GENIES: a natural-language processing system for the extraction of molecular pathways from journal articles

Systems that extract structured information from natural language passages have been highly successful in specialized domains. The time is opportune for developing analogous applications for molecular biology and genomics. We present a system, GENIES, that extracts and structures information about cellular pathways from the biological literature in accordance with a knowledge model that we developed earlier. We implemented GENIES by modifying an existing medical natural language processing system, MedLEE, and performed a preliminary evaluation study. Our results demonstrate the value of the underlying techniques for the purpose of acquiring valuable knowledge from biological journals.     

Chlorophyll fluorescence as a tool for evaluation of drought stress in strawberry

The effect of water deficit on chlorophyll fluorescence, sugar content, and growth parameters of strawberry (Fragaria×ananassa Duch. cv. Elsanta) was studied. Drought stress caused significant reductions in leaf water potential, fresh and dry masses, leaf area, and leaf number. A gradual reduction of photochemical quenching (q_P) and quantum efficiency (Φ_PS2) was observed under drought stress while non-photochemical quenching (q_N) increased. Maximum efficiency of photosystem 2 (F_v/F_m) was not affected by drought stress.     

Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.     

Chlorophyll fluorescence as a tool for evaluation of viability in freeze-stressed grapevine buds

This study examined the utility of the ratio of variable fluorescence to maximum fluorescence (F_v/F_m) to detect freezing injury on buds of two Vitis vinifera cultivars: Pinot noir and Pinot gris. Freezing treatments on buds caused a decrease both in F_v/F_m and percentage of budburst, more severely on Pinot gris than Pinot noir, specifically at the lower temperature (−20°C). F_v/F_m ratio showed a close correlation with percentage of budburst, and a threshold of the lethal F_v/F_m was proposed as an indicator of bud mortality.     

A new diagnostic tool for neurocysticercosis is a member of a cestode specific hydrophobic ligand binding protein family

A protein of unknown function has been identified as a key serological tool for diagnosis of human tapeworm neurocysticercosis, a major worldwide neurological disease. Our own sequence analysis predicts that this protein is a member of a newly identified cestode specific oligomeric hydrophobic ligand binding protein family. In this report, using a rat cestode model, we confirm that homologues of this protein can bind fatty acids and their derivatives, and thus suggest a biological function for this key diagnostic tool.     

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a-mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.     

p16INK4A is a robust in vivo biomarker of cellular aging in human skin

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.     

Unbroken: RADseq remains a powerful tool for understanding the genetics of adaptation in natural populations

Recently, Lowry et al. addressed the ability of RADseq approaches to detect loci under selection in genome scans. While the authors raise important considerations, such as accounting for the extent of linkage disequilibrium in a study system, we strongly disagree with their overall view of the ability of RADseq to inform our understanding of the genetic basis of adaptation. The family of RADseq protocols has radically improved the field of population genomics, expanding by several orders of magnitude the number of markers available while substantially reducing the cost per marker. Researchers whose goal is to identify regions of the genome under selection must consider the LD of the experimental system; however, there is no magical LD cutoff below which researchers should refuse to use RADseq. Lowry et al. further made two major arguments: a theoretical argument that modeled the likelihood of detecting selective sweeps with RAD markers, and gross summaries based on an anecdotal collection of RAD studies. Unfortunately, their simulations were off by two orders of magnitude in the worst case, while their anecdotes merely showed that it is possible to get widely divergent densities of RAD tags for any particular experiment, either by design or due to experimental efficacy. We strongly argue that RADseq remains a powerful and efficient approach that provides sufficient marker density for studying selection in many natural populations. Given limited resources, we argue that researchers should consider a wide range of trade‐offs among genomic techniques, in light of their study question and the power of different techniques to answer it.     

DNase I digestion as a tool for the quantitative evaluation of C-heterochromatic-DNA in situ.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of the pre-treatments C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic-DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse metaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in chromosomes.     

Modeling with a view to target identification in metabolic engineering: A critical evaluation of the available tools

The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The natural variability of morphogenesis: a tool for exploring the mechanics of gastrulation movements in amphibian embryos

The investigation of natural variability of metric morphological characters in frog gastrulation revealed that in genetically and environmentally homogeneous samples of embryos their variability is of a higher order of magnitude than that known for quantitative metric characters in adult organisms. Matching the coefficients of variation of characters under consideration to the specific rates of their changes in normal development revealed a strong positive correlation between the rates of morphological change and the amount of morphological variance. The increase in the variance is mainly in characters concerned with shaping of moving embryonic areas and arises as a result of a positive feedback between the movement of a given area and recruitment of cells from surrounding areas into the movement. The account of natural variation suggests a new model of amphibian gastrulation whose essential feature is the intimate connection between the movement and shaping of the dorsal blastopore lip of the gastrula.     

Lipolysis index: evaluation of a new tool for metabolic assessment in epidemiological studies on obesity

Lipid mobilization through adipocyte lipolysis is central for energy metabolism and is decreased in obesity. However, the factors of importance for lipolytic activity in the general population are not known. To further examine this we performed a cross-sectional study on teenagers and adults. We constructed and evaluated a simple index of lipolytic activity (ratio of fasting p-glycerol and body fat %) in population based samples in 316 teenagers (BMI 16-51 kg/m (2)) and 3,039 adults (BMI 16-70 kg/m (2)). In the adults, multiple regression analysis showed that waist and BMI but not age, plasma insulin, plasma noradrenaline or waist-to-hip ratio contributed independently and inversely to lipolytic activity (partial r=-0.37 and -0.28, respectively, p<0.0001). Together waist and BMI explained about 45% of the variability of lipolysis. Waist was a stronger factor than BMI in stepwise regression. The same analysis in teenagers showed that only BMI contributed independently and inversely to lipolytic activity (partial r=-0.90, p<0.0001) and explained about 55% of lipolysis variation. BMI had the strongest effect on lipolysis in lean teenagers. The results were the same for men and women. At all levels of lipolytic activity plasma fatty acid levels were elevated in obese subjects (p<0.0001). We conclude that during adolescence BMI is the major factor negatively influencing lipolytic activity, in particular among lean young subjects. In adulthood central fat accumulation together with increasing BMI decreases lipolysis. In spite of low lipolytic activity circulating fatty acid levels are increased in obesity, probably due to an adipose mass effect.     

A policy tool for establishing a balance between wildlife habitat preservation and the use of natural resources by rural people in South Africa

In this paper, a model is set up to determine the size of the wildlife population compatible with the extraction of the maximum output by rural people from natural resources. It is found that when the size of human population increases, to obtain the maximum output of food, the size of wildlife population decreases if the human population is growing faster than or at the same rate as that of the wildlife population; whereas the size of the wildlife population increases if the human population is growing slower than that of the wildlife. Furthermore, in the event that the increase in the size of the wildlife population is unable to reach the level compatible with the extraction of the maximum output of food, the improvement of the wildlife habitat and supplementing rural people's income with the proceeds of tourism are proposed as policies to maintain a balance between the preservation of the wildlife habitat and the use of natural resources by rural people.     

Gustatory intensity discrimination in rat NTS: a tool for the evaluation of neural coding theories

Summary The neural coding of taste quality in vertebrates currently is addressed by the labeledline and the across-fiber pattern theory. Experimental tests that have tried to distinguish between these theories by manipulating taste quality have been problematic in that both are fairly successful. However, the two theories maintain that different numbers of neurons participate in coding the presence of a substance. In the labeled-line theory, only those neurons labeled by that substance are supposed to be involved, whereas in the across-fiber pattern theory, all neurons responding to the substance are taken into account. We therefore expected that the theories might predict different intensity discriminating abilities among the four classical taste stimuli, which could provide a less equivocal test between the theories.A previous paper (Maes 1984) described a model for the neural basis of intensity discrimination, based on signal-detection theory. In the present paper, experimental data obtained from single neurons in the nucleus of the solitary tract of rat are analyzed in terms of this model. Whole-mouth stimulation with the four classical taste substances was used. For both theories, predictions of the intensity discriminating acuity for the four substances were derived, using different response analysis intervals, and including two plausible non-linearities in neural processing.The acuity profile across the four stimuli (consisting of the reciprocals of the four differential thresholds) turned out to be sensitive to the choice of coding theory and non-linearity. The interval chosen for response analysis had a strong effect. The predicted profiles should be checked against behavioral data on intensity discrimination in rat, but since such data are not yet available, a comparison was made with human psychophysical data, for illustrative purposes. It appeared that a unique combination of variables (tonic part of the response, across-fiber pattern coding with a contrast enhancement type of non-linearity) could be selected to match the psychophysical profile.In the Discussion, special attention is paid to the fundamental differences between the two coding theories, to the relevance of whole-mouth stimulation, and to the informational significance of various parts of the phasic/tonic response.Abbreviations AFP across-fiber pattern - CT chorda tympani - DA discrimination acuity - DT differential threshold - H sour - LL labeled line - N salty - NTS nucleus tractus solitarius - PTA pontine taste area - Q bitter - S sweet     

Photochromic agents as tools for protein structure study: lapachenole is a photoaffinity ligand of cytochrome P450 3A4

Cytochrome P450 3A4 is a drug-metabolizing enzyme of extraordinarily broad substrate specificity. This quality imparts upon the enzyme special importance in understanding its determinants of activity and substrate recognition. Limited successes in P450 3A4 active-site structure studies have been achieved by use of mechanism-based inactivators and photoaffinity ligands. We report here the potential of photochromic agents, compounds with the ability to undergo light-induced, reversible reactions, to be used as effective photoaffinity ligands. Four such compounds of the chromene family were shown by ultraviolet and visible spectroscopy to undergo photoinduced rearrangements to highly conjugated and reactive products in buffered aqueous solution. While some of these intermediates were very long-lived (>12 h, photoactivated lapachenole), others existed for milliseconds in their opened forms (precocene I and 2,2-dimethyl-5,6-benzo-2H-chromene) and were observed by laser flash photolysis. Each of the tricyclic structures studied rapidly underwent Michael addition reactions with the test nucleophile glutathione upon irradiation to form single conjugated products. The smaller precocene I reacted more extensively to form multiple products. These attributes of the chromenes inspired testing of their potential to label cytochrome P450 3A4 in a light-dependent fashion. Access to the protein active site by lapachenole was demonstrated with the molecule's ability to competitively inhibit P450 3A4-mediated oxidative metabolism of midazolam with an IC(50) value of 71 microM. This inhibition became irreversible upon irradiation of the enzyme-ligand complex with ultraviolet light. These results clearly demonstrate that chromenes are effective photoaffinity reagents for the cytochrome P450 superfamily of enzymes and probably other proteins as well.