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Hepatic cryosurgery precision: evaluation of ultrasonography, thermometry, and impedancemetry in a pig model.

One of the main problems of the use of liver cryosurgery is to be sure that a defined hepatic volume has been completely destroyed. We undertook an experimental pig study to determine histopathological evolution of cryolesions, to evaluate the value of intraoperative sonography, thermometry, and impedancemetry to monitor necrosis and to evaluate clinical and biological repercussions of hepatic cryosurgery. Forty-eight cryolesions were obtained by freezing each liver lobe of 12 experimental pigs during a 5-min contact with a flat cryoprobe cooled with liquid nitrogen. Cryolesions and the surrounding liver were monitored during cryosurgery by six thermocouple electrodes, five impedance electrodes, and intraoperative sonography. Animals were sacrificed immediately, 6 hr and between day 1 and day 32 after the procedure. Cryolesions were excised, and a full size pathological study was carried out. No morbidity or mortality was observed. At the end of the freezing time, cryolesions were hemispheric in shape, and their radius measured by sonography was 17.7 +/- 1.2 mm (mean +/- SD). Microscopic study showed sequential tissue alterations with edema, ischemic necrosis, tissue slough, and granulation. Cryolesions were sharply delineated from the normal liver tissue. The radius of necrosis at days 2 and 3 was 17 +/- 0.3 mm (mean +/- SD). It showed good correlation with the cryolesion size measured by intraoperative sonography. The temperature threshold to obtain complete normal liver necrosis was -15 degrees C. We found impedancemetry too difficult to use and not precise enough to monitor cryonecrosis. We conclude that intraoperative sonography and thermometry are useful means to monitor the extent of cryonecrosis during liver cryosurgery.

Animals↗

Impact of electroosmosis on isotachophoresis in open-tubular fused-silica capillaries: analysis of the evolution of a stationary steady-state zone structure by computer simulation and experimental validation.

A dynamic computer model for simulation of open-tubular capillary electrophoresis that includes in situ calculation of electroosmosis along the fused-silica capillary column has been applied to the characterization of an anionic isotachophoretic system in presence of a cathodic electroosmotic flow. For each column segment, electroosmosis is calculated with the use of a wall mobility, the voltage gradient and the degree of dissociation of the silanol surface groups of the capillary wall. Then, the bulk capillary flow is taken to be the average of all of the segment flows and considered to represent a plug flow. This simple approach enables the combined simulation of the temporal behavior of an isotachophoretic zone structure in presence of electroosmosis. For a model anionic isotachophoretic configuration at pH 6, simulation data reveal the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic and electroosmotic zone displacements are opposite and of equal magnitude. The position of the stationary boundaries are predicted to be dependent on the selected wall pK and mobility values. For two different instruments, qualitative agreement between experimental data and simulation results obtained with a wall pK between 5 and 6 is demonstrated. However, for the two experimental setups, significant differences in electroosmotic pumping (i.e. wall mobility values) are noted.

Anions↗

Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype.

Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.

Alleles↗

[The evolution of a contusion focus after the microsurgical autotransplantation of the omentum (an experimental study)].

The purpose of treatment of head trauma cannot be limited by simple life saving, it also should be targeted at the improvement of the quality of life. Twenty dogs with graded local head trauma underwent microsurgical revascularization of the area adjacent to the contused brain with an autologous omental graft to provide functional improvement. The effectiveness of surgery was demonstrated in series of 5 experiments when the operation was performed on posttraumatic days 7-14, with the follow-up of as long as 180 days. After the procedure, no macroscopic signs of contusion could be demonstrated. Microscopically the contused area resembled a slit two times smaller than in controls. Microscopic and histochemical studies revealed both the morphologic recovery and an improvement in cellular enzyme activity. The data obtained adds some more information regarding clinical applications of microsurgical revascularization technique.

Animals↗

C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway.

In recent years, a 'new' pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing 'foreign' carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to 'target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being 'new' to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.

Amino Acid Sequence↗

[Evolution of tool handling activity in monkeys].

Main experimental data are presented which have been obtained on lower and higher monkeys. New understanding is suggested of regularities of interaction of primates with the environmental objects. The data are discussed in relation to historic and ontogenetic development of working activity in man.

Aging↗

Evolution of eusociality and the soldier caste in termites: influence of intraspecific competition and accelerated inheritance.

We present new hypotheses and report experimental evidence for powerful selective forces impelling the evolution of both eusociality and the soldier caste in termites. Termite ancestors likely had a nesting and developmental life history similar to that of the living family Termopsidae, in which foraging does not occur outside the host wood, and nonsoldier helpers retain lifelong options for differentiation into reproductives. A local neighborhood of families that live exclusively within a limited resource results in interactions between conspecific colonies, high mortality of founding reproductives, and opportunities for accelerated inheritance of the nest and population by offspring that differentiate into nondispersing neotenic reproductives. In addition, fertile reproductive soldiers, a type of neotenic previously considered rare and docile, frequently develop in this intraspecific competitive context. They can be highly aggressive in subsequent interactions, supporting the hypothesis that intercolonial battles influenced the evolution of modern sterile termite soldier weaponry and behaviors.

Animals↗

Signal identification in NMR spectra with coupled evolution periods.

Novel multidimensional NMR experiments rely on modified time-domain sampling schemes to provide significant savings of experimental time. Several approaches are based on the coupling of evolution times resulting in a reduction of the dimensionality of the recorded spectra, and a concomitant saving of experimental time. We present a consistent and general tool, called EVOCOUP, for the analysis of these reduced dimensionality spectra. The approach is flexible in the sense that the input can consist of various forms of reduced dimensionality spectra, that any piece of information can be removed (provided enough information is left), e.g., signals undetectable due to poor signal-to-noise or covered by artifacts, and that it can be applied to spectra involving any number of nuclei. The use of a general optimization procedure and an appropriate target function provides for a robust approach with well-defined results and ensures optimal use of redundant information normally present in the input. Spectral overlap in the directly detected dimension is resolved in a fully automated manner, avoiding the assessment of signal quality and its use in combinatorial trials. The positions of all peaks in a corresponding full-dimensional spectrum are obtained without need for reconstruction of this spectrum. In a systematic analysis of a complete spectrum recorded for the 14 kDa protein azurin and involving five different nuclei, only four spin systems were missed and no false spins systems were detected.

Artifacts↗

Current status and challenges in connecting models of erythrocyte metabolism to experimental reality.

Detailed kinetic models of human erythrocyte metabolism have served to summarize the vast literature and to predict outcomes from laboratory and "Nature's" experiments on this simple cell. Mathematical methods for handling the large array of nonlinear ordinary differential equations that describe the time dependence of this system are well developed, but experimental methods that can guide the evolution of the models are in short supply. NMR spectroscopy is one method that is non-selective with respect to analyte detection but is highly specific with respect to their identification and quantification. Thus time courses of metabolism are readily recorded for easily changed experimental conditions. While the data can be simulated, the systems of equations are too complex to allow solutions of the inverse problem, namely parameter-value estimation for the large number of enzyme and membrane-transport reactions operating in situ as opposed to in vitro. Other complications with the modelling include the dependence of cell volume on time, and the rates of membrane transport processes are often dependent on the membrane potential. These matters are discussed in the light of new modelling strategies.

Animals↗

Genetic constraints on the evolution of mate recognition under natural selection.

Field populations of Drosophila serrata display reproductive character displacement in cuticular hydrocarbons (CHCs) when sympatric with Drosophila birchii. We have previously shown that the naturally occurring pattern of reproductive character displacement can be experimentally replicated by exposing field allopatric populations of D. serrata to experimental sympatry with D. birchii. Here, we tested whether the repeated evolution of reproductive character displacement in natural and experimental populations was a consequence of genetic constraints on the evolution of CHCs. The genetic variance-covariance (G) matrices for CHCs were determined for populations of D. serrata that had evolved in either the presence or absence of D. birchii under field and experimental conditions. Natural selection on mate recognition under both field and experimental sympatric conditions increased the genetic variance in CHCs consistent with a response to selection based on rare alleles. A close association between G eigenstructure and the eigenstructure of the phenotypic divergence (D) matrix in natural and experimental populations suggested that G matrix eigenstructure may have determined the direction in which reproductive character displacement evolved during the reinforcement of mate recognition.

Animals↗

Stochastic modeling of oligodendrocyte generation in cell culture: model validation with time-lapse data.

BACKGROUND: The purpose of this paper is two-fold. The first objective is to validate the assumptions behind a stochastic model developed earlier by these authors to describe oligodendrocyte generation in cell culture. The second is to generate time-lapse data that may help biomathematicians to build stochastic models of cell proliferation and differentiation under other experimental scenarios. RESULTS: Using time-lapse video recording it is possible to follow the individual evolutions of different cells within each clone. This experimental technique is very laborious and cannot replace model-based quantitative inference from clonal data. However, it is unrivalled in validating the structure of a stochastic model intended to describe cell proliferation and differentiation at the clonal level. In this paper, such data are reported and analyzed for oligodendrocyte precursor cells cultured in vitro. CONCLUSION: The results strongly support the validity of the most basic assumptions underpinning the previously proposed model of oligodendrocyte development in cell culture. However, there are some discrepancies; the most important is that the contribution of progenitor cell death to cell kinetics in this experimental system has been underestimated.

Algorithms↗

A morphologic study of opportunistic cerebral toxoplasmosis.

The morphology of toxoplasma and its interaction with the cellular elements of the brain were studied in a patient who dies of extensive cerebral toxoplasmosis superimposed on Hodgkin's disease. The cerebral lesions were devoid of inflammatory cellular response and contained numerous organisms mostly in isolated multiplying forms in neurons, glia and vascular walls. Encysted forms containing multiplying organisms were seen infrequently. Intracellular parasite was identified in normal-appearing neuropil. The mode of multiplication and cyst formation of toxoplasma appeared basically similar to that described under experimental conditions. In addition, a rapid evolution of the cerebral lesions was suggested by computerized tomography. This study suggests that tissue necrosis in human cerebral toxoplasmosis is the result of an increased rate of multiplication and enhanced cellular invasiveness of the parasite most likely related to impaired cellular immunity as has been postulated by clinical and experimental data.

Aged↗

Entangled and disentangled evolution for a single atom in a driven cavity.

For an atom in an externally driven cavity, we show that special initial states lead to near-disentangled atom-field evolution, and superpositions of these can lead to near maximally entangled states. Somewhat counterintutively, we find that (moderate) spontaneous emission in this system actually leads to a transient increase in entanglement beyond the steady-state value. We also show that a particular field correlation function could be used, in an experimental setting, to track the time evolution of this entanglement.

Journal Article↗

Sampling measurement of soft-x-ray-pulse shapes by femtosecond sequential ionization of Kr+ in an intense laser field.

We propose a sampling technique for measuring the shape of ultrashort soft-x-ray pulses. The technique uses the transient state of Kr+ ions that is produced by the femtosecond sequential evolution of Kr ions during optical-field-induced ionization as an ultrafast x-ray-absorption sampling gate. We demonstrate the technique by measuring the pulse shape of the 51st harmonic (15.6 nm) generated by a 100-fs titanium:sapphire laser pulse. The measured pulse duration is 220 fs. Our experimental result confirms that the sequential evolution of Kr+ ions from neutral Kr to Kr2+ is the dominant contribution to the ionization process from the aspect of time-domain measurement.

Journal Article↗

[The evolution of the synapses in the vertebrate central nervous system (a topical paper)].

The results of comparative morphological and ultrastructural research of the synaptic organization of spinal cord motoneurons from fishes, amphibian, reptiles and mammals are presented in the review. Fundamental regularities of the formation of the synaptic organization of motoneurons in vertebrate phylogenesis were established. The results of electron microscopical analysis showed that all basic elements of the synaptic ultrastructure preserved in the motoneuronal synapse of vertebrates during the evolution. On the basis of large experimental material it was found that the synaptic organization of individual motoneuron as a whole underwent structural reorganization, presented as corresponding transformations in mechanisms of the synaptic transmission.

Amphibians↗

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗

Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies?

A number of chemically unrelated neurotoxic compounds and several types of metabolic abnormalities cause strikingly similar patterns of distal symmetrical polyneuropathy in humans and animals. Experimental studies with laboratory species have demonstrated that many toxic polyneuropathies are associated with distal and retrograde axonal degeneration occurring in vulnerable nerve fiber tracts in the central as well as the peripheral nervous system. This has been termed central-peripheral distal axonopathy. Recent observations from the authors' laboratories regarding (1) the spatial-temporal evolution of nerve fiber degeneration in experimental toxic neuropathies and (2) the inhibition of glycolytic enzymes by chemically unrelated neurotoxic compounds point to a common metabolic basis for many distal axonopathies. It is postulated that neurotoxic compounds deplete energy supplies in the axon by inhibiting nerve fiber enzymes required for the maintenance of energy synthesis. Resupply of enzymes from the neuronal soma fails to meet the increased demand for enzyme replacement in the axon, causing the concentration of enzymes to drop in distal regions. This leads to a local blockade of energy-dependent axonal transport, which produces a series of pathological changes culminating in distal nerve fiber degeneration. The idea provides a working hypothesis with which to study the cause of inherited and acquired human and animal polyneuropathies.

Axonal Transport↗

Evolution of light-induced anchoring in dye-doped nematics: experiment and model.

A series of experiments was carried out to describe the evolution of light-induced anchoring in dye-doped nematic liquid crystals (LCs) at irradiation with polarized light. The experiments included cells filled with a pure pentyl-cyanobiphenyl (5CB) and containing a layer of azo dye deposited on an aligning film, as well as cells filled with azo dye doped 5CB, which allowed us to distinguish the role of "surface" and "bulk" dye molecules in the evolution of light-induced anchoring. Modifications of the spectra of spontaneously adsorbed dye molecules under illumination enabled us to assert that light-induced desorption is a mechanism responsible for producing an easy orientation axis in a dark-adsorbed layer. We found that the evolution of light-induced anchoring involves a competition between light-induced desorption and adsorption of the dye molecules on the aligning surface, and the final anchoring is determined by the total light irradiation dose. These data allowed introducing a theoretical model of light-induced anchoring of dye-doped nematic LCs that quantitatively described the experimental results and portrayed the whole evolution of the dye-doped LC cell at irradiation.

Journal Article↗