Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes

Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties Of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (Delta C(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in Delta C(p) on should include effects arising from the prot inlsolvent interface.

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

P>According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

Hepatic function evolution in dogs anesthetized with Zolazepam/Tiletamine

The hepatic effects of the anesthetic association zolazepam/tiletamine were investigated in dogs by analyzing the serum concentration of hepatic enzymes. Ten healthy dogs were divided into two groups of five, group I (GI) and group II (GII). The animals of GI received a single dose of 6,6 mg/kg of zolazepam/tiletamine, by intramuscular (IM) injection. GII dogs received 6,6 mg/kg of zolazepam/tiletamine by the IM route; after a period of 50 - 80 minutes the animals received two additional doses (3,3 mg/kg) by intravenous administration[SAH1]. The hepatic function were analyzed by monitoring the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl-transferase (GGT). Four blood samples were collected in different moments during the analyses: M0, before the first application of the drug; and M1 to M4. M1 through M3 was collected with intervals of 20 minutes before M0, while M4 was obtained 24 hours after M1. The normality of the obtained results was analyzed by Kolmogorov-Smirnov Test; while the Tukey`s test compared the means, using a level of significance of 5% for both statistical analyses. The mean values of all enzymes evaluated were within normal limits for both experimental groups, without any significant statistical alteration being observed between and within these groups. These results demonstrated that the association of zolazepam/tiletamine at the dosage of 6.6 mg/kg, followed by two applications additional of 3.3 mg/kg resulted in elevation of the evaluated hepatic enzymes without exceeding the physiologic values. Additionally, a single application of 6.6 mg/kg of zolazepam/tiletamine by the intramuscular route resulted in lower values when compared to three applications.

Central mucoepidermoid carcinoma: Report of a case with 11 years` evolution and peculiar macroscopical and clinical characteristics

Central mucoepidermoid carcinomas (CMC) are uncommon tumours, comprising 2-3% of all mucoepidermoid carcinomas reported. They have been reported in patients of all ages, ranging from 1 to 78-years, with the overwhelming majority occurring in the 4th and 5th decades of life. They are histologically low-grade cancers, usually affecting the mandible as uniocular or multiocular radiographic lesions. The authors report a case of CMC of the mandible with a long evolution, and peculiar clinical and macroscopical features related with the long term evolution of the disease. A 53-year-old male patient had expansion of buccal and lingual cortices of the anterior region of the mandible, covered by ulcerated mucosa, with 11 years evolution. An incisional biopsy was performed, and the histopathological findings confirm low-grade mucoepidermoid carcinoma. The patient was treated with a mandibulectomy, followed by supraomohyoid neck dissection. There was no evidence of local recurrence, regional or distant metastasis revealed; and the patient was alive and without disease after a follow-up interval of 36 months.

Mental evolution and development: Evidence for secondary representation in children, great apes, and other animals

Recent interest in the development and evolution of theory of mind has provided a wealth of information about representational skills in both children and animals, According to J, Perrier (1991), children begin to entertain secondary representations in the 2nd year of life. This advance manifests in their passing hidden displacement tasks, engaging in pretense and means-ends reasoning, interpreting external representations, displaying mirror self-recognition and empathic behavior, and showing an early understanding of mind and imitation. New data show a cluster of mental accomplishments in great apes that is very similar to that observed in 2-year-old humans. It is suggested that it is most parsimonious to assume that this cognitive profile is of homologous origin and that great apes possess secondary representational capacity. Evidence from animals other than apes is scant. This analysis leads to a number of predictions for future research.

An architectonic comparison of the ventrobasal complex of two Megachiropteran and one Microchiropteran bat: implications for the evolution of Chiroptera

The architectonic features of the thalamic ventrobasal complex (Vb) of two species of Megachiropteran (Grey-headed flying fox, Pteropus poliocephalus, and the Eastern tube-nosed bat, Nyctimene robinsoni) are compared with those of a Microchiropteran (Australian ghost bat, Macroderma gigas). The somatosensory system was chosen for comparison as it represents a sensory system that has undergone analogous modifications in both Chiropteran lineages (the evolution of the wing). The components of Vb were examined as there are taxon-specific features in this region of the brain. Within the Megachiropteran Vb, four subnuclei were recognized: the ventral posterior medial (VPM), the ventral posterior lateral (VPL), the ventral posterior inferior (VPI), and the basal ventral medial (VMb). In the ghost bat only VPM and VPL were identified with certainty. No VPI was evident in the ghost bat, however a putative VMb was observed. Vb of the ghost bat also lacked the arcuate lamina, which distinguishes VPM from VPL in the Megachiropterans and many other mammals. These taxon-specific differences lend support to the proposal that the order Chiroptera has a diphyletic origin.

The evolution of controlled multitasked gene networks: The role of introns and other noncoding RNAs in the development of complex organisms

Eukaryotic phenotypic diversity arises from multitasking of a core proteome of limited size. Multitasking is routine in computers, as well as in other sophisticated information systems, and requires multiple inputs and outputs to control and integrate network activity. Higher eukaryotes have a mosaic gene structure with a dual output, mRNA (protein-coding) sequences and introns, which are released from the pre-mRNA by posttranscriptional processing. Introns have been enormously successful as a class of sequences and comprise up to 95% of the primary transcripts of protein-coding genes in mammals. In addition, many other transcripts (perhaps more than half) do not encode proteins at all, but appear both to be developmentally regulated and to have genetic function. We suggest that these RNAs (eRNAs) have evolved to function as endogenous network control molecules which enable direct gene-gene communication and multitasking of eukaryotic genomes. Analysis of a range of complex genetic phenomena in which RNA is involved or implicated, including co-suppression, transgene silencing, RNA interference, imprinting, methylation, and transvection, suggests that a higher-order regulatory system based on RNA signals operates in the higher eukaryotes and involves chromatin remodeling as well as other RNA-DNA, RNA-RNA, and RNA-protein interactions. The evolution of densely connected gene networks would be expected to result in a relatively stable core proteome due to the multiple reuse of components, implying,that cellular differentiation and phenotypic variation in the higher eukaryotes results primarily from variation in the control architecture. Thus, network integration and multitasking using trans-acting RNA molecules produced in parallel with protein-coding sequences may underpin both the evolution of developmentally sophisticated multicellular organisms and the rapid expansion of phenotypic complexity into uncontested environments such as those initiated in the Cambrian radiation and those seen after major extinction events.

Time evolution in the unimolecular master equation at low temperatures: full spectral solution with scalable iterative methods and high precision

A new method is presented to determine an accurate eigendecomposition of difficult low temperature unimolecular master equation problems. Based on a generalisation of the Nesbet method, the new method is capable of achieving complete spectral resolution of the master equation matrix with relative accuracy in the eigenvectors. The method is applied to a test case of the decomposition of ethane at 300 K from a microcanonical initial population with energy transfer modelled by both Ergodic Collision Theory and the exponential-down model. The fact that quadruple precision (16-byte) arithmetic is required irrespective of the eigensolution method used is demonstrated. (C) 2001 Elsevier Science B.V. All rights reserved.

Subspace wavepacket evolution with Newton polynomials

Time-dependent wavepacket evolution techniques demand the action of the propagator, exp(-iHt/(h)over-bar), on a suitable initial wavepacket. When a complex absorbing potential is added to the Hamiltonian for combating unwanted reflection effects, polynomial expansions of the propagator are selected on their ability to cope with non-Hermiticity. An efficient subspace implementation of the Newton polynomial expansion scheme that requires fewer dense matrix-vector multiplications than its grid-based counterpart has been devised. Performance improvements are illustrated with some benchmark one and two-dimensional examples. (C) 2001 Elsevier Science B.V. All rights reserved.