant of PG1334 was found to adhere to and to invade better than the parent strain, but did not persist as well in human coronary artery endothelial cells. Additionally, the mutant did not persist as well in a mouse abscess model. This gene appears to be important for the virulence of P. gingivalis, both in vivo and in vitro.

Phase II study of gemcitabine in children with relapsed acute lymphoblastic leukemia or acute myelogenous leukemia (ADVL0022): a Children's Oncology Group Report.

BACKGROUND: To determine the response rate and toxicity to gemcitabine administered as 10 mg/m2/min x 360 min weekly for 3 weeks in children with relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML). Gemcitabine is a deoxycytidine analog that inhibits DNA synthesis and repair and has a broad spectrum of antitumor activity. PROCEDURE: From April 2001 to April 2003, 23 male and 9 female eligible patients were recruited for the Children's Oncology Group (COG) phase II study of Gemcitabine (ADVL0022). RESULTS: One of 20 evaluable patients with ALL and none of 10 evaluable patients with AML had complete responses to gemcitabine; there were no partial responses. Grade 3 or 4 hematologic toxicity and liver toxicity were common during therapy. Only one patient was alive 1 year after entry. The estimated 1-year overall survival probability for the 32 patients was 4% (SE = 3%). CONCLUSIONS: Gemcitabine at the dose and schedule in this trial was not effective for children with relapsed AML or ALL.

The influence of rainfall on the incidence of microbial faecal indicators and the dominant sources of faecal pollution in a Florida river.

AIMS: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. METHODS AND RESULTS: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. CONCLUSIONS: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources.

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.

Disruption of the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute lymphoblastic leukemias with t(5;14)(q34;q11).

The t(5;14)(q33-34;q11) translocation constitutes a recurrent rearrangement in acute lymphoblastic leukemia involving the T cell receptor (TCR) delta locus on chromosome 14. Breakpoint sequences of the derivative chromosome 5 were isolated by application of a ligation-mediated PCR technique using TCR delta-specific primers to amplify genomic DNA from the leukemic cells of a patient with t(5;14). Through exon trap analysis, we identified various putative exons of the chromosome 5 target gene of the translocation; compilation of sequence information of trapped exons and available expressed sequence tags (ESTs) from the GenBank database allowed us to assemble 1.2 kb of the cDNA. Full-length cDNAs were isolated from a human testis cDNA library and sequence analysis predicted a putative Ran binding protein, a novel member of the importin-beta superfamily of nuclear transport receptors, called RanBP17. The t(5;14) breakpoint maps to the 3' coding region of the gene. The breakpoint of a second t(5;14) positive patient was mapped about 8 kb downstream of the most 3' RanBP17 exon and 2 kb upstream of the first exon of the orphan homeobox gene, Hox11L2. In both cases TCR delta enhancer sequences are juxtaposed downstream of the truncated or intact RanBP17 gene, respectively on the derivative chromosome.

Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States.

Vancomycin-resistant Enterococcus spp. (VRE) were isolated from sewage and chicken feces but not from other animal fecal sources (dog, cow, and pig) or from surface waters tested. VRE from hospital wastewater were resistant to > or =20 microg of vancomycin/ml and possessed the vanA gene. VRE from residential wastewater and chicken feces were resistant to 3 to 5 microg of vancomycin/ml and possessed the vanC gene.

Imaging of metastatic breast cancer: distribution and radiological assessment at presentation.

This prospective study was performed to document the distribution of sites of disease in breast cancer patients with newly diagnosed metastatic disease, and to identify those with assessable or measurable disease by International Union Against Cancer (UICC) criteria. Data were collected on a consecutive series of 100 patients presenting with metastatic breast cancer. Imaging findings recorded included whether patients had assessable or measurable disease and which potentially assessable sites were rendered unassessable by radiotherapy. Radiologically diagnosable complications were recorded. Skeletal metastases comprised the majority, with 67 patients having skeletal involvement, although of these only 33 (49%) had assessable disease and 24 (36%) measurable disease. Sixteen (24%) patients had radiographically occult metastases. Liver ultrasound examination showed metastatic disease in 32 patients, of whom 28 (88%) had measurable lesions and 12% diffuse disease. Chest radiographs demonstrated metastatic disease in 42 patients, with assessable disease in 39 (93%) and measurable disease in 18 (43%). In total, 80 patients had radiologically assessable disease, with five rendered unassessable by the administration of radiotherapy to the only assessable site. Therefore, of the 100 patients, 75% (95% confidence interval (CI) 65-83) had radiologically assessable disease, with 55% (95% 45-65 CI) having measurable lesions by UICC criteria.

Accessibility and activity of the promoter for a dioxin-inducible ecto-ATPase gene.

We have analyzed the core promoter for a dioxin-inducible ecto-ATPase gene in mouse hepatoma cells. The transcriptional initiation site maps to a region that contains neither a TATA sequence nor a consensus initiator sequence nor a downstream promoter element. The core promoter has constitutive activity that does not require either the aromatic hydrocarbon receptor or its heterodimerization partner Arnt. Two GC-rich regions contribute approximately equally to the constitutive activity. Proteins constitutively occupy the GC-rich regions in chromatin. The promoter assumes a non-nucleosomal configuration in its native chromosomal setting in both uninduced and dioxin-induced cells. Our findings imply that the GC-rich regions together with their cognate binding proteins carry out core promoter functions for the ecto-ATPase gene. The promoter is constitutively accessible in situ, and chromatin structure is not a limiting factor for dioxin-inducible ecto-ATPase transcription in intact cells.

Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression.

One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.

Dioxin-inducible transactivation in a chromosomal setting. Analysis of the acidic domain of the Ah receptor.

We analyzed the transactivation function of the acidic segment of the Ah receptor (amino acids 515-583) by reconstituting AhR-defective mouse hepatoma cells with mutants. Our data reveal that both hydrophobic and acidic residues are important for transactivation and that these residues are clustered in two regions of the acidic segment of AhR. Both regions are crucial for function, because disruption of either one substantially impairs transactivation of the chromosomal CYP1A1 target gene. Neither region contains an amino acid motif that resembles those reported for other acidic activation domains. Furthermore, proline substitutions in both regions do not impair transactivation in vivo, a finding that implies that alpha-helix formation is not required for function.

Conserved worldwide linkage disequilibrium in the human factor XI gene.

We have identified, in four diverse human populations, five common single-nucleotide polymorphisms (SNPs) in the coding region of the gene for the blood coagulation protease factor XI. Each SNP has an allele frequency >5% in at least one population. Three of the SNPs (C472T, A844G, and T1234C), spread out over approximately 10 kb of genomic DNA, are in marked linkage disequilibrium (LD) with one another (P < 10(-4)). Interestingly, haplotypes associated with the linked SNPs are conserved across all populations studied, despite significantly different allele frequencies between populations. The presence of such common, widely dispersed haplotypes could complicate the interpretation of LD studies and emphasizes the need for a better understanding of general patterns of LD to facilitate identification of genes for common disorders.

Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in subtropical waters.

The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Survey and host fitness effects of red-cockaded woodpecker blood parasites and nest cavity arthropods.

Blood parasites and nest cavity arthropods associated with the red-cockaded woodpecker (Picoides borealis) were surveyed and the impact of blood-feeding arthropods on woodpecker fitness traits was assessed. Five woodpeckers (8%) were infected with unidentified microfilariae, and 1 woodpecker (2%) was infected with 2 species of haemoproteid (Haemoproteus velans and Haemoproteus borgesi). This is the first record of haemoproteids in this species and the first observation of H. borgesi in North America. We collected representatives of at least 6 families of mites and 12 families of primarily commensal insects from woodpecker cavities. Only a few specimens of blood-feeding insects were recovered. The mite Androlaelaps casalis was the most common hematophagous arthropod (prevalence = 76%, mean density = 51+/-7 mites/cavity). The number of A. casalis mites increased with cavity age but there was no association between the number of mites and the number of woodpecker eggs laid or the number of hatchlings or fledglings. In conclusion, the prevalence of blood parasites in the red-cockaded woodpecker is low, woodpecker cavities are not heavily infested with blood-feeding insects, and there is no evidence that A. casalis mites affect woodpecker fitness.