Bioeconomic synergy between tactics for insect eradication in the presence of Allee effects

Preventing the establishment of invading pest species can be beneficial with respect to averting future environmental and economic impacts and also in preventing the accumulation of control costs. Allee effects play an important role in the dynamics of newly established, low-density populations by driving small populations into self-extinction, making Allee effects critical in influencing outcomes of eradication efforts. We consider interactions between management tactics in the presence of Allee effects to determine cost-effective and time-efficient combinations to achieve eradication by developing a model that considers pesticide application, predator augmentation and mating disruption as control tactics, using the gypsy moth as a case study. Our findings indicate that given a range of constant expenditure levels, applying moderate levels of pesticides in conjunction with mating disruption increases the Allee threshold which simultaneously substantially decreases the time to eradication relative to either tactic alone. In contrast, increasing predation in conjunction with other tactics requires larger economic expenditures to achieve similar outcomes for the use of pesticide application or mating disruption alone. These results demonstrate the beneficial synergy that may arise from nonlinearities associated with the simultaneous application of multiple eradication tactics and offer new prospects for preventing the establishment of damaging non-native species.     

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.     

The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells

Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.     

Second generation 2-pyridyl biphenyl amide inhibitors of the hedgehog pathway

Potent and efficacious inhibitors of the hedgehog pathway for the treatment of cancer have been prepared using the 2-pyridyl biphenyl amide scaffold common to the clinical lead GDC-0449. Analogs with polar groups in the para-position of the aryl amide ring optimized potency, had minimal CYP inhibition, and possessed good exposure in rats. Compounds 9d and 14f potently inhibited hedgehog signaling as measured by Gli1 mRNA and were found to be equivalent or more potent than GDC-0449, respectively, when studied in a Ptch^+/? medulloblastoma allograft model, that is, highly dependent on hedgehog signaling.     

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.     

Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.     

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Phylum- and class-specific PCR primers for general microbial community analysis

Amplification of a particular DNA fragment from a mixture of organisms by PCR is a common first step in methods of examining microbial community structure. The use of group-specific primers in community DNA profiling applications can provide enhanced sensitivity and phylogenetic detail compared to domain-specific primers. Other uses for group-specific primers include quantitative PCR and library screening. The purpose of the present study was to develop several primer sets targeting commonly occurring and important groups. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes and for parts of both the 18S ribosomal sequence and the internal transcribed spacer region of Basidiomycota were examined. Primers were tested by comparison to sequences in the ARB 2003 database, and chosen primers were further tested by cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to be placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.     

A 6.9-Mb high-resolution BAC/PAC contig of human 4p15.3-p16.1, a candidate region for bipolar affective disorder

Bipolar affective disorder (BPAD) is a complex disease with a significant genetic component and a population lifetime risk of 1%. Our previous work identified a region of human chromosome 4p that showed significant linkage to BPAD in a large pedigree. Here, we report the construction of an accurate, high-resolution physical map of 6.9 Mb of human chromosome 4p15.3-p16.1, which includes an 11-cM (5.8 Mb) critical region for BPAD. The map consists of 460 PAC and BAC clones ordered by a combination of STS content analysis and restriction fragment fingerprinting, with a single approximately 300-kb gap remaining. A total of 289 new and existing markers from a wide range of sources have been localized on the contig, giving an average marker resolution of 1 marker/23 kb. The STSs include 57 ESTs, 9 of which represent known genes. This contig is an essential preliminary to the identification of candidate genes that predispose to bipolar affective disorder, to the completion of the sequence of the region, and to the development of a high-density SNP map.