Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses.

Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G2-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G2-phase. We report that in the absence of functional HRR, activation of the G2-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G2-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G2-checkpoint in HRR-deficient cells, suggesting that the G2-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G2-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G2-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G2-phase activate a weak but readily detectable ATM/ATR-dependent G2-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G2-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G2-checkpoint are closely intertwined in cells exposed to low IR-doses in G2-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G2-phase, and cells irradiated in S-phase utilize a different mechanistic setup.

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.

Change on apoptosis, autophagy and mitochondria of the Harderian gland in Cricetulus barabensis during age.

Harderian gland (HG) plays an important role in the physiological adaptation to terrestrial life, however, the mechanisms underlying the changes in the structure and function of the HG during aging remain unclear. This study investigated autophagy and apoptosis in the HG of striped dwarf hamsters (Cricetulus barabensis) of different ages (sub-adult, adult and aged groups) in both males and females. The results showed that LC3II/LC3I and puncta of LC3 were significantly higher in adult and aged individuals than sub-adults, whereas P62 decreased with age. Bax/bcl2was the highest in sub-adults of male and female individuals. Caspase3 activity was the highest in sub-adults of male and female individuals, and the citrate synthase activity was highest in sub-adults of females. ATP synthase, citrate synthase, dynamin-related protein 1 and mitochondrial fission factor (Mff) were the highest in sub-adults of females. Peptidylglycine α-amidating monooxygenase were the highest in the aged group, and those of gonadotropin-releasing hormone was the highest in the adult group. LC3II/LC3I, P62, Drp1, Fis, and bax/bcl2 were higher in males than that in females. These results suggest that apoptosis mainly affects growth and development in the HG, whereas autophagy affects aging. The difference of the HG weight and mitochondrial function between sexes is mainly related to the apoptosis.

Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells.

OBJECTIVES: To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. RESULTS: Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. CONCLUSIONS: The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.

Chromosome breaks generated by low doses of ionizing radiation in G2-phase are processed exclusively by gene conversion.

Four repair pathways process DNA double-strand breaks (DSBs). Among these pathways the homologous recombination repair (HRR) subpathway of gene conversion (GC) affords error-free processing, but functions only in S- and G2-phases of the cell cycle. Classical non-homologous end-joining (c-NHEJ) operates throughout the cell cycle, but causes small deletions and translocations. Similar deficiencies in exaggerated form, combined with reduced efficiency, are associated with alternative end-joining (alt-EJ). Finally, single-strand annealing (SSA) causes large deletions and possibly translocations. Thus, processing of a DSB by any pathway, except GC, poses significant risks to the genome, making the mechanisms navigating pathway-engagement critical to genome stability. Logically, the cell ought to attempt engagement of the pathway ensuring preservation of the genome, while accommodating necessities generated by the types of DSBs induced. Thereby, inception of DNA end-resection will be key determinant for GC, SSA and alt-EJ engagement. We reported that during G2-phase, where all pathways are active, GC engages in the processing of almost 50 % of DSBs, at low DSB-loads in the genome, and that this contribution rapidly drops to nearly zero with increasing DSB-loads. At the transition between these two extremes, SSA and alt-EJ compensate, but at extremely high DSB-loads resection-dependent pathways are suppressed and c-NHEJ remains mainly active. We inquired whether in this processing framework all DSBs have similar fates. Here, we analyze in G2-phase the processing of a subset of DSBs defined by their ability to break chromosomes. Our results reveal an absolute requirement for GC in the processing of chromatid breaks at doses in the range of 1 Gy. Defects in c-NHEJ delay significantly the inception of processing by GC, but leave processing kinetics unchanged. These results delineate the essential role of GC in chromatid break repair before mitosis and classify DSBs that underpin this breakage as the exclusive substrate of GC.

Chromosomal aberration dynamics through the cell cycle.

DNA double-strand breaks are the crucial lesions underlying the formation of chromosomal aberrations, their formation and kinetics have been extensively studied, although dynamics of the repair process has not been fully understood. By using a combination of different cytogenetic techniques to analyze cells in G0, G2 and M phase, in the present study we perform a follow up study of the dynamics of different radiation induced chromosomal aberrations. Data here presented show that in G0 phase chromosome fragments lacking telomere signals (incomplete chromosome elements, ICE) show a slow repair, but when repair occurs tend to reconstitute the original chromosomes, and those that do not repair seem to be selected by interphase cell death and cell cycle checkpoints. In contrast, complete chromosome aberrations, as dicentrics, show a very fast formation kinetics. Similar frequencies of dicentrics were observed in G0, G2 and M cells, indicating that this chromosome-type of aberration can progress through the cell cycle without negative selection. Our study reinforce the hypothesis that ICE are strongly negatively selected from G2 to M phase. However, the G2/M checkpoint seems to be not involved in this selection. The ICE frequencies observed after G2/M abrogation by caffeine are similar to the ones without abrogation, and clearly lower to the ones observed in G2.

Proteogenomic Annotation of Chinese Hamsters Reveals Extensive Novel Translation Events and Endogenous Retroviral Elements.

A high-quality genome annotation greatly facilitates successful cell line engineering. Standard draft genome annotation pipelines are based largely on de novo gene prediction, homology, and RNA-Seq data. However, draft annotations can suffer from incorrect predictions of translated sequence, inaccurate splice isoforms, and missing genes. Here, we generated a draft annotation for the newly assembled Chinese hamster genome and used RNA-Seq, proteomics, and Ribo-Seq to experimentally annotate the genome. We identified 3529 new proteins compared to the hamster RefSeq protein annotation and 2256 novel translational events (e.g., alternative splices, mutations, and novel splices). Finally, we used this pipeline to identify the source of translated retroviruses contaminating recombinant products from Chinese hamster ovary (CHO) cell lines, including 119 type-C retroviruses, thus enabling future efforts to eliminate retroviruses to reduce the costs incurred with retroviral particle clearance. In summary, the improved annotation provides a more accurate resource for CHO cell line engineering, by facilitating the interpretation of omics data, defining of cellular pathways, and engineering of complex phenotypes.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Is the Center of Origin of Long-tailed Hamster Cricetulus longicaudatus Milne-Edwards 1867 (Rodentia, Cricetidae) Located in Tibet?

First data on morphological and mitochondrial variation in the long-tailed hamster Cricetulus longicaudatus is presented. In contrast to genetically monomorphic populations of Mongolia and Tuva, the northeastern part of the Qinghai-Tibetian Plateau was found to harbor several divergent mtDNA lineages. This pattern suggests a recent expansion of the long-tailed hamster to the northern part of its recent range, which started from Tibet, presumably, in the late Middle Pleistocene. Several populations from the northern edge of the species range were found to be morphologically but not genetically distinct. The apparent disagreement between genetic and morphological data can be explained by rapid morphological evolution in peripheral isolates.

A reference genome of the Chinese hamster based on a hybrid assembly strategy.

Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read-based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells.

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.

Genetic characterization of the Turkish gray hamster (Cricetulus migratorius) based on mitochondrial cytochrome b and 12S rRNA sequences.

Although genetic diversity and phylogenetic status of the gray hamster (Cricetulus migratorius) have been investigated from different regions in previous studies, genetic data on this species from Turkey are still lacking, since previous data have been based on a limited number of gray hamsters sampled across the Anatolian part of Turkey. The aim of this study was to determine the genetic diversity of the Anatolian population and to reveal the phylogenetic relationships among the Anatolian population and conspecific populations of the gray hamster. The complete and partial fragments of mitochondrial Cyt b and 12S rRNA from the 20 Turkish samples were amplified and sequenced. Ten 12S rRNA (901 bp) and 15 Cyt b (1140 bp) haplotypes found in this work were not previously reported. Based on Bayesian, Maximum Likelihood, Neighbour-Joining and Median-Joining network analyses by using mitochondrial data under the name Cricetulus, the results of phylogenetic and network analyses indicated that there was a deep separation among the distinct lineages within the genus Cricetulus. When considering the position of the Turkish haplotypes in median joining network, the Anatolian part of Turkey may have hosted a source population of the gray hamster for expansion to adjacent regions in the past period. Additionally, the Anatolian population of gray hamster had relatively high haplotype diversity and the present study propounded the importance of data obtained from the Anatolian population of gray hamster to reveal the phylogenetic relationships among conspecific populations of the gray hamster.

Lessons from the Hamster: Cricetulus griseus Tissue and CHO Cell Line Proteome Comparison.

Chinese hamster ovary cells represent the dominant host for therapeutic recombinant protein production. However, few large-scale data sets have been generated to characterize this host organism and derived CHO cell lines at the proteomics level. Consequently, an extensive label-free quantitative proteomics analysis of two cell lines (CHO-S and CHO DG44) and two Chinese hamster tissues (liver and ovary) was used to identify a total of 11 801 unique proteins containing at least two unique peptides. 9359 unique proteins were identified specifically in the cell lines, representing a 56% increase over previous work. Additionally, 6663 unique proteins were identified across liver and ovary tissues, providing the first Chinese hamster tissue proteome. Protein expression was more conserved within cell lines during both growth phases than across cell lines, suggesting large genetic differences across cell lines. Overall, both gene ontology and KEGG pathway analysis revealed enrichment of cell-cycle activity in cells. In contrast, upregulated molecular functions in tissue include glycosylation and lipid transporter activity. Furthermore, cellular components including Golgi apparatus are upregulated in both tissues. In conclusion, this large-scale proteomics analysis enables us to delineate specific changes between tissues and cells derived from these tissues, which can help explain specific tissue function and the adaptations cells incur for applications in biopharmaceutical productions.

A type I combi-targeting approach for the design of molecules with enhanced potency against BRCA1/2 mutant- and O6-methylguanine-DNA methyltransferase (mgmt)- expressing tumour cells.

BACKGROUND: Mutations of the DNA repair proteins BRCA1/2 are synthetically lethal with the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which when inhibited, leads to cell death due to the absence of compensatory DNA repair mechanism. The potency of PARP inhibitors has now been clinically proven. However, disappointingly, acquired resistance mediated by the reactivation of wild type BRCA1/2 has been reported. In order to improve their efficacy, trials are ongoing to explore their combinations with temozolomide (TMZ). Here, in order to enhance potency in BRCA1/2-mutant cells, we report on the design of single molecules termed "combi-molecules" capable of not only inhibiting PARP but also damaging DNA like TMZ, which is known to induce a large number of DNA adducts. The majority of these lesions are processed through PARP-dependent base-excision repair machinery. Paradoxically, the least abundant lesion, the O6-methylguanine adduct is the most cytotoxic. Its repair by the O6-methylguanine DNA methyl transferase (MGMT) confers robust resistance to TMZ. Thus, we surmise that a combi-molecule designed to generate the same DNA adducts as TMZ, with an additional ability to block PARP, could induce BRCA1/2 mutant selective potency and a growth inhibitory profile independent of MGMT status. METHODS: The hydrolysis of EG22 and its stabilized form ZSM02 was analyzed by HPLC and fluorescence spectroscopy. Growth inhibitory potency was determined by SRB assay. PARP inhibition was determined by an enzyme assay and DNA damage by the comet assay. Subcellular distribution was visualized by confocal microscopy. RESULTS: Studies on EG22 showed that: (a) it inflicted anomalously higher levels of DNA damage than TMZ (b) it induced PARP inhibitory potency in the same range as ANI, a known PARP inhibitor (IC50 = 0.10 µM) (c) it showed strong potency in both BRCA1/2 wild type and mutated cells with 6-fold selectivity for the mutants and it was 65-303-fold more potent than TMZ and 4-63-fold than ANI alone and 3-47-fold than their corresponding equimolar combinations and (d) its potency was independent of MGMT expression. CONCLUSION: The results in toto suggest that a combi-molecular approach directed at blocking PARP and damaging DNA can lead to single molecules with selective and enhanced potency against BRCA1/2 mutant and with activity independent of MGMT, the major predictive biomarker for resistance to TMZ.

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination.

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It's well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.

Karyotype Evolution and Phylogenetic Relationships of Cricetulus sokolovi Orlov et Malygin 1988 (Cricetidae, Rodentia) Inferred from Chromosomal Painting and Molecular Data.

Sokolov's dwarf hamster (Cricetulus sokolovi) is the least studied representative of the striped hamsters (Cricetulus barabensis species group), the taxonomy of which remains controversial. The species was described based on chromosome morphology, but neither the details of the karyotype nor the phylogenetic relationships with other Cricetulus are known. In the present study, the karyotype of C. sokolovi was examined using cross-species chromosome painting. Molecular and cytogenetic data were employed to determine the phylogenetic position of Sokolov's hamster and to analyze the potential pathways of chromosome evolution in Cricetulus. Both the chromosome and molecular data support the species status of Sokolov's hamster. Phylogenetic analysis of the CYTB data placed C. sokolovi as sister to all other striped hamsters (sequence divergence of 8.1%). FISH data revealed that the karyotype of C. sokolovi is highly rearranged, with the most parsimonious scenario of its origin implying at least 4 robertsonian events and a centromere shift. Comparative cytogenetic data on Cricetinae suggest that their evolutionary history includes both periods of chromosomal conservatism and episodes of rapid chromosomal change.

CHOmine: an integrated data warehouse for CHO systems biology and modeling.

The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. Database URL: http://www.chogenome.org.

Parameter estimation in large-scale systems biology models: a parallel and self-adaptive cooperative strategy.

BACKGROUND: The development of large-scale kinetic models is one of the current key issues in computational systems biology and bioinformatics. Here we consider the problem of parameter estimation in nonlinear dynamic models. Global optimization methods can be used to solve this type of problems but the associated computational cost is very large. Moreover, many of these methods need the tuning of a number of adjustable search parameters, requiring a number of initial exploratory runs and therefore further increasing the computation times. Here we present a novel parallel method, self-adaptive cooperative enhanced scatter search (saCeSS), to accelerate the solution of this class of problems. The method is based on the scatter search optimization metaheuristic and incorporates several key new mechanisms: (i) asynchronous cooperation between parallel processes, (ii) coarse and fine-grained parallelism, and (iii) self-tuning strategies. RESULTS: The performance and robustness of saCeSS is illustrated by solving a set of challenging parameter estimation problems, including medium and large-scale kinetic models of the bacterium E. coli, bakerés yeast S. cerevisiae, the vinegar fly D. melanogaster, Chinese Hamster Ovary cells, and a generic signal transduction network. The results consistently show that saCeSS is a robust and efficient method, allowing very significant reduction of computation times with respect to several previous state of the art methods (from days to minutes, in several cases) even when only a small number of processors is used. CONCLUSIONS: The new parallel cooperative method presented here allows the solution of medium and large scale parameter estimation problems in reasonable computation times and with small hardware requirements. Further, the method includes self-tuning mechanisms which facilitate its use by non-experts. We believe that this new method can play a key role in the development of large-scale and even whole-cell dynamic models.

Factors affecting the in vitro micronucleus assay for evaluation of nanomaterials.

A number of in vitro methodologies have been used to assess the genotoxicity of different nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs) and silver nanoparticles (AgNPs). The in vitro micronucleus assay is one of the most commonly used test methods for genotoxicity evaluation of nanomaterials. However, due to the novel features of nanomaterials, such as high adsorption capacity and fluorescence properties, there are unexpected interactions with experimental components and detection systems. In this study, we evaluate the interference by two nanoparticles, AgNPs and TiO2 NPs, with the in vitro micronucleus assay system and possible confounding factors affecting cytotoxicity and genotoxicity assessment of the nanomaterials including cell lines with different p53 status, nanoparticle coatings and fluorescence, cytochalasin B, fetal bovine serum in cell treatment medium and different measurement methodologies for detecting micronuclei. Our results showed that micronucleus induction by AgNPs was similar when evaluated using flow cytometry or microscope, whereas the induction by TiO2 NPs was different using the two methods due to TiO2's fluorescence interference with the cytometry equipment. Cells with the mutated p53 gene were more sensitive to micronucleus induction by AgNPs than the p53 wild-type cells. The presence of serum during treatment increased the toxicity of AgNPs. The coatings of nanoparticles played an important role in the genotoxicity of AgNPs. These collective data highlight the importance of considering the unique properties of nanoparticles in assessing their genotoxicity using the in vitro micronucleus assay.