Local multiple sequence alignment using dead-end elimination

MOTIVATION: Local multiple sequence alignment is a basic tool for extracting functionally important regions shared by a family of protein sequences. We present an effectively polynomial-time algorithm for rigorously solving the local multiple alignment problem. RESULTS: The algorithm is based on the dead-end elimination procedure that makes it possible to avoid an exhaustive search. In the framework of the sum-of-pairs scoring system, certain rejection criteria are derived in order to eliminate those sequence segments and segment pairs that can be mathematically shown to be inconsistent (dead-ending) with the globally optimal alignment. Iterative application of the elimination criteria results in a rapid reduction of combinatorial possibilities without considering them explicitly. In the vast majority of cases, the procedure converges to a unique globally optimal solution. In contrast to the exhaustive search, whose computational complexity is combinatorial, the algorithm is computationally feasible because the number of operations required to eliminate the dead-ending segments and segment pairs grows quadratically and cubically, respectively, with the total number of sequence elements. The method is illustrated on a set of protein families for which the globally optimal alignments are well recognized. AVAILABILITY: The source code of the program implementing the algorithm is available upon request from the authors. CONTACT: alex_lukashin@biogen.com.     

Capsaicin Modulates Proliferation,Migration, and Activation of Hepatic Stellate Cells

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Occurrence and Diversity of <Emphasis Type="Italic">Legionellaceae</Emphasis> in Polar Lakes of the Antarctic Peninsula

Legionellaceae is a family of Gram-negative, mesophilic, and facultative intracellular parasitic bacteria that inhabits freshwater environments. In this article, the Legionella population of water samples from the North and South Lake, located close to the Brazilian Scientific Station on King George Island, Keller Peninsula, Antarctica has been characterized. Culture onto selective medium and a independent-culture method were applied to the samples. In our attempt to isolate Legionella species from Antarctic lakes, we were able to obtain one L. pneumophila colony by an amoebic coculture procedure followed by plate culture onto a selective medium. In addition, results obtained from phylogenetic inference showed the presence of noncharacterized specimens of Legionella spp. These findings indicated the presence of legionellae in Antarctica and suggest that these bacteria can adapt to extreme conditions and open new possibilities for understanding the survival strategies of mesophilic Legionellaceae living in Antarctic environments. Furthermore, the isolation of these symbiotic bacteria in Antarctic lakes will allow future studies on cold-resistant mechanisms of legionellae in polar environments.     

Effect of Organic Residues and Liming Materials on Metal Extraction from a Mining-Contaminated Soil

Incubation tests were used to assess the effectiveness of three different organic residues and three different liming materials, alone or in combination, in the remediation of a mine contaminated soil. The organic residues tested were sewage sludge from a municipal wastewater treatment plant (SS), compost from the organic fraction of unsorted municipal solid waste (MSWC), and garden waste compost (GWC), applied at 100 and 200 Mg ha^{? 1}. The liming materials tested were agriculture limestone (6.4 Mg ha^{? 1}), calcium oxide (3.7 Mg ha^{? 1}), and sugar beet sludge (12.2 Mg ha^{? 1}) from the sugar manufacturing process. The soil and mixtures of soil and amendments were adjusted to 70% of the maximum water holding capacity and incubated for 28 days in a controlled-temperature room at 20°C ± 1°C. At the end of the incubation, samples were analyzed for pH, electrical conductivity, organic matter content, CaCl₂-extractable, and' NH4Ac/HAc+ EDTA–extractable metal fractions (Cu, Zn, and Pb). Correlations among the variables and/or similarities among the treatments were identified by principal component analysis and hierarchical cluster analysis. The amendments tested decreased the CaCl₂-extractable Cu and Zn fractions, considered as mobile metal fractions, to below analytical detectable limits, providing organic matter to the soil with levels between 1% and 2% at the end of the experiment, significantly different relatively to the original soil. pH and electrical conductivity reached high values when using 200 Mg ha^{? 1} SS or 200 Mg ha^{? 1} MSWC, with any of the liming materials tested, making these application rates excessive for this particular situation. Furthermore, the treatments using MSWC increased the NH4Ac/HAc+ EDTA–extractable Cu, Pb, and Zn fractions, considered as mobilizable metal fractions, as did the 200 Mg ha^{? 1} SS for Pb and Zn. Considering the overall results, the compost made from garden waste decreased metal solubility in the soil and increased soil pH and organic matter content, without the addition of large amounts of soluble salts, and without increasing the mobilizable metal content. Of the organic materials tested, this was the only one that can be considered adequate for remediation of the contaminated soil under study, at the application rates tested.     

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.     

An expert system model for mapping tropical wetlands and peatlands reveals South America as the largest contributor

Wetlands are important providers of ecosystem services and key regulators of climate change. They positively contribute to global warming through their greenhouse gas emissions, and negatively through the accumulation of organic material in histosols, particularly in peatlands. Our understanding of wetlands’ services is currently constrained by limited knowledge on their distribution, extent, volume, interannual flood variability and disturbance levels. We present an expert system approach to estimate wetland and peatland areas, depths and volumes, which relies on three biophysical indices related to wetland and peat formation: (1) long‐term water supply exceeding atmospheric water demand; (2) annually or seasonally water‐logged soils; and (3) a geomorphological position where water is supplied and retained. Tropical and subtropical wetlands estimates reach 4.7 million km² (Mkm²). In line with current understanding, the American continent is the major contributor (45%), and Brazil, with its Amazonian interfluvial region, contains the largest tropical wetland area (800,720 km²). Our model suggests, however, unprecedented extents and volumes of peatland in the tropics (1.7 Mkm² and 7,268 (6,076–7,368) km³), which more than threefold current estimates. Unlike current understanding, our estimates suggest that South America and not Asia contributes the most to tropical peatland area and volume (ca. 44% for both) partly related to some yet unaccounted extended deep deposits but mainly to extended but shallow peat in the Amazon Basin. Brazil leads the peatland area and volume contribution. Asia hosts 38% of both tropical peat area and volume with Indonesia as the main regional contributor and still the holder of the deepest and most extended peat areas in the tropics. Africa hosts more peat than previously reported but climatic and topographic contexts leave it as the least peat‐forming continent. Our results suggest large biases in our current understanding of the distribution, area and volumes of tropical peat and their continental contributions.     

The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.