Variability of the distribution of differentiation pathway choices regulated by a multipotent delayed stochastic switch

We study the plasticity of a delayed stochastic model of a genetic toggle switch as a multipotent differentiation pathway switch, at the single cell and cell population levels, by observing distributions of differentiation pathways choices of genetically homogeneous cell populations. Assuming a model of stochastic pathway determination of cell differentiation that is regulated by the proteins of the switch, we vary the proteins’ expression level and degradation rates, which cells are known to be able to regulate, to vary mean level, noise, and bias of the proteins’ expression levels. It is shown that small changes in each of these dynamical features significantly and distinctively affects the dynamics of the switch at the single cell level and thus, the cell differentiation patterns. The regulation of these features allows cells to regulate their pluripotency and cell populations’ distribution of lineage choice, suggesting that the stochastic switch has high plasticity regarding differentiation pathway choice regulation, thus providing adaptability to environmental stresses and changes.     

Phylogenetic beta diversity in bacterial assemblages across ecosystems: deterministic versus stochastic processes

Increasing evidence has emerged for non-random spatial distributions of microbes, but knowledge of the processes that cause variation in microbial assemblage among ecosystems is lacking. For instance, some studies showed that deterministic processes such as habitat specialization are important, while other studies hold that bacterial communities are assembled by stochastic forces. Here we examine the relative influence of deterministic and stochastic processes for bacterial communities from subsurface environments, stream biofilm, lake water, lake sediment and soil using pyrosequencing of the 16S ribosomal RNA gene. We show that there is a general pattern in phylogenetic signal in species ecological niches across recent evolutionary time for all studied habitats, enabling us to infer the influences of community assembly processes from patterns of phylogenetic turnover in community composition. The phylogenetic dissimilarities among-habitat types were significantly higher than within them, and the communities were clustered according to their original habitat types. For communities within-habitat types, the highest phylogenetic turnover rate through space was observed in subsurface environments, followed by stream biofilm on mountainsides, whereas the sediment assemblages across regional scales showed the lowest turnover rate. Quantifying phylogenetic turnover as the deviation from a null expectation suggested that measured environmental variables imposed strong selection on bacterial communities for nearly all sample groups. For three sample groups, spatial distance reflected unmeasured environmental variables that impose selection, as opposed to spatial isolation. Such characterization of spatial and environmental variables proved essential for proper interpretation of partial Mantel results based on observed beta diversity metrics. In summary, our results clearly indicate a dominant role of deterministic processes on bacterial assemblages and highlight that bacteria show strong habitat associations that have likely emerged through evolutionary adaptation.     

Reconstructing the single-cell-level behavior of a toggle switch from population-level measurements

Single-cell-level behaviors of cells are typically inferred from ensemble measurements. However, such inferences implicitly assume a biological version of ergodicity: the percentage of cells in a state is identical to the probability to find a cell in that state. While the ergodicity does not always hold, it has been rarely tested. Here, we reveal that the ergodicity does not necessarily hold even for simple toggle switches and that apparent stabilities of the switches are due to a balance between single-cell-level biased stabilities and growth rates differences. Therefore, verification of the ergodicity and reconstructing single-cell-level behaviors are crucial for understanding intracellular systems.     

The stabilizing effect of a random environment

It is shown that the lottery competition model permits coexistence in a stochastic environment, but not in a constant environment. Conditions for coexistence and competitive exclusion are determined. Analysis of these conditions shows that the essential requirements for coexistence are overlapping generations and fluctuating birth rates which ensure that each species has periods when it is increasing. It is found that a species may persist provided only that it is favored sufficiently by the environment during favorable periods independently of the extent to which the other species is favored during its favorable periods.Coexistence is defined in terms of the stochastic boundedness criterion for species persistence. Using the lottery model as an example this criterion is justified and compared with other persistence criteria. Properties of the stationary distribution of population density are determined for an interesting limiting case of the lottery model and these are related to stochastic boundedness. An attempt is then made to relate stochastic boundedness for infinite population models to the behavior of finite population models.     

The effect of slow allosteric transitions in a coupled biochemical oscillator model.

The effect of slowed allosteric transitions in a coupled biochemical oscillator model showing complex dynamic behavior is investigated. When the allosteric transitions are sufficiently fast one can obtain a low-dimensional asymptotic approximation for the dynamics of the species that evolve on a slow time-scale. Such low-dimensional models are common in studies of biological control systems and little attention has, so far, been given to the dynamic effect of the large number of species usually eliminated from more biochemically detailed models. Here we investigate the dynamic effect of explicit inclusion of allosteric transitions having finite time-scales of equilibration. It is found that slowed allosteric transitions suppress complex dynamic modes such a bursting, quasi-periodicity and chaos. The effect arises as the enzyme of consideration becomes trapped in an active state where it is unable to respond to changes in effector concentration on the time-scale necessary to support the modes of complex dynamics. Slow allosteric transitions may be favourable in biological systems in which complex oscillations are not desirable but which, at the same time, may benefit from the presence of positive feedbacks. Our findings suggest that slow allosteric transitions and finite internal rates in general may contribute significantly to the dynamics of biological control mechanisms.     

A stochastic model of cancer initiation including a bystander effect

A stochastic model of cancer initiation is considered. The model is used to evaluate whether a bystander effect may be important in the pre-malignant and malignant stages of carcinogenesis, and furthermore, on the basis of epidemiological data, to estimate the mutation rates of genes involved in the development of oral leukoplakias. The bystander effect is defined here as the capability of oncogenic mutations to increase the mutation probability of neighbouring (bystander) cells, thus leading potentially to a cascade of neighbouring mutated and neoplastic cells as a pre-stage in the development to leukoplakias and cancer. We find that incidence data for oral cancer are indeed in accordance with a significant bystander effect, operating either alone or in combination with genomic instability in the early stages of carcinogenesis, i.e. the development of neoplasia. Simulations performed gave a picture of how mutations and neoplasia may spread in a tissue, to form characteristic leukoplakias with a core of neoplastic cells. The model also showed that the probability of finding at least one neoplastic cell in the tissue after a given number of years is more sensitive to changes in genomic instability within the cell itself than to changes in a bystander effect. Based on epidemiological data we also calculate the maximum number of oncogenic genes that may be involved in the bystander effect and development of genomic instability. Even if capable of explaining the initial development of oncogenic mutations towards neoplastic cells, the bystander model could not reproduce the observed incidence rates of leukoplakia without assuming a carcinogen mutation probability per cell per year of neoplastic cells practically equal to one. This means that the bystander effect, to be of substantial importance in the final development of neoplastic cells towards leukoplakias, requires a very significant increase in mutation probabilities for bystanders to neoplastic cells. Alternatively, additional mechanisms such as abnormal cell differentiation and uncontrolled proliferation and apoptotis in the neoplastic stage may be of major importance during the development to cancerization.     

Cyto-Sim: a formal language model and stochastic simulator of membrane-enclosed biochemical processes

MOTIVATION: Compartments and membranes are the basis of cell topology and more than 30% of the human genome codes for membrane proteins. While it is possible to represent compartments and membrane proteins in a nominal way with many mathematical formalisms used in systems biology, few, if any, explicitly model the topology of the membranes themselves. Discrete stochastic simulation potentially offers the most accurate representation of cell dynamics. Since the details of every molecular interaction in a pathway are often not known, the relationship between chemical species in not necessarily best described at the lowest level, i.e. by mass action. Simulation is a form of computer-aided analysis, relying on human interpretation to derive meaning. To improve efficiency and gain meaning in an automatic way, it is necessary to have a formalism based on a model which has decidable properties. RESULTS: We present Cyto-Sim, a stochastic simulator of membrane-enclosed hierarchies of biochemical processes, where the membranes comprise an inner, outer and integral layer. The underlying model is based on formal language theory and has been shown to have decidable properties (Cavaliere and Sedwards, 2006), allowing formal analysis in addition to simulation. The simulator provides variable levels of abstraction via arbitrary chemical kinetics which link to ordinary differential equations. In addition to its compact native syntax, Cyto-Sim currently supports models described as Petri nets, can import all versions of SBML and can export SBML and MATLAB m-files. AVAILABILITY: Cyto-Sim is available free, either as an applet or a stand-alone Java program via the web page (http://www.cosbi.eu/Rpty_Soft_CytoSim.php). Other versions can be made available upon request.     

Proteome profiling of heat shock of human primary breast epithelial cells,a dataset report

Exposure to elevated temperatures has a strong effect on cell functions, and is used in clinical practice. Hyperthermia may affect multiple regulatory mechanisms in cells. To understand better the response to hyperthermia of immortalized primary human breast epithelial cells, we performed a proteomics study of these cells cultured at 34°C or 39°C. Twenty-four proteins were shown to be differentially expressed due to hyperthermia. Analysis of these proteins showed the potential involvement of various biological processes in response to hyperthermia, e.g., cell adhesion, cell communication, and cell cycle. Transforming growth factor-β2 (TGF-β2) and heat shock protein 27 (HSP27) were found to be upregulated at 39°C. TGF-β2 was found to affect expression of HSP27, and to have a protective role in hyperthermia-induced cell death. Thus, the dataset described here of hyperthermia-related proteins in human primary breast epithelial cells predicts a number of cellular activities affected by exposure to high temperatures and provides a set of proteins for further studies.     

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein-RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.     

Lymphocyte HSP72 following exercise in hyperthermic runners: The effect of temperature

Heat shock protein 72 (HSP72) is the most inducible HSP, but is not always increased in lymphocytes following exercise. This field study examined whether lymphocyte HSP72 was increased in hyperthermic (T_rec>39.0 °C) male athletes following a 14 km competitive race in cool conditions (ambient temperature 11.2 °C). A comparison was also made between control runners (n=7) and those treated for exertional heat illness (n=9). Lymphocyte HSP72 was not increased in control runners immediately post- compared with pre-race, and there was no difference between both groups of runners. A second study of the race (ambient temperature 14.6 °C) found that lymphocyte HSP72 in control (n=7) and treated (n=9) athletes was higher 2 days post- compared with immediately post-race (p<0.01) and these increases were correlated with post-exercise T_rec (p<0.05).     

Dynamic processes that reflect anti-apoptotic strategies set up by HspB1 (Hsp27)

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis.     

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.     

A protocol comparison for the analysis of heat shock protein A1B +A1538G SNP

Heat shock proteins act as molecular chaperones, assist in peptide maturation, and transport nascent peptides across membranes. One commonly studied single nucleotide polymorphism (SNP) for one of the proteins is HSPA1B (+A1538G). However, several studies of this polymorphism have failed to achieve Hardy–Weinberg equilibrium (HWE) for their sample. We compared various published procedures for analyzing the HSPA1B +A1538G SNP and report reasons for HWE discrepancies. Samples from 141 apparently healthy, physically active, volunteers (99 men and 42 women) were analyzed. The first protocol, initially described by Schröder et al., resulted in a genotypic distribution of 22 GG (15.6%), 119 AG (84.4%), and 0 AA; results were confirmed by reanalysis and sequencing. Two other published protocols, one described by Klausz et al. and another by Fekete et al., were used to confirm these results: both resulted in 22 GG (15.6%), 46 AA (32.6%), and 73 AG (51.7%). Additionally, the results were within HWE and confirmed by sequence analysis. Of the original 119 subjects genotyped as AG by the Schröder protocol, 46 of those were confirmed as AA with the Klausz and Fekete methods. Mixing primers from the Schröder and Klausz protocol resulted in 100% concordance with the data generated by the Klausz and Fekete protocols. Some published data on HSP genotyping deviate from HWE; thus, primers used for analyzing these highly homologous genes must be carefully considered. Our results highlight the importance of reinvestigating data when HWE is not achieved for the HSPA1B, or another, polymorphism.     

The effect of hyperbaric oxygen preconditioning on heat shock protein 72 expression following in vitro stress in human monocytes

Hyperbaric oxygen (HBO) is thought to confer protection to cells via a cellular response to free radicals. This process may involve increased expression of heat shock proteins, in particular the highly inducible heat shock protein 72 (Hsp72). Healthy male volunteers (n = 16) were subjected to HBO for 1 h at 2.8 ATA. Inducible Hsp72 expression was measured by flow cytometry pre-, post- and 4 h-post HBO. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood via density centrifugation pre-, post- and 4 h post-HBO. PBMC were then subjected to an in vitro heat shock at 40°C or hypoxia at 37°C (5% O₂) with a control at 37°C. Cells were then analysed for Hsp72 expression by flow cytometry. Monocytes showed no significant changes in Hsp72 expression following HBO. No detectable Hsp72 was seen in lymphocytes or neutrophils. Following in vitro hypoxic exposure, a significant increase in Hsp72 expression was observed in monocytes isolated immediately post- (p = 0.006) and 4 h post-HBO (p = 0.010) in comparison to control values. HBO does not induce Hsp72 expression in PBMC. The reported benefits of HBO in terms of pre-conditioning are not due to inducement of Hsp72 expression in circulating blood cells, but may involve an enhancement of the stress response.     

The heat-shock response in higher plants: a biochemical model

Abstract A compilation of existing data on higher plant responses to heat-shock temperatures has been utilized to produce a biochemically based model of integrated cellular responses to elevated temperatures. This model describes a potential mechanism for the triggering of several biochemical responses to a thermally induced leakage of extracellular or vacuolar ions into the cytoplasm. It seems possible that many of the observed heat-shock responses are involved in the protection of (a) enzymes from inactivation and (b) nucleic acids from cleavage induced by the presence of elevated levels of specific metals.     

The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.