An integrative approach for the identification of quantitative trait loci

The genetic dissection of complex traits is one of the most difficult and most important challenges facing science today. We discuss here an integrative approach to quantitative trait loci (QTL) mapping in mice. This approach makes use of the wealth of genetic tools available in mice, as well as the recent advances in genome sequence data already available for a number of inbred mouse strains. We have developed mapping strategies that allow a stepwise narrowing of a QTL mapping interval, prioritizing candidate genes for further analysis with the potential of identifying the most probable candidate gene for the given trait. This approach integrates traditional mapping tools, fine mapping tools, sequence-based analysis, bioinformatics and gene expression.     

An approach for studying the mediators of pathogenesis inMycobacterium tuberculosis

Mycobacterium tuberculosis is an example of an intracellular pathogen that mediates the disease state through complex interactions with the host’s immune system. Not only does this organism replicate in the hostile environment prevailing within the infected macrophage, but it has also developed intricate mechanisms to inhibit several defence mechanisms of the host’s immune system. It is postulated here that the mediators of these interactions with the host are products of differentially expressed genes in the pathogen. B and T cell responses of the host are hence to be used as tools to identify such gene products from an expression library of theMycobacterium tuberculosis genome. The various pathways of generating a productive immune response that may be targeted by the pathogen are discussed     

An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort

Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL) response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine. To investigate such a potential, we analyzed HIV-1 gag from 468 HIV-1-positive Kenyan women by using several bioinformatic approaches that allowed the identification of positively selected amino acids in the HIV-1 gag region and study of the effects that these mutations could have on the various stages of antigen processing. Correlations between positively selected residues and mean CD4 counts also allowed study of the effect of mutation on HIV disease progression. A number of mutations that could create or destroy proteasomal cleavage sites or reduce binding affinity of the transport antigen processing protein, effectively hindering epitope presentation, were identified. Many mutations correlated with the presence of specific HLA alleles and with lower or higher CD4 counts. For instance, the mutation V190I in subtype A1-infected individuals is associated with HLA-B*5802 (P = 4.73 x 10(-4)), a rapid-progression allele according to other studies, and also to a decreased mean CD4 count (P = 0.019). Thus, V190I is a possible HLA escape mutant. This method classifies many positively selected mutations across the entire gag region according to their potential for immune escape and their effect on disease progression.     

An integrative approach to understanding mechanosensation

The ability for a living organism to sense and respond to its external environment is crucial to its survival. Understanding mechanosensation, the mechanism by which organisms react in response to mechanical stimuli, presents many interesting and challenging problems for both experimental and computational biologists. A major difficulty in studying mechanosensors is their inherent multiscale nature. The systems involved in mechanosesnsing can span eight orders of magnitude in length scale and up to 10 orders of magnitude in time scale. Trying to ascertain information across these length and time scales simultaneously is challenging. This problem has led to the need to approach these types of problems using an integrative approach, combining both computational and experimental biology. This review classifies the major types of mechanosensors and explains methods that have been employed in understanding their behavior, both using modeling and experimental techniques. Multiscale modeling methods combined with experimental techniques in an integrative approach are suggested as ways of undertaking the study of such systems.     

An integrative approach for the optical sequencing of single DNA molecules

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.     

An artificial neural network approach for studying phytoplankton succession

Artificial neural networks are used to model phytoplankton succession and gain insight into the relative strengths of bottom-up and top-down forces shaping seasonal patterns in phytoplankton biomass and community composition. Model comparisons indicate that patterns in chlorophyll aconcentrations response instantaneously to patterns in nutrient concentrations (phosphorous (P), nitrite and nitrate (NO₂/NO₃–N) and ammonium (NH₄–H) concentrations) and zooplankton biomass (daphnid cladocera and copepoda biomass); whereas lagged responses in an index of algal community composition are evident. A randomization approach to neural networks is employed to reveal individual and interacting contributions of nutrient concentrations and zooplankton biomass to predictions of phytoplankton biomass and community composition. The results show that patterns in chlorophyll aconcentrations are directly associated with P, NO₂/NO₃–N and daphnid cladocera biomass, as well as related to interactions between daphnid cladocera biomass, and NO₂/NO₃–N and P. Similarly, patterns in phytoplankton community composition are associated with NO₂/NO₃–N and daphnid cladocera biomass; however show contrasting patterns in nutrient– zooplankton and zooplankton–zooplankton interactions. Together, the results provide correlative evidence for the importance of nutrient limitation, zooplankton grazing and nutrient regeneration in shaping phytoplankton community dynamics. This study shows that artificial neural networks can provide a powerful tool for studying phytoplankton succession by aiding in the quantification and interpretation of the individual and interacting contributions of nutrient limitation and zooplankton herbivory on phytoplankton biomass and community composition under natural conditions.     

An integrative functional genomics approach for discovering biomarkers in schizophrenia

Schizophrenia (SZ) is a complex disorder resulting from both genetic and environmental causes with a lifetime prevalence world-wide of 1%; however, there are no specific, sensitive and validated biomarkers for SZ. A general unifying hypothesis has been put forward that disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association study (GWAS) are more likely to be associated with gene expression quantitative trait loci (eQTL). We will describe this hypothesis and review primary methodology with refinements for testing this paradigmatic approach in SZ. We will describe biomarker studies of SZ and testing enrichment of SNPs that are associated both with eQTLs and existing GWAS of SZ. SZ-associated SNPs that overlap with eQTLs can be placed into gene-gene expression, protein-protein and protein-DNA interaction networks. Further, those networks can be tested by reducing/silencing the gene expression levels of critical nodes. We present pilot data to support these methods of investigation such as the use of eQTLs to annotate GWASs of SZ, which could be applied to the field of biomarker discovery. Those networks that have association with SNP markers, especially cis-regulated expression, might lead to a more clear understanding of important candidate genes that predispose to disease and alter expression. This method has general application to many complex disorders.     

Molecular genetic approaches for studying the etiology of diabetic nephropathy

A critical challenge faced by clinical nephrologists today is the escalating number of patients developing end stage renal disease, a major proportion of which is attributed to diabetic nephropathy (DN). The need for new measures to prevent and treat this disease cannot be overemphasized. To this end, modern genetic approaches provide powerful tools to investigate the etiology of DN. Human studies have already established the importance of genetic susceptibility for DN. Several major susceptibility loci have been identified using linkage studies. In addition, linkage studies in rodents have pinpointed promising chromosomal segments that influence renal traits. Besides augmenting our understanding of disease pathogenesis, these animal studies may facilitate the cloning of disease susceptibility genes in man through the identification of homologous regions that contribute to renal disease. In human diabetes, various genes have been evaluated for their risk contribution to DN. This widespread strategy has been propelled by our knowledge of the glucose-activated pathways underlying DN. Evidence has emerged that a true association does indeed exist for some candidate genes. Furthermore, the in vivo manipulation of gene expression has shown that these genes can modify features of DN in transgenic and knockout rodent models, thus corroborating the findings from human association studies. Still, the exact molecular mechanisms involving these genes remain to be fully elucidated. This formidable task may be accomplished by continuing to harness the synergy between human and experimental genetic approaches. In this respect, our review provides a first synthesis of the current literature to facilitate this challenging effort.     

An integrative approach to environmental engineering in zoos

Zoos have shifted recently from the historical tendency to display as many species as possible to an emphasis on improved education and captive propagation. Many enlightened zoo administrations attempt to achieve these goals through creation of exhibits that mimic animals' natural physical and social environments, as extrapolated from field studies. An adjunct is the increased use of technology as a tool in environmental engineering, the term used herein to refer to the design of habitats for captive animals. Recently there has been discussion about the degree of technological control that is appropriate in zoo displays. One progressive group has proposed that natural resources and technology should only be used to enhance displays passively. Their efforts are concentrated on simulation of natural physical and social environments. Another group has suggested that technology should play an active role in the design of captive environments. They attempt to modify displays so that captive animals participate actively to obtain food. This paper first reviews the literature of both groups and suggests an integration of their views. The second section illustrates how both methods may be used to solve problems zoos face with animals during reproduction, development, and adulthood.     

An integrative approach to the biomechanical function and neuromuscular control of the fingers

The exquisite mechanical functionality and versatility of the human hand emerges from complex neuro-musculo-skeletal interactions that are not completely understood. I have found it useful to work within a theoretical/experimental paradigm that outlines the fundamental neuro-musculo-skeletal components and their interactions. In this integrative paradigm, the laws of mechanics, the specifications of the manipulation task, and the sensorimotor signals define the interactions among hand anatomy, the nervous system, and manipulation function. Thus, our collaborative research activities emphasize a firm grounding in the mechanics of finger function, insistence on anatomical detail, and meticulous characterization of muscle activity. This overview of our work on precision pinch (i.e., the ability to produce and control fingertip forces) presents some of our findings around three Research Themes: Mechanics-based quantification of manipulation ability; Anatomically realistic musculoskeletal finger models; and Neural control of finger muscles. I conclude that (i) driving the fingers to some limit of sensorimotor performance is instrumental to elucidating motor control strategies; (ii) that the cross-over of tendons from flexors to extensors in the extensor mechanism is needed to produce force in every direction, and (iii) the anatomical routing of multiarticular muscles makes co-contraction unavoidable for many tasks. Moreover, creating realistic and clinically useful finger models still requires developing new computational means to simulate the viscoelastic tendinous networks of the extensor mechanism, and the muscle-bone-ligament interactions in complex articulations. Building upon this neuromuscular biomechanics paradigm is of immense clinical relevance: it will be instrumental to the development of clinical treatments to preserve and restore manual ability in people suffering from neurological and orthopedic conditions. This understanding will also advance the design and control of robotic hands whose performance lags far behind that of their biological counterparts.     

An ecogenetic approach to studying the adaptation and human health]

Genetic markers--blood groups ABO, RH, MN; serum proteins HP, PI, TF, C3; erythrocyte enzymes ACP1, ESD, AK1, PGM1, GLO1, PGD, PGP; and the other: PTC-tasting, ear wax types and color vision, were studied in two aboriginal Buryatian populations of Baikal Lake region: in Chitinskaya and Irkutskaya Provinces. Two samples were further divided into subgroups, according to their health status: "healthy", "indefinite" and "sick" by means of special regression procedure. The "healthy" subgroup of the Chitinskaya Province population is characterized by higher frequencies of PTC-tasters: 0.871 vs. 0.757 in the "sick" part (chi 2 = 5.36, p less than 0.05); higher frequency of the phenotype PI M1M1: 0.734 in "healthy" vs. 0.547 in "sick" (chi 2 = 8.89, p less than 0.01); also, lower frequency of the PI M1M2 phenotype: 0.148 and 0.299, respectively (chi 2 = 7.49, p less than 0.01); the frequencies of the phenotype TF C2C2 are: 0.015 and 0.076 (chi 2 = 5.48, p less than 0.05). In Irkutskaya Province population differences between "healthy" and "sick" subgroups were discovered for blood group AB: "healthy" 0.046 and "sick"--0.175 (chi 2 = 11.28, p less than 0.010); for GC (1F-2)--0.214 and 0.116 (chi 2 = 4.45, p less than 0.05). Some other differences between "healthy" and "sick" in both populations are not significant. Some trends concerning heterozygosity in loci--GC, PGM, TF were discovered. The results are considered from the viewpoint of higher fitness of some genetic traits in the populations studied.     

A squash technique for studying the cytology of maize endosperm and other tissues.

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. The procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromosomes. With a slight modification the technique has been applied successfully to root tips and other tissues.