Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.     

Altering the balance between bacterial production and protistan bacterivory triggers shifts in freshwater bacterial community composition

Bacterivorous protists are known to induce changes in bacterial community composition (BCC). We hypothesized that changes in BCC could be related quantitatively to a measure of grazing: the ratio of bacterial mortality to growth rate. To test this hypothesis, we analyzed time-course changes in BCC, protistan grazing rate, and bacterial production from 3 in situ studies conducted in a freshwater reservoir and three laboratory studies. In the field experiments, samples were manipulated to yield different levels of protistan bacterivory and incubated in dialysis bags. Laboratory investigations were continuous cultivation studies in which different bacterivorous protists were added to bacterial communities. BCC was assessed using 4–6 different rRNA-targeted oligonucleotide probes for community analysis. Change in BCC (Δ BCC) was estimated as the sum of changes in the proportions of the two phylogenetic groups that showed the largest shifts. Analysis of a set of 22 estimates of shifts in the ratio of grazing to production rate over periods of 48–72 h and Δ BCC showed that Δ BCC was positively and tightly correlated (r ² = 0.784) with shifts in the ratio of grazing mortality to cell production. While the nature of a shift in BCC is unpredictable, the magnitude of the change can be related to changes in the balance between bacterial production and protistan grazing. This revised version was published online in August 2006 with corrections to the Cover Date.     

Building a hair: tip growth in Arabidopsis thaliana root hairs

The Arabidopsis thaliana root hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip of Arabidopsis root hairs has been identified by patch-clamp measurements. Actin has been visualized in vivo in Arabidopsis root hairs by using a green-fluorescent-protein-talin reporter and shown to form a dense mesh in the apex of the growing tip. The kojak gene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role for LRX1, a leucine-rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.     

Magnetic field design for selecting and aligning immunomagnetic labeled cells

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.     

Cell analysis system based on compact disk technology

BACKGROUND: A cell analysis system was developed to enumerate and differentiate magnetically aligned cells selected from whole blood. The cellular information extracted is similar to the readout of musical information from a compact disk (CD). Here we describe the optical design and data processing of the system. The performance of the system is demonstrated using fluorescent-labeled cells and beads. Materials and Methods System performance was demonstrated with 6-microm polystyrene beads labeled with magnetic nanoparticles and allophycocyanin (APC) and immunomagnetically aligned leukocytes, fluorescently labeled with Oxazine750 and CD4-APC, CD8-Cy5.5, and CD14-APC/Cy7 in whole blood. RESULTS: The sensitivity of the system was demonstrated using APC-labeled beads. With this system, beads containing 333 APC molecules could easily be resolved from the background. This level of sensitivity was not achievable with a commercial flow cytometer. A maximum of 20,000 immunomagnetically labeled cells could be aligned and analyzed in between 0.6 m of Ni lines, distributed over a surface area of 18 mm(2) and extracted from a blood volume that depended on the height of the chamber. The utility of the system was demonstrated by performing a three-color CD4-CD8-CD14 assay. CONCLUSIONS: We built a cell analysis system based on immunomagnetic cell selection and alignment and analysis of fluorescent signals employing CD-technology that is as good or better than current commercial analyzers. The cell analysis can be performed in whole blood or any other type of cell suspension without extensive sample preparation.     

Simulations of membranes and other interfacial systems using P2(1) and Pc periodic boundary conditions

We demonstrate the ease and utility of simulating heterogeneous interfacial systems with P2(1) and Pc periodic boundary conditions which allow, for example, lipids in a membrane to switch leaflets. In preliminary tests, P2(1) was shown to yield equivalent results to P1 in simulations of bulk water, a water/vacuum interface, and pure DPPC bilayers with an equal number of lipids per leaflet; equivalence of Pc and P1 was also demonstrated for the former two systems. P2(1) was further tested in simulations involving the spreading of an octane film on water, and equilibration of a DPPC bilayer from an initial condition containing different numbers of lipids in the two leaflets. Lastly, a simulation in P2(1) of a DOPC/melittin membrane showed significant passage of lipids to the melittin-containing leaflet from the initial distribution, and lends insight into the condensation of lipids by melittin.     

Experience-dependent modulation of tonotopic neural responses in human auditory cortex.

Experience-dependent plasticity of receptive fields in the auditory cortex has been demonstrated by electrophysiological experiments in animals. In the present study we used PET neuroimaging to measure regional brain activity in volunteer human subjects during discriminatory classical conditioning of high (8000 Hz) or low (200 Hz) frequency tones by an aversive 100 dB white noise burst. Conditioning-related, frequency-specific modulation of tonotopic neural responses in the auditory cortex was observed. The modulated regions of the auditory cortex positively covaried with activity in the amygdala, basal forebrain and orbitofrontal cortex, and showed context-specific functional interactions with the medial geniculate nucleus. These results accord with animal single-unit data and support neurobiological models of auditory conditioning and value-dependent neural selection.     

Proactive and Reactive Response Inhibition across the Lifespan

One expression of executive control involves proactive preparation for future events, and this contrasts with stimulus driven reactive control exerted in response to events. Here we describe findings from a response inhibition task, delivered using a smartphone-based platform, that allowed us to index proactive and reactive inhibitory self-control in a large community sample (n = 12,496). Change in stop-signal reaction time (SSRT) when participants are provided with advance information about an upcoming trial, compared to when they are not, provides a measure of proactive control while SSRT in the absence of advance information provides a measure of reactive control. Both forms of control rely on overlapping frontostriatal pathways known to deteriorate in healthy aging, an age-related decline that occurs at an accelerated rate in men compared to women. Here we ask whether these patterns of age-related decline are reflected in similar changes in proactive and reactive inhibitory control across the lifespan. As predicted, we observed a decline in reactive control with natural aging, with a greater rate of decline in men compared to women (~10 ms versus ~8 ms per decade of adult life). Surprisingly, the benefit of preparation, i.e. proactive control, did not change over the lifespan and women showed superior proactive control at all ages compared to men. Our results suggest that reactive and proactive inhibitory control partially rely on distinct neural substrates that are differentially sensitive to age-related change.