Overcoming technical variation and biological variation in quantitative proteomics

Quantitative proteomics investigates physiology at the molecular level by measuring relative differences in protein expression between samples under different experimental conditions. A major obstacle to reliably determining quantitative changes in protein expression is to overcome error imposed by technical variation and biological variation. In drug discovery and development the issue of biological variation often rises in concordance with the developmental stage of research, spanning from in vitro assays to clinical trials. In this paper we present case studies to raise awareness to the issues of technical variation and biological variation and the impact this places on applying quantitative proteomics. We defined the degree of technical variation from the process of two-dimensional electrophoresis as 20-30% coefficient of variation. On the other hand, biological variation observed experiment-to-experiment showed a broader degree of variation depending upon the sample type. This was demonstrated with case studies where variation was monitored across experiments with bacteria, established cell lines, primary cultures, and with drug treated human subjects. We discuss technical variation and biological variation as key factors to consider during experimental design, and offer insight into preparing experiments that overcome this challenge to provide statistically significant outcomes for conducting quantitative proteomic research.     

Flavonoid variation in Xanthisma texanum: Infrapopulation and interpopulation variation

Fourteen flavonoids were isolated from Xanthisma texanum. Chemosystematic interpretations are made concerning the origin and distribution of the infraspecific taxa of the species.     

Niche variation and the maintenance of variation in body size in a burying beetle

1. In burying beetles (Nicrophorinae), body size is known to provide both a fecundity advantage (in females) and successful resource defence (in males and females). Despite this, considerable variation in body sizes is observed in natural populations. 2. A possible explanation for the maintenance of this variation, even with intra‐ and inter‐specific resource competition, is that individuals might assort according to body size on different‐sized breeding resources. 3. We tested prediction that ‘bigger is always better’, in the wild and in the laboratory, by experimentally manipulating combinations of available breeding‐resource size (mouse carcasses) and competitor's body size in Nicrophorus vespilloides (Herbst 1783). 4. In the field, large female beetles deserted small carcasses, without breeding, more often than they did larger carcasses, but small females used carcasses indiscriminately with respect to size. In the laboratory, large beetles reared larger broods (with more offspring) on larger carcasses than small beetles, but on small carcasses small beetles had a reproductive advantage over large ones. Offspring size covaried with carcass size independently of parental body size. 5. The present combined results suggest breeding resource value depends on an individual's body size, and variation in body size is environmentally induced: maintained by differences in available carcass sizes. This produces a mechanism by which individual specialisation leads to an increase in niche variation via body size in these beetles.     

Spatial variation of biochemical and ecological phenotypes in Drosophila: Electrophoretic and quantitative variation

Electrophoretic variation at three enzyme loci-alcohol dehydrogenase (Adh), glycerophosphate dehydrogenase (Gpdh), triosephosphate isomerase (Tpi)- is compared in Australian Drosophila melanogaster populations at three levels of spatial heterogeneity; among breeding sites, within populations, and between populations at the geographic level. Heterogeneity at the breeding site level greatly exceeds that among adults within populations, indicating greater intermixing at the mobile adult stage than at the developmentally immature and less migratory larval stage. Heterogeneity at the microspatial level is large relative to the geographic level at two of these loci. Spatial patterns of variation in ecological phenotypes are also considered. It is argued that electrophoretic variants may contribute little to an understanding of this quantitative variation, and that a more useful approach in ecological genetics is to consider ecological phenotypes as primary data.     

Relating human genetic variation to variation in drug responses

Although sequencing a single human genome was a monumental effort a decade ago, more than 1000 genomes have now been sequenced. The task ahead lies in transforming this information into personalized treatment strategies that are tailored to the unique genetics of each individual. One important aspect of personalized medicine is patient-to-patient variation in drug response. Pharmacogenomics addresses this issue by seeking to identify genetic contributors to human variation in drug efficacy and toxicity. Here, we present a summary of the current status of this field, which has evolved from studies of single candidate genes to comprehensive genome-wide analyses. Additionally, we discuss the major challenges in translating this knowledge into a systems-level understanding of drug physiology, with the ultimate goal of developing more effective personalized clinical treatment strategies.     

异质景观中水土流失的空间变异与尺度变异

综述了景观格局与水土流失过程的空间变异与尺度变异的理论和方法研究进展,提出了水土流失空间变异与尺度变异的研究方向。景观格局与生态过程的尺度变异一般处于单一尺度变异和多重尺度变异的连续体之中。尺度转换即尺度外推包括尺度上推和尺度下推,其可行性决定于尺度变异特征。水土流失不仅是多因子综合影响的一个复杂的时空变异过程,而且也是一个典型的多重尺度变异过程。传统的水土流失研究一般集中在坡面径流小区和小流域两个单一尺度上,这在很大程度上限制了水土流失的空间尺度外推和过程分析。近年来,尽管国内外很多学者开始关注水土流失的尺度变异及其影响因子．但只是对水土流失在不同大小的样地尺度以及小集水区尺度上的差异及其影响因子进行了初步的比较研究,尤其缺乏水土流失及其相关环境因子的连续尺度变异特征的机制分析。空间变异和尺度变异研究方法包括统计模型模拟法、物理模型与物理过程模拟法以及综合分析与综合预报法三大类。每种方法都有其优缺点和其特定的适宜性,最佳方法组合的选取因研究对象、研究地区和研究时间的不同而异。土壤侵蚀预报模型包括经验统计模型和物理过程模型,就解决水土流失的跨尺度关系而言,基于物理过程的空间分布式的土壤侵蚀预报模型显著优于经验模型。这些模型在关键参数的空间变异性描述和水土流失的尺度变异性分析方面非常薄弱,尤其缺乏模型分辨率和研究范围对输出结果的影响研究。完善水土流失的“尺度一格局一过程”理论,加强多重尺度上水土流失及其相关环境因子的空间变异格局和尺度变异性的实地观测与数学分析,改进土壤侵蚀预报模型这3个方面是将来的研究重点。     

Genetic variation and fitness in salmonids

Over the last quarter century, many studieshave attempted to clarify the relationshipbetween genetic variability and fitness, butfew of these have involved salmonid fishes. Examination of studies of salmonids revealsthat such a relationship varies both among andwithin species. A correlation between geneticvariability and fitness can be affected bygenetic background, environment, and age, andit also depends upon the genetic markers andphenotypes evaluated. The relationshipsbetween molecular genetic variation,quantitative genetic variation, and phenotypicvariation may be more relevant to conservationissues than those between genetic variation andaverage fitness or performance. Consequently,future work in salmonids should include moreintensive investigation of the correspondenceof molecular genetic variation within and amongpopulations to quantitative genetic andphenotypic variation for traits affectingfitness. In the absence of a more completeunderstanding of the relationship betweengenetic variation and fitness, maintenance ofgenetic and phenotypic variation within andamong conspecific populations should beconsidered a primary goal of conservingsalmonid fishes.     

Epigenetic variation and inheritance in mammals

What determines phenotype is one of the most fundamental questions in biology. Historically, the search for answers had focused on genetic or environmental variants, but recent studies in epigenetics have revealed a third mechanism that can influence phenotypic outcomes, even in the absence of genetic or environmental heterogeneity. Even more surprisingly, some epigenetic variants, or epialleles, can be inherited by the offspring, indicating the existence of a mechanism for biological heredity that is not based on DNA sequence. Recent work from mouse models, human monozygotic twin studies, and large-scale epigenetic profiling suggests that epigenetically determined phenotypes and epigenetic inheritance are more common than previously appreciated.     

Epigenetics and phenotypic variation in mammals

What causes phenotypic variation? By now it is clear that phenotype is a result of the interaction between genotype and environment, in addition to variation not readily attributable to either. Epigenetic phenomena associated with phenotypic variation at the biochemical, cellular, tissue, and organism level are now well recognized and are likely to contribute to the “intangible variation” alluded to. While it is clear that epigenetic modifications are mitotically heritable, the fidelity of this process is not well understood. Inheritance through more than one generation of meioses is even less well studied. So it remains unclear to what extent epigenetic changes contribute to phenotypic variation in natural populations. How might such evidence be obtained? What are the features of phenotypes that might suggest an epigenetic component? How much of the epigenetic component is truly independent of genetic changes? The answers to such questions must come from studies designed specifically to detect subtle, stochastically determined phenotypic variation in suitable animal models.     

Heterochromatin variation and spermatogenesis in Nesokia

A probable role of heterochromatin variation in male meiosis has been evaluated using fertile and infertile Indian mole rat males (Nesokia) with polymorphic X and/or Y chromosomes. A comprehensive study of tubular histology, meiotic progression, and X-Y chromosome pairing was undertaken. Despite heterochromatin variation, spermatogenesis was found to be complete in all individuals. Patterns of X-Y synaptonemal complex pairing varied considerably from extensive synapsis in individuals with a normal heterochromatin complement, through end-to-end synapsis, to X and Y univalents in those with different degrees of loss of heterochromatin. Changes in the gonadal histology corresponding to heterochromatin variation were also observed. Loss of some coding DNA sequences in polymorphic X-chromosomes otherwise located at specific sites in the X-chromosome heterochromatin have been linked directly to modifications of the reproductive process. This is thought to be mediated by an altered X-chromosome activity during spermatogenesis or regulation of other locus/loci involved in fertility or reproduction.     

MHC variation in birds and reptiles

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring‐necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Asymmetry and variation in keratella tropica

Keratella tropica usually has two posterior spines. Both may vary in length, but the left is always shorter than the right. Sometimes the left spine is absent. Spine length varies with the length of the lorica, but there are other, external, influences. Three separate lines of evidence indicate that factors which promote spine length are related in some way to the presence of calanoid copepods.     

Cytological and morphological variation in Bellevalia

Material from 45 populations representing 15 species of Bellevalia were analyzed as to chromosome numbers and morphology. For 3 species reports are new, viz. for B. clusiana (2x), B, tauri (2x, 4x), and B. nivalis (4x). In B. saviczii diploids, tetraploids and hexaploids were found. The basic Bellevalia karyotype prevails, but chromosome heteromorphism is rather common at the populational level. B–chromosomes were found in some populations of B. glauca s.l. and B. saviczii. Discussions on karyotype variation, polyploidy, morphologic variation and generic delimitation are presented.     

Biological species and variation in arthroderma

Summary The distinctive peridial appendages ofArthroderma were of little value for the delimitation of species. The peridial appendages as well as the conidia of certain species were so similar as to be value-less for the delimitation of species. Further, morphological and physiological variation was found to be as great within, as between, species. The usual morphological methods for classifying species were, therefore, inadequate and the concept of biological species was considered.The following points were found to favor the applicability of biological species concepts. Nine of ten species were heterothallic which assured crossing. A gene pool, the result of sexual reproduction and an essential requirement of a biological species, was maintained. This was evidenced by complete fertility between isolates of the same species from diverse geographic areas. Finally, no hybridization between species was evident in over one hundred attempted crosses.Strains which were readily compatible and produced viable ascospores were considered to be members of the same species. Conversely, incompatible strains were differentiated into separate species. In this way over fifty strains from more than one hundred isolations were found to be members of ten species. Six of the species correlated with species previously described on morphological bases, and confirmed their validity. Four species were considered to be new. All four were previously confused withA. quadrifidum orA. cuniculi because they possessed similar types of conidia. Once separated on an incompatibility basis, they were found to possess characteristics of distinct species. The only reliable method for their differentiation, however, was found to be interspecific incompatibility.Several strains, some pathogens, were crossed with soil borne species ofArthroderma. Some mated but others did not. Several incompatible strains possessedArthroderma-like appendages and partial compatibility was indicated. Partial compatibility also occurred in certain interspecific crosses.