OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis.     

Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.     

Elevated basal slippage mutation rates among the Canidae

The remarkable responsiveness of dog morphology to selection is a testament to the mutability of mammals. The genetic sources of this morphological variation are largely unknown, but some portion is due to tandem repeat length variation in genes involved in development. Previous analysis of tandem repeats in coding regions of developmental genes revealed fewer interruptions in repeat sequences in dogs than in the orthologous repeats in humans, as well as higher levels of polymorphism, but the fragmentary nature of the available dog genome sequence thwarted attempts to distinguish between locus-specific and genome-wide origins of this disparity. Using whole-genome analyses of the human and recently completed dog genomes, we show that dogs possess a genome-wide increase in the basal germ-line slippage mutation rate. Building on the approach that gave rise to the initial observation in dogs, we sequenced 55 coding repeat regions in 42 species representing 10 major carnivore clades and found that a genome-wide elevated slippage mutation rate is a derived character shared by diverse wild canids, distinguishing them from other Carnivora. A similarly heightened slippage profile was also detected in rodents, another taxon exhibiting high diversity and rapid evolvability. The correlation of enhanced slippage rates with major evolutionary radiations suggests that the possession of a "slippery" genome may bestow on some taxa greater potential for rapid evolutionary change.     

Percutaneous treatment of benign biliary strictures and biliary manometric perfusion test

BACKGROUND: Benign biliary strictures are usually treated surgically or endoscopically. When these strictures are not accessible by endoscope or when open repair is not possible, percutaneous dilatation treatment is indicated. The efficacy of treatment is usually evaluated by clinical trial which includes leaving a small non-functional catheter in situ and following liver function tests. The evaluation may be effectively done by the biliary manometric perfusion test. AIM: The aim of this paper is to emphasize the importance of percutaneous dilatation treatment of benign biliary strictures with focus on the role of the biliary manometric perfusion test and its future prospects. METHODS AND RESULTS: Based on the literature and our own experience, this article gives a short overview of percutaneous treatment of benign biliary strictures, indications, techniques, complications and results. The treatment of these strictures has an overall success rate between 60 to 90%. This article also explains the biliary manometric test, the technique and its importance in evaluation of treatment success. CONCLUSION: Benign biliary strictures of the hepatic duct junction or bilio-enteric anastomosis are difficult to treat surgically and are endoscopically inaccessible. Percutaneous treatment by balloon dilatation and long term external-internal drainage is feasible in the majority of these patients. It is minimally invasive, safe and effective. The evaluation of the treatment success may be more effectively done by the manometric perfusion test. It is easy, reliable, less time-consuming giving immediate results, and relatively safe.     

Polyunsaturated fatty acid metabolites as novel lipidomic biomarkers for noninvasive diagnosis of nonalcoholic steatohepatitis

Lipotoxicity is a key mechanism thought to be responsible for the progression of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). Noninvasive diagnosis of NASH is a major unmet clinical need, and we hypothesized that PUFA metabolites, in particular arachidonic acid (AA)-derived eicosanoids, in plasma would differentiate patients with NAFL from those with NASH. Therefore, we aimed to assess the differences in the plasma eicosanoid lipidomic profile between patients with biopsy-proven NAFL versus NASH versus normal controls without nonalcoholic fatty liver disease (NAFLD; based on MRI fat fraction <5%). We carried out a cross-sectional analysis of a prospective nested case-control study including 10 patients with biopsy-proven NAFL, 9 patients with biopsy-proven NASH, and 10 non-NAFLD MRI-phenotyped normal controls. We quantitatively compared plasma eicosanoid and other PUFA metabolite levels between NAFL versus NASH versus normal controls. Utilizing a uniquely well-characterized cohort, we demonstrated that plasma eicosanoid and other PUFA metabolite profiling can differentiate between NAFL and NASH. The top candidate as a single biomarker for differentiating NAFL from NASH was 11,12-dihydroxy-eicosatrienoic acid (11,12-diHETrE) with an area under the receiver operating characteristic curve (AUROC) of 1. In addition, we also found a panel including 13,14-dihydro-15-keto prostaglandin D₂ (dhk PGD2) and 20-carboxy arachidonic acid (20-COOH AA) that demonstrated an AUROC of 1. This proof-of-concept study provides early evidence that 11,12-diHETrE, dhk PGD2, and 20-COOH AA are the leading eicosanoid candidate biomarkers for the noninvasive diagnosis of NASH.     

miRNA response to DNA damage

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that miRNAs play a crucial role in regulation of DNA damage response. In this review, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.     

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology^1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry⁵ (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells^7-10.Download video file.^{(41M, mov)}