On the number of experiments required to find the causal structure of complex systems

The need to capture the complexity of biological systems in a simpler formalism is the underlying impetus of biological sciences. Understanding the function of many biological complex systems, such as genetic networks or molecular signalling pathways, requires precise identification of the interactions between their individual components. A number of questions in the study of complex systems are then important-in particular, what can be inferred about the interactions in a complex system from an arbitrary set of experiments, and, what is the minimum number of experiments required to characterize the system? This paper shows that the problem of finding the minimal causal structure of a system based on a set of observations is computationally intractable for even moderately sized systems (it is NP-hard), but a reasonable approximation can be found in a relatively short (polynomial) time. Next, it is shown that the number of experiments required to characterize a complex system grows exponentially with the upper bound on the number of immediate upstream influences of each element, but only logarithmically with the number of elements in the system. This makes it possible to study biological systems with extremely large number of interacting elements and relatively sparse interconnections, such as gene regulatory and cell signalling networks. Finally, the construction of a randomized experimental sequence which achieves this bound is discussed.     

Pharmacogenetics and drug development: the path to safer and more effective drugs

Pharmacogenetics provides opportunities for informed decision-making along the pharmaceutical pipeline. There is a growing literature of retrospective studies of marketed medicines that describe efficacy or safety on the basis of patient genotypes. These studies emphasize the potential prospective use of genome information to enhance success in finding new medicines. An example of a prospective efficacy pharmacogenetic Phase-IIA proof-of-concept study is described. Inserting a rapidly performed efficacy pharmacogenetic step after initial clinical data are obtained can provide confidence for a commitment to full drug development. The rapid identification of adverse events during and after drug development using genomic mapping tools is also reviewed.     

Opponent colour coding is a universal strategy to evaluate the photoreceptor inputs in Hymenoptera

Summary Behavioural tests were carried out with 9 hymenopteran insect species, which ranked certain sets of coloured stimuli according to their subjective similarity to a previously memorized stimulus. Kendall's coefficient is employed for the analysis of correlation between these similarity rankings and the colour distance rankings predicted by various models of neural colour computation. The models are based on the measured spectral sensitivities of photoreceptor colour types and use a variety of simple colour coding systems to derive hypothetical colour distances. The correlation between the predictions of the models and the behavioural results serves as a measure for the likelihood of existence of a colour coding system. In all species, the similarity rankings can be best explained by assuming that colour is coded on a perceptual level by two colour opponent mechanisms. Brightness differences are ignored, indicating that an intensity-coding sub-system is not used in colour discrimination by the insects investigated. The weighting factors of the colour opponent mechanisms differ between species in detail, but not in the principles involved. It is thus possible to employ a standard measure of perceptual colour distance (colour hexagon distance) to predict the capacities of colour discrimination adequately in all the tested insects.     

The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

Silkworm as a model animal to evaluate drug candidate toxicity and metabolism

To evaluate the feasibility of using the silkworm as a model animal for screening drug candidates, we examined whether the lethal dose of cytotoxic chemicals in silkworm, Bombyx mori, were consistent with those in mammals, and compared the metabolic pathways of these drugs between silkworms and mice. The lethal dose levels of cytotoxic chemicals in silkworms were consistent with those in mammals. We examined the fate of model drugs, 4-methyl umbelliferone, umbelliferone, and 7-ethoxycoumarine, in silkworm larvae. The half-life of 4-methyl umbelliferone in the silkworm larvae hemolymph was 7.0+/-0.1 min, similar to that in mouse blood. In silkworm larvae, 4-methyl umbelliferone was conjugated with glucose, whereas in mammals it is conjugated with glucuronate or sulfate. These results are consistent with a previous report that UDP-glucosyltransferase catalyzes the conjugation of 4-methyl umbelliferone. The glucose-conjugation reaction of 4-methyl umbelliferone was observed in microsomal fractions of fat bodies isolated from silkworms. Furthermore, most umbelliferone and 7-ethoxycoumarine injected into the hemolymph of silkworms was eliminated through the feces in the glucose-conjugated form. These findings suggest that chemicals are metabolized through a pathway common to both mammals and silkworms: reaction with cytochrome P450, conjugation with hydroxylated compounds, and excretion.     

A kinematic model of the shoulder complex to evaluate the arm-reachable workspace

Upper-arm evaluation including shoulder motion in physiotherapy has no three-dimensional tool for an arm-functioning evaluation, which hampers an uniform, objective comparison. Human shoulder complex models suffer from lack of shoulder girdle kinematic data. A kinematic shoulder-complex model with six degrees of freedom is proposed as the composition of the inner joint representing the shoulder-girdle joints and outer joint representing the glenohumeral joint. The outer shoulder joint has three perpendicular rotations: adduction/abduction, retroflexion/flexion and internal/external rotation of the humerus. The inner shoulder joint has two rotations, depression/elevation and retraction/protraction, and one translation, which are all dependent on the elevation angle of the humerus. The human arm-reachable workspace that represents the area within reach of the wrist is calculated on the basis of the shoulder-complex model and the additional elbow-joint direct kinematics. It was demonstrated that cross-sections of the calculated workspace are in agreement with the measured arm-reachable workspace in all three anatomical planes. The arm-reachable workspace volume and graphics were calculated and a comparison of the arm's workspaces during a patient's shoulder treatment was made. The obtained numerical and graphical arm-reachable workspaces can be used for arm-functioning evaluations in rehabilitation and ergonomics.     

Use of procalcitonin and C-reactive protein to evaluate vaccine efficacy against pneumonia

Background

Pneumonia remains the leading cause of death in young children. The poor specificity of chest radiographs (CXRs) to diagnose pneumococcal pneumonia may underestimate the efficacy of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.

Methods and Findings

The efficacy of nine-valent pneumococcal conjugate vaccine among children not infected with HIV (21%; 95% confidence interval, 1%–37%) increased when CXR-confirmed pneumonia was associated with serum C-reactive protein of 120 mg/l (12mg/dl) or more and procalcitonin of 5.0 ng/ml or more (64%; 95% confidence interval, 23%–83%). Similar results were observed in children infected with HIV.

Conclusion

C-reactive protein and procalcitonin improve the specificity of CXR to diagnose pneumococcal pneumonia and may be useful for the future evaluation of the effectiveness of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.     

A simple animal can use a complex stimulus pattern to find a location: Nematode thermotaxis in soil

Newly hatched infective juveniles of the plant-parasitic nematode Meloidogyne incognita have recently been found to migrate in very shallow thermal gradients to a preferred temperature that is several °C above the temperature to which they were acclimated. Possible functions of this unusual behavior were explored by computer modeling of the movement of such nematodes in the dynamic thermal environment typical of soil. Reliable estimates were available for all the required parameters. The model predicts that, as the diurnal temperature fluctuations at the surface penetrate into the soil, a nematode located below the surface will move upward during part of the day and downward during the rest of the day. However, the distances moved upward and downward do not balance and there is a net change in depth over a period of days. The net change is upward or downward depending on the physiological parameters of the nematode and its depth. Using the best estimates available for the parameters, it is predicted that nematodes starting at any depth between 0 and 15 cm would move toward an intermediate depth of about 5 cm. It is hypothesized that, in the absence of chemical cues, this response leads the nematodes toward a level that is optimal for locating the roots of host plants. This suggests that simple organisms may make use of much more complex stimulus patterns than was previously realized.     

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.     

GPDTI: a Genetic Programming Decision Tree induction method to find epistatic effects in common complex diseases

MOTIVATION: The identification of risk-associated genetic variants in common diseases remains a challenge to the biomedical research community. It has been suggested that common statistical approaches that exclusively measure main effects are often unable to detect interactions between some of these variants. Detecting and interpreting interactions is a challenging open problem from the statistical and computational perspectives. Methods in computing science may improve our understanding on the mechanisms of genetic disease by detecting interactions even in the presence of very low heritabilities. RESULTS: We have implemented a method using Genetic Programming that is able to induce a Decision Tree to detect interactions in genetic variants. This method has a cross-validation strategy for estimating classification and prediction errors and tests for consistencies in the results. To have better estimates, a new consistency measure that takes into account interactions and can be used in a genetic programming environment is proposed. This method detected five different interaction models with heritabilities as low as 0.008 and with prediction errors similar to the generated errors. AVAILABILITY: Information on the generated data sets and executable code is available upon request.     

A microcomputer program to evaluate the saturation of complex solutions with respect to biominerals

The program described is written in BASIC and enables calculationof ionic activity products and degrees of saturation for solutionswith respect to minerals of biological interest. The programtakes into account ion-pair formation by association of anionsand cations. The number of constituent species, whether or notinvolved in mineral formation or in ion-pair formation, appearsto be unlimited. The computation fulfils the conditions of conservationof matter and electrical neutrality. Successful computationis achieved even for systems with highly unfavourable ratiosbetween cations and anions with high affinities. The programis easily re-orientated towards any mineral/solution system. Received on March 1, 1988; accepted on April 19, 1988     

A simple but highly effective approach to evaluate the prognostic performance of gene expression signatures

Background

Highly parallel analysis of gene expression has recently been used to identify gene sets or ‘signatures’ to improve patient diagnosis and risk stratification. Once a signature is generated, traditional statistical testing is used to evaluate its prognostic performance. However, due to the dimensionality of microarrays, this can lead to false interpretation of these signatures.

Principal Findings

A method was developed to test batches of a user-specified number of randomly chosen signatures in patient microarray datasets. The percentage of random generated signatures yielding prognostic value was assessed using ROC analysis by calculating the area under the curve (AUC) in six public available cancer patient microarray datasets. We found that a signature consisting of randomly selected genes has an average 10% chance of reaching significance when assessed in a single dataset, but can range from 1% to ∼40% depending on the dataset in question. Increasing the number of validation datasets markedly reduces this number.

Conclusions

We have shown that the use of an arbitrary cut-off value for evaluation of signature significance is not suitable for this type of research, but should be defined for each dataset separately. Our method can be used to establish and evaluate signature performance of any derived gene signature in a dataset by comparing its performance to thousands of randomly generated signatures. It will be of most interest for cases where few data are available and testing in multiple datasets is limited.     

Evolution of antiretroviral drug costs in Brazil in the context of free and universal access to AIDS treatment

Background

Little is known about the long-term drug costs associated with treating AIDS in developing countries. Brazil''s AIDS treatment program has been cited widely as the developing world''s largest and most successful AIDS treatment program. The program guarantees free access to highly active antiretroviral therapy (HAART) for all people living with HIV/AIDS in need of treatment. Brazil produces non-patented generic antiretroviral drugs (ARVs), procures many patented ARVs with negotiated price reductions, and recently issued a compulsory license to import one patented ARV. In this study, we investigate the drivers of recent ARV cost trends in Brazil through analysis of drug-specific prices and expenditures between 2001 and 2005.

Methods and Findings

We compared Brazil''s ARV prices to those in other low- and middle-income countries. We analyzed trends in drug expenditures for HAART in Brazil from 2001 to 2005 on the basis of cost data disaggregated by each ARV purchased by the Brazilian program. We decomposed the overall changes in expenditures to compare the relative impacts of changes in drug prices and drug purchase quantities. We also estimated the excess costs attributable to the difference between prices for generics in Brazil and the lowest global prices for these drugs. Finally, we estimated the savings attributable to Brazil''s reduced prices for patented drugs. Negotiated drug prices in Brazil are lowest for patented ARVs for which generic competition is emerging. In recent years, the prices for efavirenz and lopinavir–ritonavir (lopinavir/r) have been lower in Brazil than in other middle-income countries. In contrast, the price of tenofovir is US$200 higher per patient per year than that reported in other middle-income countries. Despite precipitous price declines for four patented ARVs, total Brazilian drug expenditures doubled, to reach US$414 million in 2005. We find that the major driver of cost increases was increased purchase quantities of six specific drugs: patented lopinavir/r, efavirenz, tenofovir, atazanavir, enfuvirtide, and a locally produced generic, fixed-dose combination of zidovudine and lamivudine (AZT/3TC). Because prices declined for many of the patented drugs that constitute the largest share of drug costs, nearly the entire increase in overall drug expenditures between 2001 and 2005 is attributable to increases in drug quantities. Had all drug quantities been held constant from 2001 until 2005 (or for those drugs entering treatment guidelines after 2001, held constant between the year of introduction and 2005), total costs would have increased by only an estimated US$7 million. We estimate that in the absence of price declines for patented drugs, Brazil would have spent a cumulative total of US$2 billion on drugs for HAART between 2001 and 2005, implying a savings of US$1.2 billion from price declines. Finally, in comparing Brazilian prices for locally produced generic ARVs to the lowest international prices meeting global pharmaceutical quality standards, we find that current prices for Brazil''s locally produced generics are generally much higher than corresponding global prices, and note that these prices have risen in Brazil while declining globally. We estimate the excess costs of Brazil''s locally produced generics totaled US$110 million from 2001 to 2005.

Conclusions

Despite Brazil''s more costly generic ARVs, the net result of ARV price changes has been a cost savings of approximately US$1 billion since 2001. HAART costs have nevertheless risen steeply as Brazil has scaled up treatment. These trends may foreshadow future AIDS treatment cost trends in other developing countries as more people start treatment, AIDS patients live longer and move from first-line to second and third-line treatment, AIDS treatment becomes more complex, generic competition emerges, and newer patented drugs become available. The specific application of the Brazilian model to other countries will depend, however, on the strength of their health systems, intellectual property regulations, epidemiological profiles, AIDS treatment guidelines, and differing capacities to produce drugs locally.     

Genetic and environment-induced pathways to innovation: on the possibility of a universal relationship between robustness and adaptation in complex biological systems

Recently, there has been considerable interest in the idea that mutational robustness enhances the propensity for future adaptations, i.e. evolvability, if evolution proceeds over a neutral network that extends far throughout a fitness landscape. While the genetic neutral network (NN-G) model may have important implications to our understanding of evolution, little has been done to integrate these theoretical developments with empirical evidence that heritable phenotypes can also originate and become fixated as a result of changes in the environment. In this brief commentary, I reconsider the role of environmental change in the adaptation of species and ask whether positive robustness-evolvability relationships might exist not only for genetic but also environmental buffering. In particular, I ask whether the insensitivity of species fitness towards variability in its environment can have a positive influence on the likelihood of future environment-induced adaptations (i.e. ecological opportunities) in a manner analogous to that proposed by the NN-G model. After outlining scenarios where such a counter-intuitive relationship appears plausible, I comment on the merits of evolutionary theories that can integrate complementary pathways to adaptation under static and time-variant environments. I also speculate on some of the features that such a theory might have.     

The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex

Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.     

Considerations in the design of possible cell cycle effective drugs

Antineoplastic agents are known to induce differential cytotoxic and cytostatic effects throughout the cell cycle. Many drugs have greater toxicity for cycling cells and act selectively at one or more phases of the cycle and may cause partial synchrony of surviving cells. However, these observations have been generally carried out on in vitro systems only and present a variety of complexities and pitfalls. Furthermore, human tumours are often characterized by a relatively low fraction of proliferating cells and present a large cellular heterogeneity as far as their cytogenetic, cytokinetic, and clonogenic features and their responses to drugs are concerned. Therefore, resistance to chemotherapy is due to various factors characterizing, in some instances, each individual tumour. In spite of the advent of technological advances such as flow cytometry, it is still difficult to design kinetic-orientated therapies especially for the treatment of solid tumours. Consequently, it is also difficult to design protocols based on cell cycle effective drugs. The possibility remains, at least for the moment, to stratify tumours according to their cellular heterogeneity. Different protocols could then be assigned to classes of tumours. Such an approach could be completed by further advances in the cellular monitoring of individual tumours.     

Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy.

BACKGROUND: Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2). METHODS: Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene. RESULTS: Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells. CONCLUSIONS: A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.