A rugged free energy landscape separates multiple functional RNA folds throughout denaturation

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.     

An approach of orthology detection from homologous sequences under minimum evolution

In the field of phylogenetics and comparative genomics, it is important to establish orthologous relationships when comparing homologous sequences. Due to the slight sequence dissimilarity between orthologs and paralogs, it is prone to regarding paralogs as orthologs. For this reason, several methods based on evolutionary distance, phylogeny and BLAST have tried to detect orthologs with more precision. Depending on their algorithmic implementations, each of these methods sometimes has increased false negative or false positive rates. Here, we developed a novel algorithm for orthology detection that uses a distance method based on the phylogenetic criterion of minimum evolution. Our algorithm assumes that sets of sequences exhibiting orthologous relationships are evolutionarily less costly than sets that include one or more paralogous relationships. Calculation of evolutionary cost requires the reconstruction of a neighbor-joining (NJ) tree, but calculations are unaffected by the topology of any given NJ tree. Unlike tree reconciliation, our algorithm appears free from the problem of incorrect topologies of species and gene trees. The reliability of the algorithm was tested in a comparative analysis with two other orthology detection methods using 95 manually curated KOG datasets and 21 experimentally verified EXProt datasets. Sensitivity and specificity estimates indicate that the concept of minimum evolution could be valuable for the detection of orthologs.     

A compatible exon-exon junction database for the identification of exon skipping events using tandem mass spectrum data

Background  

Alternative splicing is an important gene regulation mechanism. It is estimated that about 74% of multi-exon human genes have alternative splicing. High throughput tandem (MS/MS) mass spectrometry provides valuable information for rapidly identifying potentially novel alternatively-spliced protein products from experimental datasets. However, the ability to identify alternative splicing events through tandem mass spectrometry depends on the database against which the spectra are searched.     

Humicola insolens cutinase-catalyzed lactone ring-opening polymerizations: kinetic and mechanistic studies

This paper explores reaction kinetics and mechanism for immobilized Humicola insolenscutinase (HIC), an important new biocatalyst that efficiently catalyzes non-natural polyester synthetic reactions. HIC, immobilized on Lewatit, was used as catalyst for epsilon-caprolactone (CL) and omega-pentadecalactone (PDL) ring-opening polymerizations (ROPs). Plots of percent CL conversion vs time were obtained in the temperature range from 50 to 90 degrees C. The kinetic plot of ln([M]0/[M]t) vs time (r2 = 0.99) for HIC-catalyzed bulk ROP of CL was linear, indicating that chain termination did not occur and the propagation rate is first order with respect to monomer concentration. Furthermore, linearity to 90% conversion for M(n) vs fractional CL conversion is consistent with a chain-end propagation mechanism. Deviation from linearity above 90% conversion indicates that a competition between ring-opening chain-end propagation and chain growth by steplike polycondensations takes place at high monomer conversion. HIC was inactive for catalysis of L-lactide and (R,S)-beta-butyrolactone ROP. HIC-catalyzed ROP of epsilon-CL and PDL in toluene were successfully performed, giving high molecular weight poly(epsilon-caprolactone) and omega-poly(pentadecalactone). In addition, the relative activities of immobilized Candida antarctica lipase B (CALB) and HIC for epsilon-CL and PDL polymerizations are reported herein.     

A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.     

Modification of kidney barrier function by the urokinase receptor

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.     

A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology

Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.     

Preparation and characteristics of moulded biodegradable cellulose fibers/MPU-20 composites (CFMCs) by steam injection technology

In this work, the moulded cellulose fibers/MPU-20 composites (CFMCs) with apparent specific gravity lower than 100 kg/m³ and thickness of 20–200 mm have been successfully manufactured using a new design of steam injection technology and equipment. It was found that the CFMCs have good cushioning properties, with a cushion factor lower than 4. Two yield deformation stages were observed in the compressive process. Compressive stress–strain and cushion factor-stain curves were measured as a function of steam injection pressure, transmission time, holding time, MPU-20 resin dosage and apparent specific gravity. Chemical groups, crystallinity, and thermal properties of samples were studied through the use of FTIR spectroscopy, X-ray diffraction (XRD), and DTA–TGA. In addition, the microstructure and morphology were investigated by scanning electron microscope (SEM) and atomic force microscope (AFM).     

Effects of nitrogen deposition on forest biodiversity

Humans have altered global and regional cycles of nitrogen (N) more than any other elements. Increasing N emissions to the atmosphere from accelerating industrialization and production and use of fertilizer N now make N deposition significant not only in densely populated regions of Europe and North America, but also in other parts of the world (e.g., Asia and Latin America). Increased atmospheric N deposition is known to be able to reduce biodiversity in natural and semi-natural ecosystems. It is suggested that N deposition will be the third greatest driver of biodiversity loss on the global scale in this century, after land use and climate change. Based on published study results, we reviewed the impacts of N deposition on forest biodiversity by emphasizing 3 aspects: (1) plant diversity, including arborous plants, understory plants and cryptogam plants; (2) soil microorganism diversity; (3) animal diversity, including underground soil fauna and aboveground herbivores. In general, it was found that N deposition could alter species diversity, and excessive N could reduce species diversity, such as richness and abundance, and even lose special species. We also identified specific mechanisms on how excessive N deposition affected forest biodiversity. Finally, we summarized the current status of research on N deposition in China and in other countries, and proposed potential research activities and recommendations.