Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395.

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii.

BACKGROUND: Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin. RESULTS: In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L- 1) and content (7.28 mg g- 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L- 1. CONCLUSIONS: A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.

Identification and Functional Analysis of the psaD Promoter of Chlorella vulgaris Using Heterologous Model Strains.

Chlorella has great potential as a bio-factory for production of value-added compounds. To produce the desired chemicals more efficiently in Chlorella, genetic tools for modification of Chlorella need to be developed, especially an endogenous promoter. In this study, the promoter of photosystem I protein D (psaD) from Chlorella vulgaris UTEX395 was identified. Computational analysis revealed the presence of several putative cis-acting elements, including a potential core element, and light-responsive or stress-responsive elements. Gene expression analysis in heterologous expression system in Chlamydomonasreinhardtii and Nicotianabenthamiana showed that CvpsaD promoter can be used to drive the expression of genes. Functional analysis of this promoter suggested that the initiator element (Inr) is important for its function (i.e., TATA-less promoter) and that an additional factor (e.g., downstream of the transcriptional start site) might be needed for light response. We have shown that the CvpsaD promoter is functional, but not sufficiently strong, both in microalgae and higher plant.

Design of an embedded inverse-feedforward biomolecular tracking controller for enzymatic reaction processes.

Feedback control is widely used in chemical engineering to improve the performance and robustness of chemical processes. Feedback controllers require a 'subtractor' that is able to compute the error between the process output and the reference signal. In the case of embedded biomolecular control circuits, subtractors designed using standard chemical reaction network theory can only realise one-sided subtraction, rendering standard controller design approaches inadequate. Here, we show how a biomolecular controller that allows tracking of required changes in the outputs of enzymatic reaction processes can be designed and implemented within the framework of chemical reaction network theory. The controller architecture employs an inversion-based feedforward controller that compensates for the limitations of the one-sided subtractor that generates the error signals for a feedback controller. The proposed approach requires significantly fewer chemical reactions to implement than alternative designs, and should have wide applicability throughout the fields of synthetic biology and biological engineering.

Recursive Self-Composite Approach Towards Structural Understanding of Boolean Networks.

Boolean networks have been widely used in systems biology to study the dynamical characteristics of biological networks such as steady-states or cycles, yet there has been little attention to the dynamic properties of network structures. Here, we systematically reveal the core network structures using a recursive self-composite of the logic update rules. We find that all Boolean update rules exhibit repeated cyclic logic structures, where each converged logic leads to the same states, defined as kernel states. Consequently, the period of state cycles is upper bounded by the number of logics in the converged logic cycle. In order to uncover the underlying dynamical characteristics by exploiting the repeating structures, we propose leaping and filling algorithms. The algorithms provide a way to avoid large string explosions during the self-composition procedures. Finally, we present three examples-a simple network with a long feedback structure, a T-cell receptor network and a cancer network-to demonstrate the usefulness of the proposed algorithm.

Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas.

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.

Heterologous Gene Expression System Using the Cold-Inducible CnAFP Promoter in Chlamydomonas reinhardtii.

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method.

Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.

Untargeted Metabolomics of Fermented Rice Using UHPLC Q-TOF MS/MS Reveals an Abundance of Potential Antihypertensive Compounds.

Enzyme treatment and fermentation of cereals are known processes that enhance the release of bound bioactive compounds to make them available for bioactivity. In this study, we tested the angiotensin converting enzyme (ACE) inhibitory ability of destarched rice, Prozyme 2000p treated destarched rice (DP), and fermented DP samples. Prozyme 2000p treatment increased the ACE inhibitory ability from 15 ± 5% to 45 ± 3%. Fermentation of the Prozyme 2000p treated samples with Enterococcus faecium EBD1 significantly increased the ACE inhibitory ability to 75 ± 5%, while captopril showed an ACE inhibition of 92 ± 4%. An untargeted metabolomics approach using Ultra-high-performance liquid tandem chromatography quadrupole time of flight mass spectrometry revealed the abundance of vitamins, phenolic compounds, antioxidant peptides, DPP IV inhibitory peptides, and antihypertensive peptides in the fermented samples which may account for its strong ACE inhibition. Although fermented DP had decreased fatty acid levels, the amount of essential amino acid improved drastically compared to destarched rice. Our results show that fermenting Prozyme-treated destarched rice with Enterococcus faecium EBD1 generates abundant bioactive compounds necessary for developing antihypertensive functional foods.

Microbial Etiology and Prevention of Dental Caries: Exploiting Natural Products to Inhibit Cariogenic Biofilms.

Dental caries is one of the most common microbe-mediated oral diseases in human beings. At present, the accepted etiology of caries is based on a four-factor theory that includes oral microorganisms, oral environment, host, and time. Excessive exposure to dietary carbohydrates leads to the accumulation of acid-producing and acid-resistant microorganisms in the mouth. Dental caries is driven by dysbiosis of the dental biofilm adherent to the enamel surface. Effective preventive methods include inhibiting the cariogenic microorganisms, treatment with an anti-biofilm agent, and sugar intake control. The goal is to reduce the total amount of biofilm or the levels of specific pathogens. Natural products could be recommended for preventing dental caries, since they may possess fewer side effects in comparison with synthetic antimicrobials. Herein, the mechanisms of oral microbial community development and functional specialization are discussed. We highlight the application of widely explored natural products in the last five years for their ability to inhibit cariogenic microorganisms.

Phytochemical characterization, and antioxidant and antimicrobial activities of white cabbage extract on the quality and shelf life of raw beef during refrigerated storage.

Brassica vegetables are well-characterized, containing a wide-spectrum of phenolic compounds that are responsible for their diverse biological activities like antioxidant and antimicrobial activities. This study explored the preservative effect of Brassica oleracea var. capitate f. alba (white cabbage; WC) on beef under refrigerated conditions for 16 days. The antimicrobial activities of WC were evaluated against foodborne pathogenic bacteria and fungi. The antioxidant activity was determined on the basis of total phenolic and flavonoid contents, through employing DPPH and ABTS assays. The chemical composition was analyzed by GC-MS analysis. The results indicated that among the different solvent extracts, white cabbage chloroform extract [WCCE] exhibited outstanding bioactive properties due to the presence of 4-nitro-3-(trifluoromethyl)phenol, and the effects of WCCE at different levels (A and B) on the quality and shelf life of beef in storage were evaluated. The color parameters (lightness, yellowness, and redness), texture analysis, and pH values were monitored constantly with 4 days interval, and microbial analysis was conducted. The results showed that WCCE-A treatment significantly reduced the total viable counts, psychrotrophic bacteria, and yeast-molds when compared with WCCE-B and control during refrigeration storage, with the activity varying in a dose-dependent manner (p < 0.05). Significantly, the WCCE-A treatments had better appearance compared with the control after 16 days of storage. All results confirmed that WCCE which is rich in bioactive compounds, effectively maintains the quality of beef compared to the control by retarding lipid oxidation and microbial growth at refrigeration temperature and also emphasize the potential applications of this plant in different industrial sectors.

Bioactive Potential of 2-Methoxy-4-vinylphenol and Benzofuran from Brassica oleracea L. var. capitate f, rubra (Red Cabbage) on Oxidative and Microbiological Stability of Beef Meat.

In the future, plant based phytochemicals will be considered as efficient replacement sources of chemical preservatives, to act as potential bio-preservatives. We investigated the antibacterial and antioxidant activity of red cabbage (RC) extracts using different solvents. Among all extracts, chloroform extract exhibited strong antimicrobial and antioxidant activities. Hence, the phytochemical constitutions of the RC chloroform extract was examined by GC-MS analysis, and further, based on molecular docking analysis, revealed 2-Methoxy-4-vinylphenol and benzofuran as two major compounds found to be possessing higher degrees of interaction with DNA gyrase (4PLB; -8.63 Kcal.mol-1) and lipoprotein (LpxC-8.229 Kcal.mol-1), respectively, of the bacterial cell wall, which leads to higher antimicrobial efficacy. Further, it was confirmed with that the in vivo Caenorhabditis elegans model (but no cytotoxic effect) was exhibited in the MCF-7 cell line. Thus, we investigated the influence of this extract on the shelf life of meat under refrigeration storage. The physicochemical properties were observed periodically, and microbial analysis was conducted. The shelf life of the beef was enhanced (up to eight days) in terms of microbial and physiochemical properties, at 4 ± 2 °C when compared to control. We concluded that chloroform extract of RC has potential as a natural preservative in the meat processing industry.

Enhanced lipid productivity in AGP knockout marine microalga Tetraselmis sp. using a DNA-free CRISPR-Cas9 RNP method.

A marine green microalga, Tetraselmis sp., has been studied for the production of biomass and lipids in seawater culture. Since carbohydrate and lipid biosynthesis are competitive metabolic pathways, we attempted to increase lipid synthesis in Tetraselmis by inhibiting carbohydrate synthesis. The main regulatory enzyme in the starch synthesis pathway is ADP-glucose pyrophosphorylase (AGP). AGP loss-of-function mutants were developed using the CRISPR-Cas9 ribonucleoprotein (RNP) delivery system. AGP mutants showed a slight decrease in growth. However, the lipid content in two AGP mutants was significantly enhanced by 2.7 and 3.1 fold (21.1% and 24.1% of DCW), respectively, compared to that in the wild type (7.68% of DCW) under nitrogen starvation. This study is an example of metabolic engineering by genetic editing using the CRISPR-Cas9 RNP method in marine green microalgae. Consequently, starchless Tetraselmis mutants might be considered potential producers of lipids in seawater cultures.

Toward a Closed Loop, Integrated Biocompatible Biopolymer Wound Dressing Patch for Detection and Prevention of Chronic Wound Infections.

Chronic wound infections represent a significant burden to healthcare providers globally. Often, chronic wound healing is impeded by the presence of infection within the wound or wound bed. This can result in an increased healing time, healthcare cost and poor patient outcomes. Thus, there is a need for dressings that help the wound heal, in combination with early detection of wound infections to support prompt treatment. In this study, we demonstrate a novel, biocompatible wound dressing material, based on Polyhydroxyalkanoates, doped with graphene platelets, which can be used as an electrochemical sensing substrate for the detection of a common wound pathogen, Pseudomonas aeruginosa. Through the detection of the redox active secondary metabolite, pyocyanin, we demonstrate that a dressing can be produced that will detect the presence of pyocyanin across clinically relevant concentrations. Furthermore, we show that this sensor can be used to identify the presence of pyocyanin in a culture of P. aeruginosa. Overall, the sensor substrate presented in this paper represents the first step toward a new dressing with the capacity to promote wound healing, detect the presence of infection and release antimicrobial drugs, on demand, to optimized healing.

Linear time-varying models can reveal non-linear interactions of biomolecular regulatory networks using multiple time-series data.

MOTIVATION: Inherent non-linearities in biomolecular interactions make the identification of network interactions difficult. One of the principal problems is that all methods based on the use of linear time-invariant models will have fundamental limitations in their capability to infer certain non-linear network interactions. Another difficulty is the multiplicity of possible solutions, since, for a given dataset, there may be many different possible networks which generate the same time-series expression profiles. RESULTS: A novel algorithm for the inference of biomolecular interaction networks from temporal expression data is presented. Linear time-varying models, which can represent a much wider class of time-series data than linear time-invariant models, are employed in the algorithm. From time-series expression profiles, the model parameters are identified by solving a non-linear optimization problem. In order to systematically reduce the set of possible solutions for the optimization problem, a filtering process is performed using a phase-portrait analysis with random numerical perturbations. The proposed approach has the advantages of not requiring the system to be in a stable steady state, of using time-series profiles which have been generated by a single experiment, and of allowing non-linear network interactions to be identified. The ability of the proposed algorithm to correctly infer network interactions is illustrated by its application to three examples: a non-linear model for cAMP oscillations in Dictyostelium discoideum, the cell-cycle data for Saccharomyces cerevisiae and a large-scale non-linear model of a group of synchronized Dictyostelium cells. AVAILABILITY: The software used in this article is available from http://sbie.kaist.ac.kr/software

Modelling and Control of Gene Regulatory Networks for Perturbation Mitigation.

Synthetic Biologists are increasingly interested in the idea of using synthetic feedback control circuits for the mitigation of perturbations to gene regulatory networks that may arise due to disease and/or environmental disturbances. Models employing Michaelis-Menten kinetics with Hill-type nonlinearities are typically used to represent the dynamics of gene regulatory networks. Here, we identify some fundamental problems with such models from the point of view of control system design, and argue that an alternative formalism, based on so-called S-System models, is more suitable. Using tools from system identification, we show how to build S-System models that capture the key dynamics of an example gene regulatory network, and design a genetic feedback controller with the objective of rejecting an external perturbation. Using a sine sweeping method, we show how the S-System model can be approximated by a linear transfer function and, based on this transfer function, we design our controller. Simulation results using the full nonlinear S-System model of the network show that the synthetic control circuit is able to mitigate the effect of external perturbations. Our study is the first to highlight the usefulness of the S-System modelling formalism for the design of synthetic control circuits for gene regulatory networks.

Sedimentation rate-based screening of oleaginous microalgae for utilization as a direct combustion fuel.

The co-combustion of microalgae biomass with coal has the potential to significantly reduce CO2 emissions by eliminating expensive and carbon-emitting downstream processes. In this study, the utilization of microalgal biomass as a direct combustion fuel in co-firing industries and the screening of potential oleaginous strains of high calorific value was investigated. High-lipid accumulating mutants were selected from mutant mixtures based on cell density using differential sedimentation rates. Of the mutant strains obtained in the top phase of the separation medium, 72% showed a higher lipid content than the wild-type strain. One mutant strain exhibited a 57.3% enhanced lipid content and a 9.3% lower heating value (LHV), both indicators of direct combustion fuel performance, compared to the wild-type strain. Our findings indicate that sedimentation rate-based strain selection allows for the easy and rapid screening of high-lipid content algal strains for the use of microalgae as direct combustion fuels.

Drug Diffusion Along an Intact Mammalian Cochlea.

Intratympanic drug administration depends on the ability of drugs to pass through the round window membrane (RW) at the base of the cochlea and diffuse from this location to the apex. While the RW permeability for many different drugs can be promoted, passive diffusion along the narrowing spiral of the cochlea is limited. Earlier measurements of the distribution of marker ions, corticosteroids, and antibiotics demonstrated that the concentration of substances applied to the RW was two to three orders of magnitude higher in the base compared to the apex. The measurements, however, involved perforating the cochlear bony wall and, in some cases, sampling perilymph. These manipulations can change the flow rate of perilymph and lead to intake of perilymph through the cochlear aqueduct, thereby disguising concentration gradients of the delivered substances. In this study, the suppressive effect of salicylate on cochlear amplification via block of the outer hair cell (OHC) somatic motility was utilized to assess salicylate diffusion along an intact guinea pig cochlea in vivo. Salicylate solution was applied to the RW and threshold elevation of auditory nerve responses was measured at different times and frequencies after application. Resultant concentrations of salicylate along the cochlea were calculated by fitting the experimental data using a mathematical model of the diffusion and clearing of salicylate in a tube of variable diameter combined with a model describing salicylate action on cochlear amplification. Concentrations reach a steady-state at different times for different cochlear locations and it takes longer to reach the steady-state at more apical locations. Even at the steady-state, the predicted concentration at the apex is negligible. Model predictions for the geometry of the longer human cochlea show even higher differences in the steady-state concentrations of the drugs between cochlear base and apex. Our findings confirm conclusions that achieving therapeutic drug concentrations throughout the entire cochlear duct is hardly possible when the drugs are applied to the RW and are distributed via passive diffusion. Assisted methods of drug delivery are needed to reach a more uniform distribution of drugs along the cochlea.

Stochastic noise and synchronisation during dictyostelium aggregation make cAMP oscillations robust.

Stable and robust oscillations in the concentration of adenosine 3', 5'-cyclic monophosphate (cAMP) are observed during the aggregation phase of starvation-induced development in Dictyostelium discoideum. In this paper we use mathematical modelling together with ideas from robust control theory to identify two factors which appear to make crucial contributions to ensuring the robustness of these oscillations. Firstly, we show that stochastic fluctuations in the molecular interactions play an important role in preserving stable oscillations in the face of variations in the kinetics of the intracellular network. Secondly, we show that synchronisation of the aggregating cells through the diffusion of extracellular cAMP is a key factor in ensuring robustness of the oscillatory waves of cAMP observed in Dictyostelium cell cultures to cell-to-cell variations. A striking and quite general implication of the results is that the robustness analysis of models of oscillating biomolecular networks (circadian clocks, Ca(2+) oscillations, etc.) can only be done reliably by using stochastic simulations, even in the case where molecular concentrations are very high.