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.     

An accurate definition of the status of inactive hepatitis B virus carrier by a combination of biomarkers (FibroTest-ActiTest) and viral load

Background

The combination of transaminases (ALT), biopsy, HBeAg and viral load have classically defined the inactive status of carriers of chronic hepatitis B. The use of FibroTest (FT) and ActiTest (AT), biomarkers of fibrosis and necroinflammatory activity, has been previously validated as alternatives to biopsy. We compared the 4-year prognostic value of combining FT-AT and viral load for a better definition of the inactive carrier status.

Methods and Findings

1,300 consecutive CHB patients who had been prospectively followed since 2001 were pre-included. The main endpoint was the absence of liver-related complications, transplantation or death. We used the manufacturers'' definitions of normal FT (< = 0.27), normal AT (< = 0.29) and 3 standard classes for viral load. The adjustment factors were age, sex, HBeAg, ethnic origin, alcohol consumption, HIV-Delta-HCV co-infections and treatment.

Results

1,074 patients with baseline FT-AT and viral load were included: 41 years old, 47% African, 27% Asian, 26% Caucasian. At 4 years follow-up, 50 complications occurred (survival without complications 93.4%), 36 deaths occurred (survival 95.0%), including 27 related to HBV (survival 96.1%). The prognostic value of FT was higher than those of viral load or ALT when compared using area under the ROC curves [0.89 (95%CI 0.84–0.93) vs 0.64 (0.55–0.71) vs 0.53 (0.46–0.60) all P<0.001], survival curves and multivariate Cox model [regression coefficient 5.2 (3.5–6.9; P<0.001) vs 0.53 (0.15–0.92; P = 0.007) vs −0.001 (−0.003−0.000;P = 0.052)] respectively. A new definition of inactive carriers was proposed with an algorithm combining “zero” scores for FT-AT (F0 and A0) and viral load classes. This new algorithm provides a 100% negative predictive value for the prediction of liver related complications or death. Among the 275 patients with the classic definition of inactive carrier, 62 (23%) had fibrosis presumed with FT, and 3 died or had complications at 4 year.

Conclusion

In patients with chronic hepatitis B, a combination of FibroTest-ActiTest and viral load testing accurately defined the prognosis and the inactive carrier status.     

Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites

Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.     

Conflicting mitochondrial and nuclear phylogeographic signals and evolution of host-plant shifts in the boreo-montane leaf beetle Chrysomela lapponica

We conducted a phylogeographic study on the cold-adapted leaf beetle Chrysomela lapponica, that feeds on willow or birch, by sampling several populations throughout most of the geographic distribution of the species, and by sequencing for each individual one mitochondrial and two nuclear DNA fragments. Patterns of DNA sequence variation from the mitochondrial and nuclear loci, as displayed in the median-joining networks, appear to display contradicting historical signal: a deep genealogical divergence is observed with the mitochondrial genome between the Alpine population and all other populations found in the Euro-Siberian distribution of the species, that is completely absent with both nuclear loci. We use coalescence simulations of DNA sequence evolution to test the hypothesis that this apparent conflict is compatible with a neutral model of sequence evolution (i.e., to check whether the stochastic nature of the coalescence process can explain these patterns). Because the simulations show that this is highly unlikely, we consider two alternative hypotheses: (1) introgression of the mitochondrial genome of another species and (2) the effect of natural selection. Although introgression is the most plausible explanation, we fail to identify the source species of the introgressed mitochondrial genome among all known species closely related to C. lapponica. We therefore suggest that the putative introgression event is ancient and the source species is either extinct or currently outside the geographic range of C. lapponica explored in this study. The observed DNA sequence variation also suggests that a host-plant shift from willow to birch has occurred recently and independently in each of the three birch-feeding populations. This emphasizes further the relative ease with which these beetles can escape their ancestral host-plant specialization on willow, but shows at the same time that host-plant shifts are highly constrained, as they only occur between willow and birch.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Lymphocytic Choriomeningitis Virus Infection in FVB Mouse Produces Hemorrhagic Disease

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-γ, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVB×C57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing.     

In vitro concentration dependent transport of phenytoin and phenobarbital,but not ethosuximide,by human P-glycoprotein

AimsOne possible mechanism for epilepsy drug resistance is overexpression of P-glycoprotein in the blood–brain barrier, but whether (or which) antiepileptic drugs (AEDs) are transported by P-gp remains unclear. We evaluated AEDs as P-gp substrates using cell monolayers.Main methodsBi-directional transport assays and concentration equilibrium transport assays (CETAs) were performed for phenytoin (PHT), phenobarbital (PB), and ethosuximide (ESM) using wildtype Madin–Darby Canine Kidney II cell line MDCKII and porcine renal endothelial cell line LLC–PK1 cells and these cells transfected with human MDR1 cDNA to express P-gp.Key findingsWildtype cells demonstrated no efflux transport of PHT, PB, or ESM. In CETAs, both MDR1-transfected cell lines transported PHT from basolateral to apical when PHT loading concentrations were 5 or 10, but not 20 µg/ml. MDCK–MDR1 cells transported PB when initial concentrations were 10 or 20, but not 5 µg/ml. LLC–MDR1 did not transport PB. P-gp inhibitor verapamil blocked efflux transport. MDR1-transfected cells did not transport ESM at 5.6 or 56 µg/ml. Bi-directional transport assays demonstrated weak transport for PHT but not PB or ESM.SignificanceHuman P-gp transports PHT and PB, but not ESM, in a concentration dependent manner. CETA may be more sensitive than bi-directional assays to detect transport of drugs with high passive diffusion. Potential P-gp substrates should be tested at clinically relevant concentration ranges.