Applying neutral drift to the directed molecular evolution of a β-glucuronidase into a β-galactosidase: Two different evolutionary pathways lead to the same variant

<p>Abstract</p> <p>Background</p> <p>Directed protein evolution has been used to modify protein activity and research has been carried out to enhance the production of high quality mutant libraries. Many theoretical approaches suggest that allowing a population to undergo neutral selection may be valuable in directed evolution experiments.</p> <p>Findings</p> <p>Here we report on an investigation into the value of neutral selection in a classical model system for directed evolution, the conversion of the <it>E. coli </it>β-glucuronidase to a β-galactosidase activity. We find that neutral selection, i.e. selection for retaining glucuronidase activity, can efficiently identify the majority of sites of mutation that have been identified as beneficial for galactosidase activity in previous experiments. Each variant demonstrating increased galactosidase activity identified by our neutral drift experiments contained a mutation at one of four sites, T509, S557, N566 or W529. All of these sites have previously been identified using direct selection for beta galactosidase activity.</p> <p>Conclusions</p> <p>Our results are consistent with others that show that a neutral selection approach can be effective in selecting improved variants. However, we interpret our results to show that neutral selection is, in this case, not a more efficient approach than conventional directed evolution approaches. However, the neutral approach is likely to be beneficial when the resulting library can be screened for a range of related activities. More detailed statistical studies to resolve the apparent differences between this system and others are likely to be a fruitful avenue for future research.</p

Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful

Automatic prediction of catalytic residues by modeling residue structural neighborhood

Background: Prediction of catalytic residues is a major step in characterizing the function of enzymes. In its simpler formulation, the problem can be cast into a binary classification task at the residue level, by predicting whether the residue is directly involved in the catalytic process. The task is quite hard also when structural information is available, due to the rather wide range of roles a functional residue can play and to the large imbalance between the number of catalytic and non-catalytic residues.Results: We developed an effective representation of structural information by modeling spherical regions around candidate residues, and extracting statistics on the properties of their content such as physico-chemical properties, atomic density, flexibility, presence of water molecules. We trained an SVM classifier combining our features with sequence-based information and previously developed 3D features, and compared its performance with the most recent state-of-the-art approaches on different benchmark datasets. We further analyzed the discriminant power of the information provided by the presence of heterogens in the residue neighborhood.Conclusions: Our structure-based method achieves consistent improvements on all tested datasets over both sequence-based and structure-based state-of-the-art approaches. Structural neighborhood information is shown to be responsible for such results, and predicting the presence of nearby heterogens seems to be a promising direction for further improvements.Journal ArticleResearch Support, N.I.H. Extramuralinfo:eu-repo/semantics/publishe

Asenapine effects in animal models of psychosis and cognitive function

Asenapine, a novel psychopharmacologic agent in the development for schizophrenia and bipolar disorder, has high affinity for serotonergic, α-adrenergic, and dopaminergic receptors, suggesting potential for antipsychotic and cognitive-enhancing properties. The effects of asenapine in rat models of antipsychotic efficacy and cognition were examined and compared with those of olanzapine and risperidone. Amphetamine-stimulated locomotor activity (Amp-LMA; 1.0 or 3.0 mg/kg s.c.) and apomorphine-disrupted prepulse inhibition (Apo-PPI; 0.5 mg/kg s.c.) were used as tests for antipsychotic activity. Delayed non-match to place (DNMTP) and five-choice serial reaction (5-CSR) tasks were used to assess short-term spatial memory and attention, respectively. Asenapine doses varied across tasks: Amp-LMA (0.01–0.3 mg/kg s.c.), Apo-PPI (0.001–0.3 mg/kg s.c.), DNMTP (0.01–0.1 mg/kg s.c.), and 5-CSR (0.003–0.3 mg/kg s.c.). Asenapine was highly potent (active at 0.03 mg/kg) in the Amp-LMA and Apo-PPI assays. DNMTP or 5-CSR performance was not improved by asenapine, olanzapine, or risperidone. All agents (P &lt; 0.01) reduced DNMTP accuracy at short delays; post hoc analyses revealed that only 0.1 mg/kg asenapine and 0.3 mg/kg risperidone differed from vehicle. All active agents (asenapine, 0.3 mg/kg; olanzapine, 0.03–0.3 mg/kg; and risperidone, 0.01–0.1 mg/kg) significantly impaired 5-CSR accuracy (P &lt; 0.05). Asenapine has potent antidopaminergic properties that are predictive of antipsychotic efficacy. Asenapine, like risperidone and olanzapine, did not improve cognition in normal rats. Rather, at doses greater than those required for antipsychotic activity, asenapine impaired cognitive performance due to disturbance of motor function, an effect also observed with olanzapine and risperidone

Enhanced Astrocytic Nitric Oxide Production and Neuronal Modifications in the Neocortex of a NOS2 Mutant Mouse

BACKGROUND: It has been well accepted that glial cells in the central nervous system (CNS) produce nitric oxide (NO) through the induction of a nitric oxide synthase isoform (NOS2) only in response to various insults. Recently we described rapid astroglial, NOS2-dependent, NO production in the neocortex of healthy mice on a time scale relevant to neuronal activity. To explore a possible role for astroglial NOS2 in normal brain function we investigated a NOS2 knockout mouse (B6;129P2-Nos2(tm1Lau)/J, Jackson Laboratory). Previous studies of this mouse strain revealed mainly altered immune responses, but no compensatory pathways and no CNS abnormalities have been reported. METHODOLOGY/PRINCIPAL FINDINGS: To our surprise, using NO imaging in brain slices in combination with biochemical methods we uncovered robust NO production by neocortical astrocytes of the NOS2 mutant. These findings indicate the existence of an alternative pathway that increases basal NOS activity. In addition, the astroglial mutation instigated modifications of neuronal attributes, shown by changes in the membrane properties of pyramidal neurons, and revealed in distinct behavioral abnormalities characterized by an increase in stress-related parameters. CONCLUSIONS/SIGNIFICANCE: The results strongly indicate the involvement of astrocytic-derived NO in modifying the activity of neuronal networks. In addition, the findings corroborate data linking NO signaling with stress-related behavior, and highlight the potential use of this genetic model for studies of stress-susceptibility. Lastly, our results beg re-examination of previous studies that used this mouse strain to examine the pathophysiology of brain insults, assuming lack of astrocytic nitrosative reaction

The “conscious pilot”—dendritic synchrony moves through the brain to mediate consciousness

Cognitive brain functions including sensory processing and control of behavior are understood as “neurocomputation” in axonal–dendritic synaptic networks of “integrate-and-fire” neurons. Cognitive neurocomputation with consciousness is accompanied by 30- to 90-Hz gamma synchrony electroencephalography (EEG), and non-conscious neurocomputation is not. Gamma synchrony EEG derives largely from neuronal groups linked by dendritic–dendritic gap junctions, forming transient syncytia (“dendritic webs”) in input/integration layers oriented sideways to axonal–dendritic neurocomputational flow. As gap junctions open and close, a gamma-synchronized dendritic web can rapidly change topology and move through the brain as a spatiotemporal envelope performing collective integration and volitional choices correlating with consciousness. The “conscious pilot” is a metaphorical description for a mobile gamma-synchronized dendritic web as vehicle for a conscious agent/pilot which experiences and assumes control of otherwise non-conscious auto-pilot neurocomputation

Gastrointestinal decontamination in the acutely poisoned patient

ObjectiveTo define the role of gastrointestinal (GI) decontamination of the poisoned patient.Data sourcesA computer-based PubMed/MEDLINE search of the literature on GI decontamination in the poisoned patient with cross referencing of sources.Study selection and data extractionClinical, animal and in vitro studies were reviewed for clinical relevance to GI decontamination of the poisoned patient.Data synthesisThe literature suggests that previously, widely used, aggressive approaches including the use of ipecac syrup, gastric lavage, and cathartics are now rarely recommended. Whole bowel irrigation is still often recommended for slow-release drugs, metals, and patients who "pack" or "stuff" foreign bodies filled with drugs of abuse, but with little quality data to support it. Activated charcoal (AC), single or multiple doses, was also a previous mainstay of GI decontamination, but the utility of AC is now recognized to be limited and more time dependent than previously practiced. These recommendations have resulted in several treatment guidelines that are mostly based on retrospective analysis, animal studies or small case series, and rarely based on randomized clinical trials.ConclusionsThe current literature supports limited use of GI decontamination of the poisoned patient

Descending controls modulate inflammatory joint pain and regulate CXC chemokine and iNOS expression in the dorsal horn.

Descending control of nociceptive processing, by pathways originating in the rostral ventromedial medulla (RVM) and terminating in the dorsal horn, contributes to behavioural hypersensitivity in a number of pain models. Two facilitatory pathways have been identified and are characterized by serotonin (5-HT) content or expression of the mu opiate receptor. Here we investigated the contribution of these pathways to inflammatory joint pain behaviour and gene expression changes in the dorsal horn