A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate.     

Flushuffle and Fluresort: new algorithms to identify reassorted strains of the influenza virus by mass spectrometry

ABSTRACT: BACKGROUND: Influenza is one of the oldest and deadliest infectious diseases known to man. Reassorted strains of the virus pose the greatest risk to both human and animal health and have been associated with all pandemics of the past century, with the possible exception of the 1918 pandemic, resulting in tens of millions of deaths. We have developed and tested new computer algorithms, FluShuffle and FluResort, which enable reassorted viruses to be identified by the most rapid and direct means possible. These algorithms enable reassorted influenza, and other, viruses to be rapidly identified to allow prevention strategies and treatments to be more efficiently implemented. RESULTS: The FluShuffle and FluResort algorithms were tested with both experimental and simulated mass spectra of whole virus digests. Flu Shuffle considers different combinations of viral protein identities that match the mass spectral data using a Gibbs sampling algorithm employing a mixed protein Markov chain Monte Carlo (MCMC) method. Flu Resort utilizes those identities to calculate the weighted distance of each across two or more different phylogenetic trees constructed through viral protein sequence alignments. Each weighted mean distance value is normalized by conversion to a Z-score to establish a reassorted strain. CONCLUSIONS: The new Flu Shuffle and Flu Resort algorithms can correctly identify the origins of influenza viral proteins and the number of reassortment events required to produce the strains from the high resolution mass spectral data of whole virus proteolytic digestions. This has been demonstrated in the case of constructed vaccine strains as well as common human seasonal strains of the virus. The algorithms significantly improve the capability of the proteotyping approach to identify reassorted viruses that pose the greatest pandemic risk.     

Major effect of retinal short-chain dehydrogenase reductase (RDHE2) on bovine fat colour

Beef with yellow fat is considered undesirable by consumers in most European and Asian markets. β-Carotene is the major carotenoid deposited in the adipose tissue and milk fat of cattle (Bos taurus), which can result in the yellowness. The effects of retinal short-chain dehydrogenase reductase (RDHE2) and β, β-carotene 9',10-dioxygenase (BCO2) were considered jointly as major candidate genes for causing the yellow fat colour, based on their genomic locations in the fat colour quantitative trait loci (QTL) and their roles in the metabolism of β-carotene. In a secondary pathway, BCO2 cleaves β-carotene into retinoic acid, the most potent form of vitamin A. RDHE2 converts trans-retinol to trans-retinal, a less active form of vitamin A. We evaluated the effects of two amino acid variants of the RDHE2 gene (V6A and V33A) along with a mutation in the BCO2 gene that results in a stop codon (W80X) in seven cattle populations. The RDHE2 V6A genotype affected several fat colour traits but the size of the effect varied in the populations studied. The genotype effect of the RDHE2 V33A variant was observed only in New Zealand samples of unknown breed. In general, the individual effects of RDHE2 V6A and V33A SNPs genotypes were greater in the random New Zealand samples than in samples from pedigreed Jersey-Limousin backcross progeny, accounting for 8-17 % of the variance in one population. Epistasis between the BCO2 W80X and RDHE2 variants was observed, and in some populations this explained more of the variation than the effects of the individual RDHE2 variants.     

Advancing the use of morphometric data for estimating and managing common bottlenose dolphin (Tursiops truncatus) mass

Knowledge of a dolphin's body mass is central to establishing body condition, comparing across individuals, and designing successful management programs. In the present study, sex‐specific prediction equations for estimating body mass were generated from morphometrics (i.e., length and girth) and ages of bottlenose dolphins residing under professionally managed care. Measurements of wild dolphins in Sarasota Bay, Florida, were used to generate sex‐specific body mass reference ranges. Gompertz growth models were fitted to length measurements and age to compare growth across populations. From the regression analyses, the body mass of managed females (R² = 0.937), managed males (R² = 0.953), wild females (R² = 0.979), and wild males (R² = 0.972) were predicted with high levels of accuracy. Managed adults had similar or longer asymptotic lengths compared to their wild conspecifics. To apply this information, ZooMorphTrak, a mobile software application, was developed to provide a new resource for management. The “Approximate” feature was designed to approximate body mass based on user inputs of individual morphometrics. The “Management” feature compared a managed dolphin's known body mass with respect to body mass reference ranges generated from wild dolphins. ZooMorphTrak, developed by the Chicago Zoological Society, is available for download at http://itunes.apple.com .     

Roles of Glutamine Synthetase Inhibition in Epilepsy

Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that is critically involved in several metabolic pathways in the brain, including the glutamine-glutamate-GABA cycle and detoxification of ammonia. GS is normally localized to the cytoplasm of most astrocytes, with elevated concentrations of the enzyme being present in perivascular endfeet and in processes close to excitatory synapses. Interestingly, an increasing number of studies have indicated that the expression, distribution, or activity of brain GS is altered in several brain disorders, including Alzheimer’s disease, schizophrenia, depression, suicidality, and mesial temporal lobe epilepsy (MTLE). Although the metabolic and functional sequelae of brain GS perturbations are not fully understood, it is likely that a deficiency in brain GS will have a significant biological impact due to the critical metabolic role of the enzyme. Furthermore, it is possible that restoration of GS in astrocytes lacking the enzyme could constitute a novel and highly specific therapy for these disorders. The goals of this review are to summarize key features of mammalian GS under normal conditions, and discuss the consequences of GS deficiency in brain disorders, specifically MTLE.     

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A–F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1–3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity.