Iatrogenic Cushing's syndrome on F-FDG-PET/CT: A pitfall in metabolic assessment of oncologic response.

Iatrogenic Cushing's syndrome has very well-known stigmata on physical examination but can pose a diagnostic challenge when it rarely presents radiologically. We present a classic albeit rarely encountered imaging appearance of Iatrogenic Cushing's on F-FDG-PET/CT, with diffuse subcutaneous white adipose proliferation and metabolic activation in a 7-year old patient one-month after starting a high dose steroid regimen for lymphoma. There was an extreme shift in the expected FDG biodistribution with dominant localization to the subcutaneous adipose tissue.

The Silent Sway of Splicing by Synonymous Substitutions.

Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation.

Directed DNA shuffling of retrovirus and retrotransposon integrase protein domains.

Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR.

Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53.

The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive.

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants"). Activity can be restored by second-site suppressor mutations ("rescue mutants"). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids.

All-codon scanning identifies p53 cancer rescue mutations.

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes.

Recombinant human collagen and biomimetic variants using a de novo gene optimized for modular assembly.

A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants.

Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex.

Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC.

Predicting positive p53 cancer rescue regions using Most Informative Positive (MIP) active learning.

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results.

Amino Acid Metabolism and Fluxes.

By the mid1960s, the pioneering work of Umbarger and Gerhart and Pardee had shown us that carbon flow through a biosynthetic pathway was controlled by allosteric inhibition of the first enzyme of the pathway by its end product; and, studies of the lac operon by Jacob and Monod had established that genes were controlled by an operator-repressor mechanism. During the intervening forty-plus years, knowledge and technologies have continued to explode in unanticipated ways. Today, we understand in great detail the molecular mechanisms of the many levels of metabolic and genetic regulation that control carbon flow through the amino acid biosynthetic pathways.

Computationally Optimised DNA Assembly of synthetic genes.

Gene synthesis is hampered by two obstacles: improper assembly of oligonucleotides; oligonucleotide defects incurred during chemical synthesis. To overcome the first problem, we describe the employment of a Computationally Optimised DNA Assembly (CODA) algorithm that uses the degeneracy of the genetic code to design overlapping oligonucleotides with thermodynamic properties for self-assembly into a single, linear, DNA product. To address the second problem, we describe a hierarchical assembly strategy that reduces the incorporation of defective oligonucleotides into full-length gene constructs.

Ty3 nucleocapsid controls localization of particle assembly.

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX(2)CX(4)HX(4)C zinc-binding motif found in retrovirus NC proteins.

How noisy and replicable are DNA microarry data?

This paper analyses variability in highly replicated measurements of DNA microarray data conducted on nylon filters and Affymetrix GeneChips with different cDNA targets, filters, and imaging technology. Replicability is assessed quantitatively using correlation analysis as a global measure and differential expression analysis and ANOVA at the level of individual genes.

TY3 GAG3 protein forms ordered particles in Escherichia coli.

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly.

Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering.

Translation Engineering combined with synthetic biology (gene synthesis) techniques makes it possible to deliberately alter the presumed translation kinetics of genes without altering the amino acid sequence. Here, we describe proprietary technologies that design and assemble synthetic genes for high expression and enhanced protein production, and offers new insights and methodologies for affecting protein structure and function. We have patented Translation Engineering technologies to manage the complexity of gene design to account for codon pair usage, translational pausing signals, RNA secondary structure and user-defined sequences such as restriction sites.

Ty3 capsid mutations reveal early and late functions of the amino-terminal domain.

The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles.

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface.

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al.

DNA deformation energy as an indirect recognition mechanism in protein-DNA interactions.

Proteins that bind to specific locations in genomic DNA control many basic cellular functions. Proteins detect their binding sites using both direct and indirect recognition mechanisms. Deformation energy, which models the energy required to bend DNA from its native shape to its shape when bound to a protein, has been shown to be an indirect recognition mechanism for one particular protein, Integration Host Factor (IHF).

GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine.

The present study evaluates immunogenicity and protection potency of a codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the three GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine.

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy.

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif.

HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae.

We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers.

Application of a generalized MWC model for the mathematical simulation of metabolic pathways regulated by allosteric enzymes.

In our effort to elucidate the systems biology of the model organism, Escherichia coli, we have developed a mathematical model that simulates the allosteric regulation for threonine biosynthesis pathway starting from aspartate. To achieve this goal, we used kMech, a Cellerator language extension that describes enzyme mechanisms for the mathematical modeling of metabolic pathways. These mechanisms are converted by Cellerator into ordinary differential equations (ODEs) solvable by Mathematica.

Application of a generalized MWC model for the mathematical simulation of metabolic pathways regulated by allosteric enzymes.

In our effort to elucidate the systems biology of the model organism, Escherichia coli, we have developed a mathematical model that simulates the allosteric regulation for threonine biosynthesis pathway starting from aspartate. To achieve this goal, we used kMech, a Cellerator language extension that describes enzyme mechanisms for the mathematical modeling of metabolic pathways. These mechanisms are converted by Cellerator into ordinary differential equations (ODEs) solvable by Mathematica.