Reagent decontamination to eliminate false-positives in Escherichia coli qPCR

The application of real-time quantitative PCR (qPCR) for the detection of low concentrations of Escherichia coli as well as universal 16S rDNA has been hindered by false-positives due to endogenous contamination of PCR reagents with E. coli and other bacterial DNA. We optimized a DNase I decontamination method to eliminate false-positives in a qPCR assay targeting the uidA gene in E. coli. In contrast to previous methods reported in the literature, our decontamination method did not cause PCR inhibition. We determined that residual DNase I activity was the cause of the inhibition in the previous methods, and eliminated it by ensuring complete inactivation prior to qPCR. DNase inactivation was accomplished by adding dithiothreitol (DTT) and then heating for 30 min at 80 degrees C. The optimized DNase method was compared to another decontamination method, ultrafiltration, and to untreated controls. We detected contamination in 85% of the untreated commercial PCR master mix samples at a level of about 10 copies per well (12.5 microL of master mix). Both decontamination methods could eliminate up to 100 copies of added contaminant DNA and did not cause PCR inhibition, resulting in a reduction of the detection limit to 10 copies per reaction well.     

Temporal patterns of microbial community structure in the Mid-Atlantic Bight

Although open ocean time-series sites have been areas of microbial research for years, relatively little is known about the population dynamics of bacterioplankton communities in the coastal ocean on kilometer spatial and seasonal temporal scales. To gain a better understanding of microbial community variability, monthly samples of bacterial biomass were collected in 1995-1996 along a 34-km transect near the Long-Term Ecosystem Observatory (LEO-15) off the New Jersey coast. Surface and bottom sampling was performed at seven stations along a transect line with depths ranging from 1 to 35 m (n=178). Microbial populations were fingerprinted using ribosomal 16S rRNA genes and terminal restriction fragment length polymorphism analysis. Results from cluster analysis revealed distinct temporal patterns among the bacterioplankton communities in the Mid-Atlantic Bight rather than grouping by sample location or depth. Principal components analysis models supported the temporal patterns. In addition, partial least squares regression modeling could not discern a significant correlation from traditional oceanographic physical and phytoplankton nutrient parameters on overall bacterial community variability patterns at LEO-15. These results suggest factors not traditionally measured during oceanographic studies are structuring coastal microbial communities.     

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.     

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.     

Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection microscopy. To determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement fourfold. We found that whole myosin II and myosin V both twirl actin with a relatively long (∼1 μm), left-handed pitch that is insensitive to myosin concentration, filament length, and filament velocity.     

Efficiency of histidine-associating compounds for blocking the alzheimer's Abeta channel activity and cytotoxicity

The opening of the Alzheimer's Aβ channel permits the flux of calcium into the cell, thus critically disturbing intracellular ion homeostasis. Peptide segments that include the characteristic histidine (His) diad, His¹³ and His¹⁴, efficiently block the Aβ channel activity, blocking Aβ cytotoxicity. We hypothesize that the vicinal His-His peptides coordinate with the rings of His in the mouth of the pore, thus blocking the flow of calcium ions through the channel, with consequent blocking of Aβ cytotoxicity. To test this hypothesis, we studied Aβ ion channel activity and cytotoxicity after the addition of compounds that are known to have His association capacity, such as Ni²⁺, imidazole, His, and a series of His-related compounds. All compounds were effective at blocking both Aβ channel and preventing Aβ cytotoxicity. The efficiency of protection of His-related compounds was correlated with the number of imidazole side chains in the blocker compounds. These data reinforce the premise that His residues within the Aβ channel sequence are in the pathway of ion flow. Additionally, the data confirm the contribution of the Aβ channel to the cytotoxicity of exogenous Aβ.     

Rapid evolution and the convergence of ecological and evolutionary time

Recent studies have documented rates of evolution of ecologically important phenotypes sufficiently fast that they have the potential to impact the outcome of ecological interactions while they are underway. Observations of this type go against accepted wisdom that ecological and evolutionary dynamics occur at very different time scales. While some authors have evaluated the rapidity of a measured evolutionary rate by comparing it to the overall distribution of measured evolutionary rates, we believe that ecologists are mainly interested in rapid evolution because of its potential to impinge on ecological processes. We therefore propose that rapid evolution be defined as a genetic change occurring rapidly enough to have a measurable impact on simultaneous ecological change. Using this definition we propose a framework for decomposing rates of ecological change into components driven by simultaneous evolutionary change and by change in a non‐evolutionary factor (e.g. density dependent population dynamics, abiotic environmental change). Evolution is judged to be rapid in this ecological context if its contribution to ecological change is large relative to the contribution of other factors. We provide a worked example of this approach based on a theoretical predator–prey interaction [ Abrams, P. & Matsuda, H. (1997) . Evolution, 51, 1740], and find that in this system the impact of prey evolution on predator per capita growth rate is 63% that of internal ecological dynamics. We then propose analytical methods for measuring these contributions in field situations, and apply them to two long‐term data sets for which suitable ecological and evolutionary data exist. For both data sets relatively high rates of evolutionary change have been found when measured as character change in standard deviations per generation (haldanes). For Darwin's finches evolving in response to fluctuating rainfall [ Grant, P.R. & Grant, B.R. (2002) . Science, 296, 707], we estimate that evolutionary change has been more rapid than ecological change by a factor of 2.2. For a population of freshwater copepods whose life history evolves in response to fluctuating fish predation [ Hairston, N.G. Jr & Dillon, T.A. (1990) . Evolution, 44, 1796], we find that evolutionary change has been about one quarter the rate of ecological change – less than in the finch example, but nevertheless substantial. These analyses support the view that in order to understand temporal dynamics in ecological processes it is critical to consider the extent to which the attributes of the system under investigation are simultaneously changing as a result of rapid evolution.