Reconstruction of a genome-scale metabolic model and in-silico flux analysis of Aspergillus tubingensis: a non-mycotoxinogenic citric acid-producing fungus.

BACKGROUND: Aspergillus tubingensis is a citric acid-producing fungus that can utilize sugars in hydrolysate of lignocellulosic biomass such as sugarcane bagasse and, unlike A. niger, does not produce mycotoxins. To date, no attempt has been made to model its metabolism at genome scale. RESULTS: Here, we utilized the whole-genome sequence (34.96 Mb length) and the measured biomass composition to reconstruct a genome-scale metabolic model (GSMM) of A. tubingensis DJU120 strain. The model, named iMK1652, consists of 1652 genes, 1657 metabolites and 2039 reactions distributed over four cellular compartments. The model has been extensively curated manually. This included removal of dead-end metabolites and generic reactions, addition of secondary metabolite pathways and several transporters. Several mycotoxin synthesis pathways were either absent or incomplete in the genome, providing a genomic basis for the non-toxinogenic nature of this species. The model was further refined based on the experimental phenotypic microarray (Biolog) data. The model closely captured DJU120 fermentative data on glucose, xylose, and phosphate consumption, as well as citric acid and biomass production, showing its applicability to capture citric acid fermentation of lignocellulosic biomass hydrolysate. CONCLUSIONS: The model offers a framework to conduct metabolic systems biology investigations and can act as a scaffold for integrative modelling of A. tubingensis.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses.

BACKGROUND: Marine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO2, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses. RESULTS: In this work, we cultured the marine cyanobacterium Synechococcus sp. PCC 7002 in a 3-L externally illuminated bioreactor with working volume of 2 L with a biomass productivity of ~ 0.8 g L-1 day-1. Hydrolysis of the biomass with acids released proteins and hydrolyzed glycogen while hydrolysis of the biomass with base released only proteins but did not hydrolyze glycogen. Among the different acids tested, treatment with HNO3 led to the highest release of proteins and glucose. Cyanobacterial biomass hydrolysate (CBH) prepared in HNO3 was used as a medium to produce cellulase enzyme by the Penicillium funiculosum OAO3 strain while CBH prepared in HCl and treated with charcoal was used as a medium for citric acid by Aspergillus tubingensis. Approximately 50% higher titers of both products were obtained compared to traditional media. CONCLUSIONS: These results show that the hydrolysate of marine cyanobacteria is an effective source of nutrients/proteins for fungal bioprocesses.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate.

BACKGROUND: Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. RESULTS: The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. CONCLUSIONS: In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

In silico evolution of Aspergillus niger organic acid production suggests strategies for switching acid output.

BACKGROUND: The fungus Aspergillus niger is an important industrial organism for citric acid fermentation; one of the most efficient biotechnological processes. Previously we introduced a dynamic model that captures this process in the industrially relevant batch fermentation setting, providing a more accurate predictive platform to guide targeted engineering. In this article we exploit this dynamic modelling framework, coupled with a robust genetic algorithm for the in silico evolution of A. niger organic acid production, to provide solutions to complex evolutionary goals involving a multiplicity of targets and beyond the reach of simple Boolean gene deletions. We base this work on the latest metabolic models of the parent citric acid producing strain ATCC1015 dedicated to organic acid production with the required exhaustive genomic coverage needed to perform exploratory in silico evolution. RESULTS: With the use of our informed evolutionary framework, we demonstrate targeted changes that induce a complete switch of acid output from citric to numerous different commercially valuable target organic acids including succinic acid. We highlight the key changes in flux patterns that occur in each case, suggesting potentially valuable targets for engineering. We also show that optimum acid productivity is achieved through a balance of organic acid and biomass production, requiring finely tuned flux constraints that give a growth rate optimal for productivity. CONCLUSIONS: This study shows how a genome-scale metabolic model can be integrated with dynamic modelling and metaheuristic algorithms to provide solutions to complex metabolic engineering goals of industrial importance. This framework for in silico guided engineering, based on the dynamic batch growth relevant to industrial processes, offers considerable potential for future endeavours focused on the engineering of organisms to produce valuable products.

An accurate description of Aspergillus niger organic acid batch fermentation through dynamic metabolic modelling.

BACKGROUND: Aspergillus niger fermentation has provided the chief source of industrial citric acid for over 50 years. Traditional strain development of this organism was achieved through random mutagenesis, but advances in genomics have enabled the development of genome-scale metabolic modelling that can be used to make predictive improvements in fermentation performance. The parent citric acid-producing strain of A. niger, ATCC 1015, has been described previously by a genome-scale metabolic model that encapsulates its response to ambient pH. Here, we report the development of a novel double optimisation modelling approach that generates time-dependent citric acid fermentation using dynamic flux balance analysis. RESULTS: The output from this model shows a good match with empirical fermentation data. Our studies suggest that citric acid production commences upon a switch to phosphate-limited growth and this is validated by fitting to empirical data, which confirms the diauxic growth behaviour and the role of phosphate storage as polyphosphate. CONCLUSIONS: The calibrated time-course model reflects observed metabolic events and generates reliable in silico data for industrially relevant fermentative time series, and for the behaviour of engineered strains suggesting that our approach can be used as a powerful tool for predictive metabolic engineering.

Examining the interaction between cognitive control and reward sensitivity in substance use dependence.

BACKGROUND: Drug dependence is characterized by altered reward processing and poor cognitive control, expressed as a preference for immediate rewards and impaired inhibitory control, respectively. To examine the interaction between reward processing (via the presence or absence of reward) and mechanisms of inhibitory control in drug dependence, the current study used the Monetary Incentive Control Task (MICT) to examine whether a group of opiate dependent persons demonstrated greater difficulty exerting control over immediate rewards compared to neutral stimuli. METHODS: The MICT is a Go/Stop paradigm that examines inhibitory control over immediate rewards. Performance of 32 opiate dependent individuals was compared to 29 healthy controls. RESULTS: Opiate users demonstrated poorer inhibitory performance than controls, irrespective of cues signaling immediate reward. Whereas control participants' responses were modulated by probability cues, the opiate group did not show a capacity to up-regulate their cognitive control performance. CONCLUSIONS: The present results suggest a general decrease in cognitive control in opiate dependence, accompanied by a reduced ability to optimally modulate behavior in accordance with external cues. Opiate users and controls did not differ in the interaction between cognitive control and reward. The study highlights important issues for future research to consider when further examining this interaction in drug dependence.

An improved cognitive model of the Iowa and Soochow Gambling Tasks with regard to model fitting performance and tests of parameter consistency.

The Iowa Gambling Task (IGT) and the Soochow Gambling Task (SGT) are two experience-based risky decision-making tasks for examining decision-making deficits in clinical populations. Several cognitive models, including the expectancy-valence learning (EVL) model and the prospect valence learning (PVL) model, have been developed to disentangle the motivational, cognitive, and response processes underlying the explicit choices in these tasks. The purpose of the current study was to develop an improved model that can fit empirical data better than the EVL and PVL models and, in addition, produce more consistent parameter estimates across the IGT and SGT. Twenty-six opiate users (mean age 34.23; SD 8.79) and 27 control participants (mean age 35; SD 10.44) completed both tasks. Eighteen cognitive models varying in evaluation, updating, and choice rules were fit to individual data and their performances were compared to that of a statistical baseline model to find a best fitting model. The results showed that the model combining the prospect utility function treating gains and losses separately, the decay-reinforcement updating rule, and the trial-independent choice rule performed the best in both tasks. Furthermore, the winning model produced more consistent individual parameter estimates across the two tasks than any of the other models.

Motivationally significant self-control: enhanced action withholding involves the right inferior frontal junction.

In everyday life, people use self-control to withhold actions. This ability is particularly important when the consequences of action withholding have an impact on the individual's well-being. Despite its importance, it is unclear as to how the neural nodes implicated in action withholding contribute to this real-world type of self-control. By modifying an action withholding paradigm, the go/no-go task, we examined how the brain exerts self-control during a scenario in which the implications of withholding an action are meaningful and motivationally significant. A successfully withheld response contributed to long-term monetary rewards, whereas failure to withhold a response incurred an immediate monetary punishment. Compared with neutral action withholding, participants significantly improved their performance when these contingencies were applied. Crucially, although the right IFG and pre-SMA were found to promote overall action withholding, the enhancement in behavioral performance relative to a neutral condition was only reflected by a physiological change in a region encompassing the right inferior frontal junction and precentral gyrus. We speculate that the ability to flexibly modulate attention to goal-relevant stimuli is crucial to enhanced, motivationally driven action withholding and that this ability is subserved by the right inferior frontal junction. These findings suggest that control-modulating factors, rather than action withholding processes per se, can be critical to improving motivationally significant action withholding outcomes.

Comparing the Iowa and soochow gambling tasks in opiate users.

The Iowa Gambling Task (IGT) is in many respects the gold standard for demonstrating decision making in drug using groups. However, it is not clear how basic task properties such as the frequency and magnitude of rewards and losses affect choice behavior in drug users and even in healthy players. In this study, we used a variant of the IGT, the Soochow Gambling Task (SGT), to observe choice behavior in opiate users and healthy decision makers in a task where reward frequency is not confounded with the long-term outcome of each alternative. In both opiate users (n = 26) and healthy controls (n = 27), we show that reward frequency strongly influences choice behavior in the IGT and SGT. Neither group showed a consistent preference across tasks for alternatives with good long-term outcomes, but rather, subjects appeared to prefer alternatives that win most frequently. We interpret this as evidence to suggest that healthy players perform better than opiate users on the IGT because they are able to utilize gain-loss frequencies to guide their choice behavior on the task. This challenges the previous notion that poorer performance on the IGT in drug users is due to an inability to be guided by future consequences.

Stop task after-effects in schizophrenia: behavioral control adjustments and repetition priming.

Stop task after-effects are behavioral consequences of response inhibition (i.e., slowed response time), and may index both behavioral control adjustments and repetition priming. Patients with schizophrenia and healthy controls completed a stop task, and responses to the go signal were analyzed according to characteristics of the immediately preceding trial. Schizophrenia was associated with reduced slowing following unsuccessful response inhibition, however there was no evidence of impairments in repetition priming. These results support neurocognitive models of schizophrenia that suggest an absence or reduction of behavioral adjustments (perhaps reflecting impaired error detection), but are inconsistent with current retrieval-based repetition priming accounts.

Propensity for risk taking and trait impulsivity in the Iowa Gambling Task.

The Iowa Gambling Task (IGT) is sensitive to decision making impairments in several clinical groups with frontal impairment. However the complexity of the IGT, particularly in terms of its learning requirements, makes it difficult to know whether disadvantageous (risky) selections in this task reflect deliberate risk taking or a failure to recognise risk. To determine whether propensity for risk taking contributes to IGT performance, we correlated IGT selections with a measure of propensity for risk taking from the Balloon Analogue Risk Task (BART), taking into account potential moderating effects of IGT learning requirements, and trait impulsivity, which is associated with learning difficulties. We found that IGT and BART performance were related, but only in the later stages of the IGT, and only in participants with low trait impulsivity. This finding suggests that IGT performance may reflect different underlying processes in individuals with low and high trait impulsivity. In individuals with low trait impulsivity, it appears that risky selections in the IGT reflect in part, propensity for risk seeking, but only after the development of explicit knowledge of IGT risks after a period of learning.

ERP correlates of response inhibition after-effects in the stop signal task.

Several studies have found that response inhibition in the stop signal task is associated with a delay in subsequent response speed, which may result from the automatic retrieval of a conflicting stimulus-goal association. This study investigated the neurophysiological correlates of this sequence effect using event related potentials (ERPs). ERPs were recorded in 17 healthy people while they performed the stop signal task. We found reduced P3b amplitude for responses following successful inhibition, but only when the stimulus was repeated from the previous trial (repetition-after-effects). For responses following failed inhibition, P3b amplitude was reduced regardless of stimulus repetition status. We also found a general increase in frontal N2 amplitude on response trials following inhibition, regardless of stimulus repetition or behavioural slowing. The complex pattern of ERP findings, dependent on stimulus repetition and success of inhibition, suggests multiple sources of behavioural slowing in the present data. ERP findings suggest that a memory retrieval processes underlies the repetition component of inhibition after effects. These findings are considered within the broader context of ERP findings in the negative priming literature.

A combined rTMS and ERP investigation of dorsolateral prefrontal cortex involvement in response inhibition.

The stop signal task is used to investigate inhibition of an initiated response. Converging evidence suggests that right inferior prefrontal cortex is involved in this behavior, although other regions in the prefrontal cortex have also been implicated. One technique used to determine the contribution of specific cortical regions to behavior is repetitive transcranial magnetic stimulation (rTMS). In the present study, fourteen subjects performed the stop signal task before and after receiving a train of rTMS to the left and right dorsolateral prefrontal cortex (DLPFC). The effects of rTMS were determined using event-related potential (ERP) measures that have been associated with response inhibition in previous studies. Stimulation of left and right DLPFC did not affect ERP measures of response inhibition. This negative finding is interpreted with caution, but is consistent with a recent study which found that stimulation of the same region had no effect on a behavioral measure of response inhibition.

Stop task after-effects: the extent of slowing during the preparation and execution of movement.

In the stop task, response time to the go signal is increased when the immediately preceding trial involves the presentation of a stop signal. A recent explanation suggests that these "after-effects" are due to mechanisms that occur prior to the completion of response selection processes, but it is possible that they instead may reflect a slowed motor response (i.e., deliberate slowing after response selection). The participants completed a novel stop task that allows a differentiation between the time taken to prepare a movement (which incorporates response selection processes) and the time taken to execute a movement (i.e., speed of motor response). If mechanisms underlying stop task after-effects occur prior to the completion of response selection processes, then slowing should only occur during movement preparation. Movement preparation and execution time during go trials were analysed according to the characteristics of the preceding trial. Slowing after a stop trial was found during movement preparation time (regardless of inhibition success on that stop trial), and it further increased during this period when the primary task stimulus was repeated. There was also evidence for general after-effects during movement execution time, but no effect of repetition. These findings support the current theoretical accounts that suggest that repetition-based stop task after-effects are attributable to a mechanism that occurs prior to the completion of response selection processes, and also indicate a possible switch to a more conservative response set (as in signal detection theory terms) that results in deliberate slowing of movement.

A study of intensity dependence of the auditory evoked potential (IDAEP) in medicated melancholic and non-melancholic depression.

BACKGROUND: Major Depressive Disorder is widely recognised to be a heterogeneous syndrome with numerous depressive phenotypes, one of which is melancholic depression. Patients with melancholic depression exhibit treatment responses and outcomes that differ from patients with non-melancholic depression. The current study aimed to assess whether differences existed between melancholic and non-melancholic subtypes of depression, as measured by the event related potential, intensity dependence of the auditory evoked potential (IDAEP). METHODS: IDAEP was assessed in 14 melancholic and 13 non-melancholic depressed subjects and 14 controls. RESULTS: The melancholic patients had a significantly shallower IDAEP slope than the non-melancholic patients not explained by depression severity or age. LIMITATIONS: Antidepressants were taken by all patients in this study and the effect of continual use of these drugs on the IDAEP slopes has yet to be confirmed. CONCLUSIONS: These results provide support for neurobiological differences between melancholic and non-melancholic depressive subtypes. Melancholic depression may be characterized by ongoing over function of the serotonin system in spite of medication treatment.

Cortical inhibition in motor and non-motor regions: a combined TMS-EEG study.

A number of studies using paired pulse transcranial magnetic stimulation (TMS) have demonstrated that cortical inhibition (CI) of the motor cortex can be recorded and also gauged through surface electromyography. However, recording CI from other brain regions that are more directly related with the pathophysiology of some neurologic and psychiatric disorders (e.g., dorsolateral prefrontal cortex (DLPFC) in schizophrenia) was previously fraught with technical difficulties. This study was therefore designed to examine, through a combination of TMS with EEG, whether CI could be measured directly from the motor cortex, DLPFC, and another non-motor region. To index CI, long interval cortical inhibition (LICI; a TMS paradigm) was used in the motor cortex and DLPFC in 14 healthy subjects, and in the parietal lobe in 5 of those subjects. In the motor cortex, LICI resulted in a significant suppression in mean cortical evoked activity on EEG (37.31 +/- 47.51%). In the DLPFC, LICI resulted in a significant suppression (32.45 +/- 47.86%) in mean cortical evoked activity and did not correlate with LICI in the motor cortex although they did not significantly differ. In the parietal lobe, LICI resulted in significant suppression (47.76 +/- 44.70%) in mean cortical evoked activity. In conclusion, CI in the dorsolateral prefrontal cortex, motor cortex and parietal cortex were similar at 120% of motor threshold. These data suggest that CI can be recorded by combining TMS with EEG and may facilitate future research attempting to ascertain the role of CI in the pathophysiology of several neurologic and psychiatric disorders.

Chemoarchitecture of the middle temporal visual area in the marmoset monkey (Callithrix jacchus): laminar distribution of calcium-binding proteins (calbindin, parvalbumin) and nonphosphorylated neurofilament.

We studied the distributions of interneurons containing the calcium-binding proteins parvalbumin and calbindin D-28k, as well as that of pyramidal neurons containing nonphosphorylated neurofilament (NNF), in the middle temporal visual area (MT) of marmoset monkeys. The distributions of these classes of cells in MT are distinct from those found in adjacent areas. Similar to the primary visual area (V1), in MT, calbindin-immunopositive neurons can be objectively classified into "dark" and "light" subtypes based on optical density of stained cell bodies. Calbindin-positive dark neurons are particularly concentrated in layers 2 and 3, whereas light neurons have a more widespread distribution. In addition, a subcategory of calbindin-positive dark neuron, characterized by a "halo" of stained processes surrounding the cell body, is found within and around layer 4 of MT and V1. These cells are rare in most other visual areas. In comparison, parvalbumin-immunopositive cells in area MT have a relatively homogeneous distribution, although with a trend toward higher spatial density in lower layer 3, and are relatively uniform in terms of density of staining. Finally, MT shows a characteristic trilaminar distribution of NNF-immunopositive pyramidal cells, with stained cell bodies evident in layers 3, 5, and 6. Although the laminar distribution of cells stained for the three markers overlap to some extent, these subcategories can be readily distinguished in terms of morphology, including cell body size. Chemoarchitectural parallels observed between MT and V1 suggest comparable physiological requirements and neuronal circuitry.