A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis.

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

Dissecting the metabolic reprogramming of maize root under nitrogen-deficient stress conditions.

The growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through the root. Hence, studying its growth, response, and associated metabolic reprogramming to stress conditions is becoming an important research direction. A genome-scale metabolic model (GSM) for the maize root was developed to study its metabolic reprogramming under nitrogen stress conditions. The model was reconstructed based on the available information from KEGG, UniProt, and MaizeCyc. Transcriptomics data derived from the roots of hydroponically grown maize plants were used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N+) and nitrogen-deficient (N-) condition. Model-predicted flux-sum variability analysis achieved 70% accuracy compared with the experimental change of metabolite levels. In addition to predicting important metabolic reprogramming in central carbon, fatty acid, amino acid, and other secondary metabolism, maize root GSM predicted several metabolites (l-methionine, l-asparagine, l-lysine, cholesterol, and l-pipecolate) playing a regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidylcholine and phosphatidylglycerol metabolites which, even though not coupled with biomass production, played a key role in the increased biomass production under N-deficient conditions. Overall, the omics-integrated GSM provides a promising tool to facilitate stress condition analysis for maize root and engineer better stress-tolerant maize genotypes.

Current State, Challenges, and Opportunities in Genome-Scale Resource Allocation Models: A Mathematical Perspective.

Stoichiometric genome-scale metabolic models (generally abbreviated GSM, GSMM, or GEM) have had many applications in exploring phenotypes and guiding metabolic engineering interventions. Nevertheless, these models and predictions thereof can become limited as they do not directly account for protein cost, enzyme kinetics, and cell surface or volume proteome limitations. Lack of such mechanistic detail could lead to overly optimistic predictions and engineered strains. Initial efforts to correct these deficiencies were by the application of precursor tools for GSMs, such as flux balance analysis with molecular crowding. In the past decade, several frameworks have been introduced to incorporate proteome-related limitations using a genome-scale stoichiometric model as the reconstruction basis, which herein are called resource allocation models (RAMs). This review provides a broad overview of representative or commonly used existing RAM frameworks. This review discusses increasingly complex models, beginning with stoichiometric models to precursor to RAM frameworks to existing RAM frameworks. RAM frameworks are broadly divided into two categories: coarse-grained and fine-grained, with different strengths and challenges. Discussion includes pinpointing their utility, data needs, highlighting framework strengths and limitations, and appropriateness to various research endeavors, largely through contrasting their mathematical frameworks. Finally, promising future applications of RAMs are discussed.

Using EuGeneCiD and EuGeneCiM computational tools for synthetic biology.

Synthetic biology often relies on the design of genetic circuits, utilizing "bioparts" (modular DNA pieces) to accomplish desired responses to external stimuli. While such designs are usually intuited, detailed here is a computational approach to synthetic biology design and modeling using optimization-based tools named Eukaryotic Genetic Circuit Design and Modeling. These allow for designing and subsequent screening of genetic circuits to increase the chances of in vivo success and contribute to the development of an application development pipeline. For complete details on the use and execution of this protocol, please refer to Schroeder, Baber, and Saha (2021).

Optimization-based Eukaryotic Genetic Circuit Design (EuGeneCiD) and modeling (EuGeneCiM) tools: Computational approach to synthetic biology.

Synthetic biology has the potential to revolutionize the biotech industry and our everyday lives and is already making an impact. Developing synthetic biology applications requires several steps including design and modeling efforts which may be performed by in silico tools. In this work, we have developed two such tools, Eukaryotic Genetic Circuit Design (EuGeneCiD) and Modeling (EuGeneCiM), which use optimization concepts and bioparts including promotors, transcripts, and terminators in designing and modeling genetic circuits. EuGeneCiD and EuGeneCiM preclude problematic designs leading to future synthetic biology application development pipelines. EuGeneCiD and EuGeneCiM are applied to developing 30 basic logic gates as genetic circuit conceptualizations which respond to heavy metal ions pairs as input signals for Arabidopsis thaliana. For each conceptualization, hundreds of potential solutions were designed and modeled. Demonstrating its time-dependence and the importance of including enzyme and transcript degradation in modeling, EuGeneCiM is used to model a repressilator circuit.

Gesture subtype-dependent left lateralization of praxis planning: an event-related fMRI study.

Ideomotor apraxia is a disorder mainly of praxis planning, and the deficit is typically more evident in pantomiming transitive (tool related) than intransitive (communicative) gestures. The goal of the present study was to assess differential hemispheric lateralization of praxis production using event-related functional magnetic resonance imaging. Voxel-based analysis demonstrated significant activations in posterior parietal cortex (PPC) and premotor cortex (PMC) association areas, which were predominantly left hemispheric, regardless of whether planning occurred for right or left hand transitive or intransitive pantomimes. Furthermore, region of interest-based calculation of mean laterality index (LI) revealed a significantly stronger left lateralization in PPC/PMC clusters for planning intransitive (LI = -0.49 + 0.10, mean + standard deviation [SD]) than transitive gestures (-0.37 + 0.08, P = 0.02, paired t-tests) irrespective of the hand involved. This differential left lateralization for planning remained significant in PMC (LI = -0.47 + 0.14 and -0.36 + 0.13, mean + SD, P = 0.04), but not in PPC (-0.56 + 0.11 and -0.45 + 0.12, P = 0.11), when both regions were analyzed separately. In conclusion, the findings point to a left-hemispheric specialization for praxis planning, being more pronounced for intransitive gestures in PMC, possibly due to their communicative nature.

Protocol for Genome-Scale Reconstruction and Melanogenesis Analysis of Exophiala dermatitidis.

Exophiala dermatitidis is a polyextremotolerant fungus with a small genome, thus suitable as a model system for melanogenesis and carotenogensis. A genome-scale model, iEde2091, is reconstructed to increase metabolic understanding and used in a shadow price analysis of pigments, as detailed here. Important to this reconstruction is OptFill, a recently developed alternative gap-filling method useful in the holistic and conservative reconstruction of genome-scale models of metabolism, particularly for understudied organisms like E. dermatitidis where gaps in metabolic knowledge are abundant. For complete details on the use and execution of this protocol, please refer to Schroeder and Saha (2020) and Schroeder et al. (2020).

Introducing an Optimization- and explicit Runge-Kutta- based Approach to Perform Dynamic Flux Balance Analysis.

In this work we introduce the generalized Optimization- and explicit Runge-Kutta-based Approach (ORKA) to perform dynamic Flux Balance Analysis (dFBA), which is numerically more accurate and computationally tractable than existing approaches. ORKA is applied to a four-tissue (leaf, root, seed, and stem) model of Arabidopsis thaliana, p-ath773, uniquely capturing the core-metabolism of several stages of growth from seedling to senescence at hourly intervals. Model p-ath773 has been designed to show broad agreement with published plant-scale properties such as mass, maintenance, and senescence, yet leaving reaction-level behavior unconstrainted. Hence, it serves as a framework to study the reaction-level behavior necessary for observed plant-scale behavior. Two such case studies of reaction-level behavior include the lifecycle progression of sulfur metabolism and the diurnal flow of water throughout the plant. Specifically, p-ath773 shows how transpiration drives water flow through the plant and how water produced by leaf tissue metabolism may contribute significantly to transpired water. Investigation of sulfur metabolism elucidates frequent cross-compartment exchange of a standing pool of amino acids which is used to regulate the proton flow. Overall, p-ath773 and ORKA serve as scaffolds for dFBA-based lifecycle modeling of plants and other systems to further broaden the scope of in silico metabolic investigation.

OptFill: A Tool for Infeasible Cycle-Free Gapfilling of Stoichiometric Metabolic Models.

Stoichiometric metabolic modeling, particularly genome-scale models (GSMs), is now an indispensable tool for systems biology. The model reconstruction process typically involves collecting information from public databases; however, incomplete systems knowledge leaves gaps in any reconstruction. Current tools for addressing gaps use databases of biochemical functionalities to address gaps on a per-metabolite basis and can provide multiple solutions but cannot avoid thermodynamically infeasible cycles (TICs), invariably requiring lengthy manual curation. To address these limitations, this work introduces an optimization-based multi-step method named OptFill, which performs TIC-avoiding whole-model gapfilling. We applied OptFill to three fictional prokaryotic models of increasing sizes and to a published GSM of Escherichia coli, iJR904. This application resulted in holistic and infeasible cycle-free gapfilling solutions. In addition, OptFill can be adapted to automate inherent TICs identification in any GSM. Overall, OptFill can address critical issues in automated development of high-quality GSMs.

Computation-Driven Analysis of Model Polyextremo-tolerant Fungus Exophiala dermatitidis: Defensive Pigment Metabolic Costs and Human Applications.

The polyextremotolerant black yeast Exophiala dermatitidis is a tractable model system for investigation of adaptations that support growth under extreme conditions. Foremost among these adaptations are melanogenesis and carotenogenesis. A particularly important question is their metabolic production cost. However, investigation of this issue has been hindered by a relatively poor systems-level understanding of E. dermatitidis metabolism. To address this challenge, a genome-scale model (iEde2091) was developed. Using iEde2091, carotenoids were found to be more expensive to produce than melanins. Given their overlapping protective functions, this suggests that carotenoids have an underexplored yet important role in photo-protection. Furthermore, multiple defensive pigments with overlapping functions might allow E. dermatitidis to minimize cost. Because iEde2091 revealed that E. dermatitidis synthesizes the same melanins as humans and the active sites of the key tyrosinase enzyme are highly conserved this model may enable a broader understanding of melanin production across kingdoms.

Comparison of spontaneous and experimentally induced canine prostatic hyperplasia.

Spontaneous prostatic hyperplasia in the beagle appears to progress with age from a glandular to a cystic histological appearance. Prostatic hyperplasia can be induced in young beagles with intact testes by treatment for 4 mo with either dihydrotestosterone or 5 alpha-androstane-3 alpha, 17 beta-diol, alone, or with either of these steroids in combination with 17 beta-estradiol. In contrast, the induction of prostatic hyperplasia in young castrated beagles, in which the gland had been allowed to involute for 1 mo, requires the administration of both 17 beta-estradiol and either 5 alpha-androstane-3 alpha, 17 beta-diol or dihydrotestosterone. Testosterone and 17 beta-estradiol, either singly or in combination, did not produce the hyperplastic condition in intact or castrated beagles. The experimentally induced prostatic hyperplasia is identical in pathology to the glandular hyperplasia that occurs naturally in the aging dog with intact testes. However, cystic hyperplasia was not produced by any of the treatments tested in young animals.

Spontaneous benign prostatic hyperplasia in the beagle. Age-associated changes in serum hormone levels, and the morphology and secretory function of the canine prostate.

This paper is a cross-sectional study of spontaneous benign prostatic hyperplasia (BPH) in a single canine species. The effects of aging and hormonal changes on the growth, histology, and glandular secretory function of the canine prostate were studied in 42 male beagles ranging in age from 8 mo to 9 yr. The beagle prostate enlarges for at least 6 yr, whether normal or hyperplastic. In contrast, prostatic secretory function, determined by ejaculate volume and total ejaculate protein, declines markedly after 4 yr of age. These reciprocal growth and functional changes in the prostate are closely associated with a progressive increase in the incidence of BPH, which is already apparent in some dogs by age two. With age there is a modest decrease in serum androgen levels with no apparent change in serum 17 beta-estradiol levels. This suggests that the growth and functional changes that are associated with the development of BPH and are initiated very early in life reflect an altered sensitivity of the prostate to serum androgens or a response to the relative decrease in the serum androgen to estrogen ratio.

Temporal mapping of the differentiation pathway of the murine erythroleukemia cell.

These studies are concerned with "mapping" the temporal order of the precommitment events in the differentiation pathway of the Friend erythroleukemia cell. We have used a single-block procedure in which a differentiation-specific inhibitor of a temperature-sensitive (ts) differentiation-defective mutation was used to block the differentiation program. Later, the block was removed, differentiation was allowed to proceed, and the time required to reach a reference marker was monitored. These studies have indicated that the mutations tsC2GP1 and tsB5A, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, and the glucocorticoid hormone dexamethasone blocked functions which are required just prior to commitment. We have also used a double-block procedure involving two consecutive restrictive conditions, which suggests that the 3-aminobenzamide- and tsC2GP1-blocked functions constitute a part of a sequentially ordered pathway leading to terminal differentiation. The convergence of the 3-aminobenzamide, dexamethasone, and ts mutational blocks just prior to commitment suggests that the blocked functions may be part of a major control mechanism for commitment. In these studies, we have introduced a cytochalasin B-based assay to monitor commitment. The use of cytochalasin B permits a direct assay for commitment and obviates the need for colony-forming assays using semisolid medium, which have inherent problems such as efficiency of plating.

The visual encoding of tool-object affordances.

The perception of tool-object pairs involves understanding their action-relationships (affordances). Here, we sought to evaluate how an observer visually encodes tool-object affordances. Eye-movements were recorded as right-handed participants freely viewed static, right-handed, egocentric tool-object images across three contexts: correct (e.g. hammer-nail), incorrect (e.g. hammer-paper), spatial/ambiguous (e.g. hammer-wood), and three grasp-types: no hand, functional grasp-posture (grasp hammer-handle), non-functional/manipulative grasp-posture (grasp hammer-head). There were three areas of interests (AOI): the object (nail), the operant tool-end (hammer-head), the graspable tool-end (hammer-handle). Participants passively evaluated whether tool-object pairs were functionally correct/incorrect. Clustering of gaze scanpaths and AOI weightings grouped conditions into three distinct grasp-specific clusters, especially across correct and spatial tool-object contexts and to a lesser extent within the incorrect tool-object context. The grasp-specific gaze scanpath clusters were reasonably robust to the temporal order of gaze scanpaths. Gaze was therefore automatically primed to grasp-affordances though the task required evaluating tool-object context. Participants also primarily focused on the object and the operant tool-end and sparsely attended to the graspable tool-end, even in images with functional grasp-postures. In fact, in the absence of a grasp, the object was foveally weighted the most, indicative of a possible object-oriented action priming effect wherein the observer may be evaluating how the tool engages on the object. Unlike the functional grasp-posture, the manipulative grasp-posture caused the greatest disruption in the object-oriented priming effect, ostensibly as it does not afford tool-object action due to its non-functional interaction with the operant tool-end that actually engages with the object (e.g., hammer-head to nail). The enhanced attention towards the manipulative grasp-posture may serve to encode grasp-intent. Results here shed new light on how an observer gathers action-information when evaluating static tool-object scenes and reveal how contextual and grasp-specific affordances directly modulate visuospatial attention.

Thromboxane as a measure of cutaneous vascular damage following irradiation.

The present study was designed to evaluate the role of thromboxane in radiation-induced cutaneous injury and to use the quantitation of cutaneous thromboxane B2 as an indicator of vascular alteration and tissue viability in canine skin. Ten adult intact male dogs underwent epithermal neutron irradiation with or without boron neutron capture. Skin biopsies were obtained from (1) within, (2) the edge of, and (3) outside the radiation field at 5, 8, 11, 14, 21, and 90 days after irradiation. Clinical changes at each sampling time were assigned a numerical score. One-half of each biopsy was assigned a numerical score based on histologic changes. Thromboxane B2 was measured from the second half by enzyme immunoabsorbent assay. Thromboxane B2 concentration paralleled the response of clinical and histologic score over time, indicating the value of thromboxane measurement for evaluation of skin changes secondary to irradiation.

Comparison of two dosages of prostaglandin F2a on canine uterine motility.

The effect of 50 ug/kg prostaglandin F2a (PGF2a) was compared with 250 ug/kg PGF2a on uterine motility in the diestrous female. Microtipped pressure transducers were surgically implanted in the uteri of 6 females at 30 days diestrus and in 6 females at 60 days diestrus. Uterine responses to intravenous PGF2a (5 ug/kg), oxytocin (0.05 USP units/kg), and intramuscular PGF2a (50 ug/kg and 250 ug/kg) were measured in the awake females on Days 1 and 2 after implantation. There was no significant difference in the increase in intrauterine pressure produced by 50 ug/kg of PGF2a compared with 250 ug/kg of PGF2a. The longest duration of the effect occurred when 250 ug/kg of PGF2a were given. Side effects were also documented. Significantly more vomiting occurred when 250 ug/kg PGF2a were given than when 50 ug/kg PGF2a were administered. The only advantage to using a higher dosage of PGF2a appears to be the longer duration of motility.

The oxytocic effect of xylazine on the canine uterus.

The effect of xylazine on intrauterine pressure was compared to that of prostaglandin and oxytocin in seven diestrual bitches. Microtipped pressure transducers were surgically implanted in the uteri of four bitches at 30 d diestrus and in three bitches at 60 d diestrus. Uterine contractile force was measured in the awake bitches on Day 1 and Day 2 following implantation. Uterine responses to intravenous prostaglandin (5 mug/kg), oxytocin (0.05 USP units/kg), and xylazine (0.22 mg/kg) were measured. In the 30-d diestrual bitches, prostaglandin and oxytocin increased intrauterine pressure to 67 and 69 mmHg, with the duration of action being 16 and 14 min, respectively. Xylazine increased intra-uterine pressure to 49 mmHg and had a duration of action of 8 min. All results were decreased but similar in the 60-d diestrual bitches. These findings indicate that xylazine, given intravenously, produces a transitory increase in intrauterine pressure in the diestrual bitch.

Evaluation of beta-endorphin and naloxone on bovine uterine motility.

Effects of beta-endorphin and naloxone on bovine uterine motility were tested both in vivo and in vitro. Six cyclic Holstein cows were used to study in vivo effects of beta-endorphin and naloxone on uterine motility during estrus and diestrus. Intrauterine pressure changes were recorded by a microtip pressure transducer before and after treatment. Blood samples were taken every 10 min during the recording periods for beta-endorphin assay. The results revealed that beta-endorphin anc naloxone had no effect on intrauterine pressure in vivo. The effects of beta-endorphin and naloxone on myometrial contractility were also examined in vitro. Beta-endorphin and naloxone were added to tissue baths containing estrous and diestrous uterine strips. The results showed no significant effect of beta-endorphin and naloxone on bovine myometrial contractility. The role of beta-endorphin in bovine reproductive physiology is still not clearly understood, and additional studies are needed.

Nuclear imaging of the stomach of healthy dogs.

To evaluate the use of technetium pertechnetate (99mTcO4) as a means of estimating gastric mucosal integrity, nuclear images of the empty stomach were obtained from 6 dogs at 20, 40, 60, 120, 180, and 240 minutes after IV administration of the radiopharmaceutical. Blood and gastric secretion samples were collected during the same time intervals. The left lateral-view image of the stomach was used to calculate the relative fraction of the dose in the stomach and the count density ratio. Between 20 and 40 minutes and 40 and 60 minutes, significant differences (P less than 0.001) were apparent in the amount of 99mTcO4 in the stomach. Blood concentration of 99mTcO4 decreased significantly (P less than 0.001), whereas gastric secretion concentration increased significantly (P less than 0.001) over time. Qualitative assessment of the gastric nuclear scans and the statistical analytic results indicated that the optimal time for imaging the canine stomach was between 40 and 60 minutes after radiopharmaceutical administration. In a second study, the same dogs were pretreated with the H2-receptor antagonist cimetidine and the cholinergic antagonist glycopyrrolate to block gastric secretions. Over time, changes in the relative dose fraction in the stomach and the density ratio were the same as values obtained during the experiment performed without use of cimetidine and glycopyrrolate. Results of the study indicate that nuclear imaging with 99mTcO4 outlines normal canine gastric mucosa and that pretreatment with cimetidine and glycopyrrolate has no effect on the quality of the gastric image.

Use of a nuclear imaging technique to detect gastric wall ischemia.

A nuclear imaging technique of the stomach, using technetium pertechnetate (99mTcO4), was evaluated in healthy dogs. The stomach was first insufflated with room air, then filled with barium sulfate to induce mild distention, outlining the gastric wall. Six dogs were imaged twice: initially without use of drugs that might affect gastric secretion of 99mTcO4, then after pretreatment with cimetidine and glycopyrrolate. These scans established the appearance of the normal (control) stomach and compared the quality of the image in the same dogs not pretreated, then pretreated with cimetidine and glycopyrrolate before administration of 99mTcO4. Avascular defects were then surgically created on the greater curvature of the stomach of the same 6 dogs, and gastroscintigraphy was performed in similar manner. Significant (P less than 0.05) quantitative differences were detected in the gastric images for scans of the avascular area, compared with various control scans. Qualitative assessment had overall accuracy of 90.28%. Results of the study reported here indicate that nuclear imaging can be a valuable diagnostic technique for detecting ischemic areas in the gastric wall of dogs.