Repeated Random Mutagenesis of alpha-Amylase from Bacillus licheniformis for Improved pH Performance

The alpha-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis alpha-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.     

An S/MAR-based L1 retrotransposition cassette mediates sustained levels of insertional mutagenesis without suffering from epigenetic silencing of DNA methylation

L1 is an insertional mutagen that is capable of mediating permanent gene disruption in mammalian genomes. However, currently available L1 retrotransposition vectors exhibit low or unstable transgene expression when expressed in somatic cells and tissues. This restriction limits their potential utility in long-term screening procedures or somatic mutagenesis applications. In this study, we addressed this problem by developing a minicircle, nonviral L1 retrotransposition vector using a scaffold/matrix attachment region (S/MAR) in the vector backbone and evaluated its utility in human cell lines. The S/MAR-based L1 retrotransposition vector provides stable, elevated levels of L1 expression compared to the currently used EBNA1-based L1 vector. In addition, the S/MAR elements effectively mediate sustained levels of L1 retrotransposition in prolonged cell culturing without suffering from epigenetic silencing by DNA methylation or from vector integration problems even in the absence of selection pressure. These findings indicate that the simple inclusion of S/MAR in the vector backbone increased levels of L1 expression and retrotransposition that can be used as an effective tool to generate insertional mutagenesis in large-scale somatic mutagenesis applications in mammalian cells.     

Interpreting Metabolomic Profiles using Unbiased Pathway Models

Human disease is heterogeneous, with similar disease phenotypes resulting from distinct combinations of genetic and environmental factors. Small-molecule profiling can address disease heterogeneity by evaluating the underlying biologic state of individuals through non-invasive interrogation of plasma metabolite levels. We analyzed metabolite profiles from an oral glucose tolerance test (OGTT) in 50 individuals, 25 with normal (NGT) and 25 with impaired glucose tolerance (IGT). Our focus was to elucidate underlying biologic processes. Although we initially found little overlap between changed metabolites and preconceived definitions of metabolic pathways, the use of unbiased network approaches identified significant concerted changes. Specifically, we derived a metabolic network with edges drawn between reactant and product nodes in individual reactions and between all substrates of individual enzymes and transporters. We searched for “active modules”—regions of the metabolic network enriched for changes in metabolite levels. Active modules identified relationships among changed metabolites and highlighted the importance of specific solute carriers in metabolite profiles. Furthermore, hierarchical clustering and principal component analysis demonstrated that changed metabolites in OGTT naturally grouped according to the activities of the System A and L amino acid transporters, the osmolyte carrier SLC6A12, and the mitochondrial aspartate-glutamate transporter SLC25A13. Comparison between NGT and IGT groups supported blunted glucose- and/or insulin-stimulated activities in the IGT group. Using unbiased pathway models, we offer evidence supporting the important role of solute carriers in the physiologic response to glucose challenge and conclude that carrier activities are reflected in individual metabolite profiles of perturbation experiments. Given the involvement of transporters in human disease, metabolite profiling may contribute to improved disease classification via the interrogation of specific transporter activities.     

CSPG is a secreted factor that stimulates neural stem cell survival possibly by enhanced EGFR signaling

Understanding how autocrine/paracrine factors regulate neural stem cell (NSC) survival and growth is fundamental to the utilization of these cells for therapeutic applications and as cellular models for the brain. In vitro, NSCs can be propagated along with neural progenitors (NPs) as neurospheres (nsphs). The nsph conditioned medium (nsph-CM) contains cell-secreted factors that can regulate NSC behavior. However, the identity and exact function of these factors within the nsph-CM has remained elusive. We analyzed the nsph-CM by mass spectrometry and identified DSD-1-proteoglycan, a chondroitin sulfate proteoglycan (CSPG), apolipoprotein E (ApoE) and cystatin C as components of the nsph-CM. Using clonal assays we show that CSPG and ApoE are responsible for the ability of the nsph-CM to stimulate nsph formation whereas cystatin C is not involved. Clonal nsphs generated in the presence of CSPG show more than four-fold increase in NSCs. Thus CSPG specifically enhances the survival of NSCs. CSPG also stimulates the survival of embryonic stem cell (ESC)-derived NSCs, and thus may be involved in the developmental transition of ESCs to NSCs. In addition to its role in NSC survival, CSPG maintains the three dimensional structure of nsphs. Lastly, CSPG's effects on NSC survival may be mediated by enhanced signaling via EGFR, JAK/STAT3 and PI3K/Akt pathways.     

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

Background

Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis.

Methods and Findings

In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.

Conclusions

Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.     

Pattern identification in biogeography

Identifying common patterns among area cladograms that arise in historical biogeography is an important tool for biogeographical inference. We develop the first rigorous formalization of these pattern-identification problems. We develop metrics to compare area cladograms. We define the maximum agreement area cladogram (MAAC) and we develop efficient algorithms for finding the MAAC of two area cladograms, while showing that it is NP-hard to find the MAAC of several binary area cladograms. We also describe a linear-time algorithm to identify if two area cladograms are identical     

Age-dependent characterization of pendrin gene expression in various tissues of deer mice

Pendrin is a membrane transport protein which functions as the transporter of chloride, bicarbonate, formate, and iodide. In this study, we characterized pendrin gene expression in various tissues of deer mice (Peromyscus maniculatus), a sentinel wildlife species. Deer mice were euthanized at post-natal day (PND) 21 (day of weaning) and PND 45 (24 days post-weaning) for tissue collection. A deer mouse-specific partial pendrin cDNA sequence was generated, from which Taqman-specific probe and primers were designed for quantification of mRNA equivalents of pendrin gene expression using real-time polymerase chain reaction (PCR). The expression profile was standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results indicate that the pendrin gene was expressed at different levels in the different tissues of developing deer mice relative to GAPDH expression. Expression in the tissues was determined to be age-dependent. Pendrin gene was highly expressed in the kidney, lungs and reproductive tissues. PND 21 expression in the kidney and testes was significantly lower than PND 45. This study represents the first identification of differential expression of pendrin gene in various deer mouse tissues.     

Disabling of receptor activator of nuclear factor-kappaB (RANK) receptor complex by novel osteoprotegerin-like peptidomimetics restores bone loss in vivo

The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-kappaB (RANK) and osteoprotegerin (OPG), are known to be regulators of development and activation of osteoclasts in bone remodeling. Sustained osteoclast activation that occurs through TRANCE-RANK causes osteopenic disorders such as osteoporosis and contributes to osteolytic metastases. Here, we report a rationally designed small molecule mimic of osteoprotegerin to inhibit osteoclast formation in vitro and limit bone loss in an animal model of osteoporosis. One of the mimetics, OP3-4, significantly inhibited osteoclast formation in vitro (IC(50) = 10 microm) and effectively inhibited total bone loss in ovariectomized mice at a dosage of 2 mg/kg/day. Unlike soluble OPG receptors, which preclude TRANCE binding to RANK, OP3-4 shows the ability to modulate RANK-TRANCE signaling pathways and alters the biological functions of the RANK-TRANCE receptor complex by facilitating a defective receptor complex. These features suggest that OPG-derived small molecules can be used as a probe to understand complex biological functions of RANK-TRANCE-OPG receptors and also can be used as a platform to develop more useful therapeutic agents for inflammation and bone disease.