A method for the enumeration of bacterial adhesion to epithelial cells using image analysis

Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.     

Computer-assisted image analysis of the distributions of peptidergic terminals in the central nucleus of the amygdala: A preliminary study

When viewed under dark-field illumination, peptidergic terminals in sections stained by the Sternberger PAP immunocytochemical method are seen as individual points of light. Under high magnification, the degree of brightness of various areas of immunoreactive terminals is seen to be a function of the density of terminals in these areas. By analyzying the relative brightness of the immunostained central nucleus of the amygdala (CNA) with an EyeCom II PDP-$\text{11}\text{34}$ image analysis system, we have obtained a relative evaluation of the density distribution of neurotensin (NT)-, substance P (SP), VIP-, angiotensin II (AII), m-enkephalin (m-ENK) and somatostatin (SS)-immunoreactive terminals in terms of normal morphology and following a brain lesion. The EyeCom II system divides the presented image into 307200 picture elements (pixels) and assigns one of 256 grey values to the average brightness with each pixel. We have aggregated the grey level frequencies into 5 levels where level 1 corresponds to the highest terminal density and level 5 to the lowest density. At level 1, only NT- and VIP-immunoreactive terminals occupy a significant percentage of the cross-sectional area of the CNA (20%). About 15% of the area of the CNA has VIP terminals with level 5 density. The distributions of the top 20% of the terminal density range of NT, SP, AII and VIP support a classical medial/lateral division of the nucleus. The distribution of the same range of SS- and ENK terminals suggests a dorsoventral division of the CNA. A preliminary study indicates that comparison of grey level frequency histograms generated by image analysis from homologous lesioned and unlesioned sections of the CNA can yield useful information regarding post-lesion changes in the distribution of immunoreactive terminals.     

A system of insect neurons sensitive to horizontal and vertical image motion connects the medulla and midbrain

Summary The response properties and gross morphologies of neurons that connect the medulla and midbrain in the butterfly Papilio aegeus are described. The neurons presented give direction-selective responses, i.e. they are excited by motion in the preferred direction and the background activity of the cells is inhibited by motion in the opposite, null, direction. The neurons are either maximally sensitive to horizontal motion or to slightly off-axis vertical upward or vertical downward motion, when tested in the frontal visual field. The responses of the cells are dependent on the contrast frequency of the stimulus with peak values at 5–10 Hz. The receptive fields of the medulla neurons are large and are most sensitive in the frontal visual field. Examination of the local and global properties of the receptive fields of the medulla neurons indicates that (1) they are fed by local elementary motion-detectors consistent with the correlation model and (2) there is a non-linear spatial integration mechanism in operation.     

Evaluating the conservation status of epiphytic lichens of Italy: A red list

Despite the fact that Italy is among the lichenologically best known areas worldwide, a national red list of lichens is still lacking. The aim of this work was to provide a red list of the epiphytic lichens of Italy which could facilitate the inclusion of lichens in national conservation plans. The evaluation of the species against International Union for Conservation of Nature (IUCN) criteria was based on data from multiple sources which represent the best available information on the epiphytic lichens of Italy. The species were assigned to the IUCN categories mainly using criteria D and B. A total of 368 species were evaluated: for 23 species, information is missing from more than 50 years and they were listed as regionally extinct, 64 as critically endangered, 75 as endangered, 74 as vulnerable, 58 as near threatened, 20 as least concern and 54 species as data deficient. Our results indicate that more than one-fourth of the epiphytic lichens of Italy are likely to be threatened, so that further research and effort are needed to include lichens in the main national conservation plans. Our results also highlight the lack of information that still hampers the rigorous evaluation of Italian lichens against IUCN criteria.     

Symmetry and entropy of biological patterns: discrete Walsh functions for 2D image analysis

To quantify symmetry and entropy inherent in the discrete patterns such as spatial self-organization in cell sorting and mussel bed ecosystems, we introduce the discrete Walsh analysis. This analysis enables us to estimate the degree of the complicated symmetry, and to extract the symmetry from the pattern that seems to be asymmetric. The results obtained in this paper are summarized as follows. (I) The geometrical patterns of the cell sorting become systematic with the predominance of the particular symmetry. This implies that not only the entropy but also the particular symmetry can decrease in the biological process. (II) The magnitude of the symmetry is related to the absolute value of the adhesion, and the type of the symmetry is related to the sign of the adhesion. That is, centro-symmetry dominates in the cell sorting pattern caused by large negative adhesion, and double symmetry dominates in the pattern caused by large positive adhesion. (III) Spatial self-organization in mussel bed is accompanied by the decreasing of the centro-symmetry. This implies that the positive "adhesion" between mussel individuals increases with time. (IV) In the biological process, the Curie symmetry breaking occurs at intervals.     

CellShape: A user‐friendly image analysis tool for quantitative visualization of bacterial cell factories inside

Visualization of the intracellular constituents of individual bacteria while performing as live biocatalysts is in principle doable through more or less sophisticated fluorescence microscopy. Unfortunately, rigorous quantitation of the wealth of data embodied in the resulting images requires bioinformatic tools that are not widely extended within the community‐let alone that they are often subject to licensing that impedes software reuse. In this context we have developed CellShape, a user‐friendly platform for image analysis with subpixel precision and double‐threshold segmentation system for quantification of fluorescent signals stemming from single‐cells. CellShape is entirely coded in Python, a free, open‐source programming language with widespread community support. For a developer, CellShape enhances extensibility (ease of software improvements) by acting as an interface to access and use existing Python modules; for an end‐user, CellShape presents standalone executable files ready to open without installation. We have adopted this platform to analyse with an unprecedented detail the tridimensional distribution of the constituents of the gene expression flow (DNA, RNA polymerase, mRNA and ribosomal proteins) in individual cells of the industrial platform strain Pseudomonas putida KT2440. While the CellShape first release version (v0.8) is readily operational, users and/or developers are enabled to expand the platform further.     

Measurement of bubble and pellet size distributions: past and current image analysis technology

Measurements of bubble and pellet size distributions are useful for biochemical process optimizations. The accuracy, representation, and simplicity of these measurements improve when the measurement is performed on-line and in situ rather than off-line using a sample. Historical and currently available measurement systems for photographic methods are summarized for bubble and pellet (morphology) measurement applications. Applications to cells, mycelia, and pellets measurements have driven key technological developments that have been applied for bubble measurements. Measurement trade-offs exist to maximize accuracy, extend range, and attain reasonable cycle times. Mathematical characterization of distributions using standard statistical techniques is straightforward, facilitating data presentation and analysis. For the specific application of bubble size distributions, selected bioreactor operating parameters and physicochemical conditions alter distributions. Empirical relationships have been established in some cases where sufficient data have been collected. In addition, parameters and conditions with substantial effects on bubble size distributions were identified and their relative effects quantified. This information was used to guide required accuracy and precision targets for bubble size distribution measurements from newly developed novel on-line and in situ bubble measurement devices.     

A novel computerised image analysis method for the measurement of production of conidia from the aphid pathogenic fungus Erynia neoaphidis

A semi-automated method has been developed for the quantification and measurement of conidia discharged by the aphid pathogen Erynia neoaphidis. This was used to compare conidiation by E. neoaphidis-mycosed pea aphid cadavers, mycelial plugs cut from agar plates, mycelial pellets from shake flasks and by mycelial pellets from different phases of liquid batch fermenter culture. Aphid cadavers discharged significantly more and significantly smaller conidia than plugs or pellets. The volume of conidia discharged was stable over the period of discharge (80 h), but more detailed analysis of the size frequency distribution showed that more very small and very large conidia were discharged after 5 h incubation than after 75 h incubation. Biomass harvested at the end of the exponential growth phase in batch fermenter culture produced significantly more conidia than biomass from any other growth phase. The implications of these findings for the development of production and formulation processes for E. neoaphidis as a biological control agent are discussed.     

A review of algorithms for medical image segmentation and their applications to the female pelvic cavity

This paper aims to make a review on the current segmentation algorithms used for medical images. Algorithms are classified according to their principal methodologies, namely the ones based on thresholds, the ones based on clustering techniques and the ones based on deformable models. The last type is focused on due to the intensive investigations into the deformable models that have been done in the last few decades. Typical algorithms of each type are discussed and the main ideas, application fields, advantages and disadvantages of each type are summarised. Experiments that apply these algorithms to segment the organs and tissues of the female pelvic cavity are presented to further illustrate their distinct characteristics. In the end, the main guidelines that should be considered for designing the segmentation algorithms of the pelvic cavity are proposed.     

The use of image analysis to study the effects of residues of esfenvalerate on the locomotory behaviour of Panonychus ulmi and Typhlodromus pyri (Acari: Tetranychidae, Phytoseiidae)

The influences of esfenvalerate on mite behaviour were investigated by image analysis. Video recordings of the locomotory behaviour of two mite species were converted into a series of x,y coordinates that, when joined, closely resembled their paths. These coordinates were used to calculate walking speed, direction of travel, turning frequency, turn bias and tortuosity. Two experimental arenas were used: (1) a leaf disc arena for the European red mite, Panonychus ulmi and (2) a glass coverslip arena for Typhlodromus pyri. The behavioural responses of P. ulmi and T. pyri to esfenvalerate (field rate) indicated that these mites did not show a preference for the unsprayed halves of the arenas during the first 48 min. However, significant differences between most of the behavioural parameters to esfenvalerate residues were found with P. ulmi when whole arenas were compared. © Rapid Science Ltd. 1998     

Modified procedures for extraction and analysis of carrageenan applied to the red alga Hypnea musciformis

Dried powder of Hypnea musciformis was extracted with water at pH 7 after an initial short pre-treatment with cold, diluted HCl. Carrageenans were isolated by alcohol precipitation after an amylase treatment and a filtration of the extracts. The yields at 25 and 90 °C were 25 and 75% (w/w) of the dry alga, with molecular weights (M_w) corresponding to 194 000 and 245 000, respectively. The chemical structure was dominated by G4S-DA-(kappa-carrageenan or carrageenose 4-sulphate). A simple fractionation procedure for kappa-carrageenase hydrolysates, based on stirring in different enthanol/water mixtures, is introduced. NMR analysis showed that oligosaccharides with a repeating DA-G4S structure were the main constituents in the enzymic hydrolysates of the carrageenans from Hypnea musciformis. These oligosaccharides were solubilized in an ethanol concentration from 96 to 48% (v/v). In some enzyme resistant fractions D6S-G4S and DA2S-G4S sequences and D2S,6S unites were detected by ¹³C-NMR.Author for correspondence     

A study of the cambial zone and conductive phloem of common beech (Fagus sylvatica L.) using an image analysis method

Quantification is a major problem when using histology to study the influence of ecological factors on tree structure. This paper presents a method to prepare and to analyse transverse sections of cambial zone and of conductive phloem in bark samples. The following paper (II) presents the automated measurement procedure. Part I here describes and discusses the preparation method, and the influence of tree age on the observed structure. Highly contrasted images of samples extracted at breast height during dormancy were analysed with an automatic image analyser. Between three young (38 years) and three old (147 years) trees, age-related differences were identified by size and shape parameters, at both cell and tissue levels. In the cambial zone, older trees had larger and more rectangular fusiform initials. In the phloem, sieve tubes were also larger, but their shape did not change and the area for sap conduction was similar in both categories. Nevertheless, alterations were limited, and demanded statistical analysis to be identified and ascertained. The physiological implications of the structural changes are discussed.     

The effectiveness of gadolinium MRI to improve target delineation for radiotherapy in hepatocellular carcinoma: A comparative study of rigid image registration techniques

To achieve consistent target delineation in radiotherapy for hepatocellular carcinoma (HCC), image registration between simulation CT and diagnostic MRI was explored.Twenty patients with advanced HCC were included. The median interval between MRI and CT was 11 days. CT was obtained with shallow free breathing and MRI at exhale phase. On each CT and MRI, the liver and the gross target volume (GTV) were drawn. A rigid image registration was taken according to point information of vascular bifurcation (Method[A]) and pixel information of volume of interest only including the periphery of the liver (Method[B]) and manually drawn liver (Method[C]). In nine cases with an indefinite GTV on CT, a virtual sphere was generated at the epicenter of the GTV. The GTV from CT (V_GTV[CT]) and MRI (V_GTV[MR]) and the expanded GTV from MRI (V_+GTV[MR]) considering geometrical registration error were defined. The underestimation (uncovered V[CT] by V[MR]) and the overestimation (excessive V[MR] by V[CT]) were calculated. Through a paired T-test, the difference between image registration techniques was analyzed.For method[A], the underestimation rates of V_GTV[MR] and V_+GTV[MR] were 16.4 ± 8.9% and 3.2 ± 3.7%, and the overestimation rates were 16.6 ± 8.7% and 28.4 ± 10.3%, respectively. For V_GTV[MR] and V_+GTV[MR], the underestimation rates and overestimation rates of method[A] were better than method[C]. The underestimation rates and overestimation rates of the V_GTV[MR] were better in method[B] than method[C]. By image registration and additional margin, about 97% of HCC could be covered. Method[A] or method[B] could be recommended according to physician preference.     

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue

Objective

The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation.

Introducing operator characteristic curves to define appropriate frequency of quality control tests: A case study involving whole breast radiotherapy image guidance

Background and purposeSampling theory and operator characteristic curves are methods that can determine an optimal schedule for quality control tests. We apply this method to positional data for whole breast radiotherapy since several surveys report inconsistent image guidance practice for this technique.Materials and methodsPositional errors were defined, for 55 consecutive breast cancer patients, by comparing the central lung distance measured on portal images with that obtained from the corresponding digitally reconstructed radiograph. From the distribution of positional errors, the probability of a setup error >5 mm in the direction of the mediastinum was established. Using operator characteristic curves, we compared the effectiveness of various image-guidance schedules in dealing with such errors. We also calculated the dosimetric impact of undetected errors.ResultsSetup errors >5 mm towards the mediastinum for this cohort were unlikely, at 2.7%. Imaging half of the fractions protects most patients against three or more undetected errors. Undetected, such an error increases, on average, the maximum dose to 10 cm³ of the heart by 50 cGy, the mean heart dose by 4 cGy, and the left lung V20Gy by 0.2%; therefore, the clinical impact is minute. Given that detected positional errors outside of tolerance are corrected, their residual likelihood decreases with the ratio of fractions being imaged.ConclusionsFor most tangential breast radiotherapy patients, setup errors >5 mm towards the mediastinum are unlikely, and their dosimetric impact is remote. Imaging half of the fractions of a course of whole breast radiotherapy prevents these errors to occur more than twice.     

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis: A comparative study

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (P _f, cm/sec × 10^–4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (P _d, cm/sec × 10^–4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the P _f and P _d values. The observed P _fP _d ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce P _d values in either species, (vi) The P _fP _d values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.This work was supported by Fundación Antorchas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Universidad de Buenos Aires (UBA). It was developed in the frame of an INSERM (France)-CONICET cooperative program.     

An information‐theoretic approach to estimating the composite genetic effects contributing to variation among generation means: Moving beyond the joint‐scaling test for line cross analysis

The pace and direction of evolution in response to selection, drift, and mutation are governed by the genetic architecture that underlies trait variation. Consequently, much of evolutionary theory is predicated on assumptions about whether genes can be considered to act in isolation, or in the context of their genetic background. Evolutionary biologists have disagreed, sometimes heatedly, over which assumptions best describe evolution in nature. Methods for estimating genetic architectures that favor simpler (i.e., additive) models contribute to this debate. Here we address one important source of bias, model selection in line cross analysis (LCA). LCA estimates genetic parameters conditional on the best model chosen from a vast model space using relatively few line means. Current LCA approaches often favor simple models and ignore uncertainty in model choice. To address these issues we introduce Software for Analysis of Genetic Architecture (SAGA), which comprehensively assesses the potential model space, quantifies model selection uncertainty, and uses model weighted averaging to accurately estimate composite genetic effects. Using simulated data and previously published LCA studies, we demonstrate the utility of SAGA to more accurately define the components of complex genetic architectures, and show that traditional approaches have underestimated the importance of epistasis.     

我国生态保护红线三维制度体系——以宁德市为例

生态保护红线是维护国土生态安全和人类健康的最低保障线,是国家为建设生态文明提出的一种底限思维,用创新的思路解决中国环境危机,处理保护资源和经济发展的关系。鉴于红线在生态保护领域中的重要地位,介绍了国内外相似的案例和实践应用,以及国内生态保护红线的推进情况,主要以生态空间保护为主。基于相关文献综述,生态红线的保护理念逐步扩展,即生态保护红线逐步从空间红线拓展到涵盖空间、资源及环境质量三方面的一个综合的红线体系,并进一步提出警告上线、调控中线及保有底线的控制要求,形成生态保护红线框架体系的初步构想。同时以宁德市为例,三维制度体系具体落实到地方控制指标,形成红线指标体系。最后,从法规、制度及市场等3个方面建立其配套的保障机制,以期为进一步完善整个理论体系提供参考。