Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.     

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm², respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization. Received: 13 October 2000 / Accepted: 17 November 2000     

Aerial manoeuvrability in wingless gliding ants (Cephalotes atratus)

In contrast to the patagial membranes of gliding vertebrates, the aerodynamic surfaces used by falling wingless ants to direct their aerial descent are unknown. We conducted ablation experiments to assess the relative contributions of the hindlegs, midlegs and gaster to gliding success in workers of the Neotropical arboreal ant Cephalotes atratus (Hymenoptera: Formicidae). Removal of hindlegs significantly reduced the success rate of directed aerial descent as well as the glide index for successful flights. Removal of the gaster alone did not significantly alter performance relative to controls. Equilibrium glide angles during successful targeting to vertical columns were statistically equivalent between control ants and ants with either the gaster or the hindlegs removed. High-speed video recordings suggested possible use of bilaterally asymmetric motions of the hindlegs to effect body rotations about the vertical axis during targeting manoeuvre. Overall, the control of gliding flight was remarkably robust to dramatic anatomical perturbations, suggesting effective control mechanisms in the face of adverse initial conditions (e.g. falling upside down), variable targeting decisions and turbulent wind gusts during flight.     

Spatial Generalization in Operant Learning: Lessons from Professional Basketball

In operant learning, behaviors are reinforced or inhibited in response to the consequences of similar actions taken in the past. However, because in natural environments the “same” situation never recurs, it is essential for the learner to decide what “similar” is so that he can generalize from experience in one state of the world to future actions in different states of the world. The computational principles underlying this generalization are poorly understood, in particular because natural environments are typically too complex to study quantitatively. In this paper we study the principles underlying generalization in operant learning of professional basketball players. In particular, we utilize detailed information about the spatial organization of shot locations to study how players adapt their attacking strategy in real time according to recent events in the game. To quantify this learning, we study how a make \ miss from one location in the court affects the probabilities of shooting from different locations. We show that generalization is not a spatially-local process, nor is governed by the difficulty of the shot. Rather, to a first approximation, players use a simplified binary representation of the court into 2 pt and 3 pt zones. This result indicates that rather than using low-level features, generalization is determined by high-level cognitive processes that incorporate the abstract rules of the game.     

Determining an economic injury level for the persea mite,Oligonychus perseae,a new pest of avocado in Israel

The persea mite, Oligonychus perseae Tuttle, Baker & Abbatiello (Acari: Tetranychidae), a pest of avocado, was first discovered in Israel in the autumn of 2001. It has since spread to most avocado growing areas in Israel. To establish an economic injury level (EIL), based on the percentage of leaf area damaged (PLAD), we conducted an extensive field study. For three consecutive seasons we created distinct pest infestation levels on the Hass avocado cv., by applying acaricides (spirodiclofen and abamectin) at 50, 100, and 250 mites per leaf levels, along with non‐sprayed controls in a replicated block design. At harvest time we evaluated the level of leaf damage and fruit yields across treatments. In two out of the 3 years, trees sprayed at 50 and 100 mites per leaf levels had similar PLAD values, differing from trees treated at the 250 mites per leaf level and the non‐treated control, the latter pair also being similar. Over the 3 years, mean yield attained at the two higher infestation levels was reduced by 20% in comparison to the mean yields recorded for plots sprayed at the lower thresholds. Accordingly, we suggest that scouts adopt an action threshold (AT) of 50–100 mites per leaf. Future research is needed to refine this AT. Mean annual cumulative mite days (CMDs) of the two higher levels was ca. 13500 ± 700 per leaf. Using the linear regression equation PLAD = 0.0009CMDs + 2.42, describing leaf damage as a function of CMDs, we estimated an EIL of ca. 15 PLAD.     

Nutrients and water availability constrain the seasonality of vegetation activity in a Mediterranean ecosystem

Anthropogenic nitrogen (N) deposition and resulting differences in ecosystem N and phosphorus (P) ratios are expected to impact photosynthetic capacity, that is, maximum gross primary productivity (GPP_max). However, the interplay between N and P availability with other critical resources on seasonal dynamics of ecosystem productivity remains largely unknown. In a Mediterranean tree–grass ecosystem, we established three landscape‐level (24 ha) nutrient addition treatments: N addition (NT), N and P addition (NPT), and a control site (CT). We analyzed the response of ecosystem to altered nutrient stoichiometry using eddy covariance fluxes measurements, satellite observations, and digital repeat photography. A set of metrics, including phenological transition dates (PTDs; timing of green‐up and dry‐down), slopes during green‐up and dry‐down period, and seasonal amplitude, were extracted from time series of GPP_max and used to represent the seasonality of vegetation activity. The seasonal amplitude of GPP_max was higher for NT and NPT than CT, which was attributed to changes in structure and physiology induced by fertilization. PTDs were mainly driven by rainfall and exhibited no significant differences among treatments during the green‐up period. Yet, both fertilized sites senesced earlier during the dry‐down period (17–19 days), which was more pronounced in the NT due to larger evapotranspiration and water usage. Fertilization also resulted in a faster increase in GPP_max during the green‐up period and a sharper decline in GPP_max during the dry‐down period, with less prominent decline response in NPT. Overall, we demonstrated seasonality of vegetation activity was altered after fertilization and the importance of nutrient–water interaction in such water‐limited ecosystems. With the projected warming‐drying trend, the positive effects of N fertilization induced by N deposition on GPP_max may be counteracted by an earlier and faster dry‐down in particular in areas where the N:P ratio increases, with potential impact on the carbon cycle of water‐limited ecosystems.     

Novel Fluorescein Angiography-Based Computer-Aided Algorithm for Assessment of Retinal Vessel Permeability

Purpose

To present a novel method for quantitative assessment of retinal vessel permeability using a fluorescein angiography-based computer algorithm.

Methods

Twenty-one subjects (13 with diabetic retinopathy, 8 healthy volunteers) underwent fluorescein angiography (FA). Image pre-processing included removal of non-retinal and noisy images and registration to achieve spatial and temporal pixel-based analysis. Permeability was assessed for each pixel by computing intensity kinetics normalized to arterial values. A linear curve was fitted and the slope value was assigned, color-coded and displayed. The initial FA studies and the computed permeability maps were interpreted in a masked and randomized manner by three experienced ophthalmologists for statistical validation of diagnosis accuracy and efficacy.

Results

Permeability maps were successfully generated for all subjects. For healthy volunteers permeability values showed a normal distribution with a comparable range between subjects. Based on the mean cumulative histogram for the healthy population a threshold (99.5%) for pathological permeability was determined. Clear differences were found between patients and healthy subjects in the number and spatial distribution of pixels with pathological vascular leakage. The computed maps improved the discrimination between patients and healthy subjects, achieved sensitivity and specificity of 0.974 and 0.833 respectively, and significantly improved the consensus among raters for the localization of pathological regions.

Conclusion

The new algorithm allows quantification of retinal vessel permeability and provides objective, more sensitive and accurate evaluation than the present subjective clinical diagnosis. Future studies with a larger patients’ cohort and different retinal pathologies are awaited to further validate this new approach and its role in diagnosis and treatment follow-up. Successful evaluation of vasculature permeability may be used for the early diagnosis of brain microvascular pathology and potentially predict associated neurological sequelae. Finally, the algorithm could be implemented for intraoperative evaluation of micovascular integrity in other organs or during animal experiments.     

Efficient calculation of interval scores for DNA copy number data analysis.

DNA amplifications and deletions characterize cancer genome and are often related to disease evolution. Microarray-based techniques for measuring these DNA copy-number changes use fluorescence ratios at arrayed DNA elements (BACs, cDNA, or oligonucleotides) to provide signals at high resolution, in terms of genomic locations. These data are then further analyzed to map aberrations and boundaries and identify biologically significant structures. We develop a statistical framework that enables the casting of several DNA copy number data analysis questions as optimization problems over real-valued vectors of signals. The simplest form of the optimization problem seeks to maximize phi(I) = Sigmanu(i)/radical|I| over all subintervals I in the input vector. We present and prove a linear time approximation scheme for this problem, namely, a process with time complexity O (nepsilon(-2)) that outputs an interval for which phi(I) is at least Opt/alpha(epsilon), where Opt is the actual optimum and alpha(epsilon) --> 1 as epsilon --> 0. We further develop practical implementations that improve the performance of the naive quadratic approach by orders of magnitude. We discuss properties of optimal intervals and how they apply to the algorithm performance. We benchmark our algorithms on synthetic as well as publicly available DNA copy number data. We demonstrate the use of these methods for identifying aberrations in single samples as well as common alterations in fixed sets and subsets of breast cancer samples.