Regional distribution of glucose utilization in the telencephalon of toads in response to configurational visual stimuli: a14C-2DG study

Summary The regional glucose utilization in the telencephalon of toadsBufo bufo during stimulation with different visual key stimuli was quantitatively mapped by means of the¹⁴C-2DG autoradiographic method: (i) a 4×28 mm² worm-like stripe (W) eliciting prey catching responses, (ii) a 84×84 mm² square (S) releasing predator avoidance responses, and (iii) a 28×4 mm² antiwormlike stripe (A) eliciting no motor response.Various telencephalic structures changed¹⁴C- 2DG uptake statistical significantly during stimulation with the above visual objects in comparison with binocular enucleated animals (brain-to-brain comparison) and in comparison between both hemispheres in monocular animals (interhemispherical comparison): (1) The ventral two-thirds of the posterior half of the medial palliumdecreased ^14C-2DG uptake during W- and S-experiments, particularly in response to W. (2) In the posterior two-thirds of the lateral pallium,¹⁴C- 2DG uptake wasdecreased in response to the worm-, andincreased in response to the square (S) and antiworm stimuli (A). (3) The ventral striatumincreased uptake of¹⁴C-2DG during the animal's response to W- and S-stimuli significantly stronger than in the A-experiment. (4) The dorsal striatum also showed a significant change in¹⁴C-2DG uptake which, on a lower level, was not correlated with the type of stimulation experiment.Various prosencephalic structures are involved in circuitries related to attentional phenomena and the gating of prey catching and predator avoidance behavior. The different functions of these structures are discussed.Abbreviations A anterior dorsal thalamus - ACC nucleus ac-cumbens - APL amygdala, pars lateralis - APM amygdala, pars medialis - aLP anterior third of the lateral pallium - aMP anterior half of the medial pallium - B Bed nucleus of the palliai commissure - Ea entopeduncular nucleus, pars anterior - dMP dorsal medial pallium - dP dorsal pallium - dSTR dorsal striatum - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, postero-dorsal division - OT optic tectum - P posterior thalamic nucleus - pLP posterior two-thirds of the lateral pallium - PO preoptic area of the hypothalamus; - RET tegmental portion of the medial reticular formation - SEP medial (MS) and lateral (LS) septum - vMP ventral two-thirds of the medial pallium (MP) - vSTR ventral striatum     

Responses of medullary neurons to moving visual stimuli in the common toad

The concept of coded 'command releasing systems' proposes that visually specialized descending tectal (and pretectal) neurons converge on motor pattern generating medullary circuits and release--in goal-specific combination--specific action patterns. Extracellular recordings from medullary neurons of the medial reticular formation of the awake immobilized toad in response to moving visual stimuli revealed the following main results. (i) Properties of medullary neurons were distinguished by location, shape, and size of visual receptive fields (ranging from relatively small to wide), by trigger features of various moving configural stimulus objects (including prey- and predator-selective properties), by tactile sensitivity, and by firing pattern characteristics (sluggish, tonic, warming-up, and cyclic). (ii) Visual receptive fields of medullary neurons and their responses to moving configural objects suggest converging inputs of tectal (and pretectal) descending neurons. (iii) In contrast to tectal monocular 'small-field' neurons, the excitatory visual receptive fields of comparable medullary neurons were larger, ellipsoidally shaped, mostly oriented horizontally, and not topographically mapped in an obvious fashion. Furthermore, configural feature discrimination was sharper. (iv) The observation of multiple properties in most medullary neurons (partly showing combined visual and cutaneous sensitivities) suggests integration of various inputs by these cells, and this is in principle consistent with the concept of command releasing systems. (v) There is evidence for reciprocal tectal/medullary excitatory pathways suitable for premotor warming-up. (vi) Cyclic bursting of many neurons, spontaneously or as a post-stimulus sustaining event, points to a medullary premotor/motor property.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Aktivierung der Verhaltensfolge beim Beutefang der Erdkröte (Bufo bufo L.) durch elektrische Mittelhirn-Reizung

Ohne ZusammenfassungHerrn Professor Dr. Dr. h. c. Karl v. Frisch zum 80. Geburtstag gewidmet.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.Laboratory diagnosis of infections with Acanthamoeba spp. is based on identification of the parasite in infected tissue. Although various techniques, including immunocytological and molecular methods, have been described, recovery of viable parasites by cultivation on agar is still the basic procedure used (16). This method is usually associated with histopathological examination of the specimen to prove tissue invasion by the parasite.Recognition of parasites in tissue sections is often difficult and depends on the expertise of the pathologist. In addition to traditional histological staining methods, immunohistology using parasite-specific antibodies, lectin conjugates, and calcofluor white have been used for visualization of parasites in tissue sections (3).Some protozoan parasites have the ability to protect themselves by forming a cyst wall, which is resistant to environmental stresses such as desiccation, lack of nutrients, and variations in temperature and pH. In most pathogenic protozoans studied, chitin is the carbohydrate polymer providing the required structural toughness to the cyst wall. Acanthamoeba spp. are exceptions, as their cysts are made up of cellulose. Recently, cellulose has also been identified as a cyst wall component in a closely related amoeba, Balamuthia mandrillaris (15). Cellulose consists of β-d-glucosyl units linked by β-1,4-glucosidic bonds. Chitin is very similar but contains N-acetylglucosamine as the monomer. Both polymers form very similar crystalline macroscopic structures. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones. This is especially true for various fluorescent brightening agents, such as calcofluor white, used as cytochemical markers in microscopic diagnosis of protozoan and fungal infections. A two-domain structural organization is often observed in cellulose-degrading enzymes. Most Trichoderma reesei cellulases consist of a catalytic domain and a cellulose-binding domain (CBD) joined by a linker. The catalytic domain contains the active site with the amino acid residues responsible for the hydrolytic mechanism. The role of the CBD is to bind to the solid cellulose. The ability of CBDs to attach to cellulose can be utilized in various applications. Individual types of CBDs can vary significantly in their properties, such as affinity, preference for crystalline or amorphous cellulose, and cross-reactivity with other similar carbohydrates (7, 8, 9, 10).We have previously described a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of T. reesei cellulase to protozoan cyst wall cellulose (12). In that study we used a recombinant dimeric CBD (D-CBD) fusion protein in an indirect immunofluorescence analysis to specifically stain the cellulose and visualize its localization in the cyst wall. In preliminary studies, this method was also shown to be useful detection of parasites in tissue sections (11).The aim of the present study was to simplify the detection method by preparing D-CBDs as fluorescent and biotinylated conjugates that could be used for direct and rapid detection of cellulose in Acanthamoeba by both fluorescence and ordinary light microscopy.     

Regional differences in prediction models of lung function in Germany

Background

Little is known about the influencing potential of specific characteristics on lung function in different populations. The aim of this analysis was to determine whether lung function determinants differ between subpopulations within Germany and whether prediction equations developed for one subpopulation are also adequate for another subpopulation.

Methods

Within three studies (KORA C, SHIP-I, ECRHS-I) in different areas of Germany 4059 adults performed lung function tests. The available data consisted of forced expiratory volume in one second, forced vital capacity and peak expiratory flow rate. For each study multivariate regression models were developed to predict lung function and Bland-Altman plots were established to evaluate the agreement between predicted and measured values.

Results

The final regression equations for FEV₁ and FVC showed adjusted r-square values between 0.65 and 0.75, and for PEF they were between 0.46 and 0.61. In all studies gender, age, height and pack-years were significant determinants, each with a similar effect size. Regarding other predictors there were some, although not statistically significant, differences between the studies. Bland-Altman plots indicated that the regression models for each individual study adequately predict medium (i.e. normal) but not extremely high or low lung function values in the whole study population.

Conclusions

Simple models with gender, age and height explain a substantial part of lung function variance whereas further determinants add less than 5% to the total explained r-squared, at least for FEV1 and FVC. Thus, for different adult subpopulations of Germany one simple model for each lung function measures is still sufficient.     

Nest site choice compensates for climate effects on sex ratios in a lizard with environmental sex determination

Theoretical models suggest that in changing environments natural selection on two traits, maternal nesting behaviour and pivotal temperatures (those that divide the sexes) is important for maintaining viable offspring sex ratios in species with environmental sex determination (ESD). Empirical evidence, however, is lacking. In this paper, we provide such evidence from a study of clinal variation in four sex-determining traits (maternal nesting behaviour, pivotal temperatures, nesting phenology, and nest depth) in Physignathus lesueurii, a wide-ranging ESD lizard inhabiting eastern Australia. Despite marked differences in air and soil temperatures across our five study sites spanning 19° latitude and 1200 m in elevation, nest temperatures did not differ significantly among sites. Lizards compensated for climatic differences chiefly by selecting more open nest sites with higher incident radiation at cooler sites. Clinal variation in the onset of nesting also compensated for climatic differences, but to a lesser extent. There was no evidence of compensation through pivotal temperatures or nest depth. More broadly, our results extend to the egg stage the life history prediction that behaviour is the chief compensatory mechanism for climatic differences experienced by species spanning environmental extremes. Furthermore, our study was unique in revealing that nest site choice influenced mainly the daily range in nest temperatures, rather than mean temperatures, in a shallow-nesting reptile. Finally, indirect evidence suggests that the cue used by nesting lizards was radiation or temperature (through basking or assessing substrate temperatures), not visual detection of canopy openness. We conclude that maternal nesting behaviour and nesting phenology are traits subject to sex ratio selection in P. lesueurii, and thus, must be considered among the repertoire of ESD species for responding to climate change.     

On-Chip Imaging of Schistosoma haematobium Eggs in Urine for Diagnosis by Computer Vision

Background

Microscopy, being relatively easy to perform at low cost, is the universal diagnostic method for detection of most globally important parasitic infections. As quality control is hard to maintain, misdiagnosis is common, which affects both estimates of parasite burdens and patient care. Novel techniques for high-resolution imaging and image transfer over data networks may offer solutions to these problems through provision of education, quality assurance and diagnostics. Imaging can be done directly on image sensor chips, a technique possible to exploit commercially for the development of inexpensive “mini-microscopes”. Images can be transferred for analysis both visually and by computer vision both at point-of-care and at remote locations.

Methods/Principal Findings

Here we describe imaging of helminth eggs using mini-microscopes constructed from webcams and mobile phone cameras. The results show that an inexpensive webcam, stripped off its optics to allow direct application of the test sample on the exposed surface of the sensor, yields images of Schistosoma haematobium eggs, which can be identified visually. Using a highly specific image pattern recognition algorithm, 4 out of 5 eggs observed visually could be identified.

Conclusions/Significance

As proof of concept we show that an inexpensive imaging device, such as a webcam, may be easily modified into a microscope, for the detection of helminth eggs based on on-chip imaging. Furthermore, algorithms for helminth egg detection by machine vision can be generated for automated diagnostics. The results can be exploited for constructing simple imaging devices for low-cost diagnostics of urogenital schistosomiasis and other neglected tropical infectious diseases.