A need for a 'whole-istic functional genomics' approach in complex human diseases: arthritis

'Genomic tools', such as gene/protein chips, single nucleotide polymorphism and haplotype analyses, are empowering us to generate staggering amounts of correlative data, from human/animal genetics and from normal and disease-affected tissues obtained from complex diseases such as arthritis. These tools are transforming molecular biology into a 'data rich' science, with subjects with an '-omic' suffix. These disciplines have to converge and integrate at a systemic level to examine the structure and dynamics of cellular and organismal function ('functionomics') simultaneously, using a multidimensional approach for cells, tissues, organs, rodents and Zebra fish models, which intertwines various approaches and readouts to study the development and homeostasis of a system. In summary, the postgenomic era of functionomics will facilitate narrowing the bridge between correlative data and causative data, thus integrating 'intercoms' of interacting and interdependent disciplines and forming a unified whole.     

Gene expression profiling of hereditary exencephaly in chickens

In this preliminary study, differentially expressed genes were investigated in cranial tissues from chickens with hereditary exencephaly using cDNA microarrays containing 1,152 genes and expressed sequence tags (ESTs). Genes showing twofold or greater differences at P < 0.05 between affected and normal cranial cells were considered to be candidates for hereditary exencephaly in chicken. Eighteen ESTs (11 known genes/homologues) were upregulated and 108 ESTs (51 known genes/homologues) were downregulated. The EST AL584231 (ROS006C9), orthologous to human MTHFD1, a known candidate gene for human neural tube defects (NTDs), was expressed at the same level both in normal and affected chicken cranial tissues. ESTs AL584253 (ROS006F7, thioredoxin reductase 1) and AL585511 (ROS024H9, thioredoxin), both involved in NTD pathogenic pathways in mice, were downregulated and had mean ratios of 0.41 and 0.04 for expression in affected vs. normal cells respectively. Expression differences of these two ESTs were confirmed by quantitative real-time polymerase chain reaction. These data indicate that ESTs AL584253 and AL585511 are candidates for hereditary exencephaly in chickens.     

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.KEY WORDS: Caenorhabditis elegans, Cell non-autonomous proteotoxicity, Prion-like spreading     

Overcoming barriers to understanding the cellular basis of aluminium resistance

There appears to be an emerging consensus that resistance to aluminium (Al) is mediated at the cellular level. Virtually all current hypotheses which seek to explain the basis of Al resistance have a cellular focus, including those which postulate that external mechanisms limit the rate of Al entry across the membrane and/or protect sensitive extracellular sites, as well as those which postulate that internal mechanisms detoxify Al in the cytoplasm. If Al resistance is a cellular phenomenon, it should be expressed in single cells. Attempts to demonstrate resistance in cell culture systems, however, have not been uniformly satisfying. Considerable uncertainty has arisen from use of experimental conditions which favour formation of insoluble or non-toxic Al species. This problem has plagued research which has attempted to select for Al resistance in cell culture systems, as well as research which has attempted to express existing patterns of differential resistance in cell culture systems. Despite technical problems such as this, work at the cellular level has provided some important contributions. Most importantly, we now know resistance to Al can be expressed at the cellular level. We have discovered also that plant cells accumulate Al much more rapidly in cell culture systems than in intact roots and that isolated cells are more sensitive to Al than complex tissues. While this type of research is still hampered by a number of technical barriers, it would appear that more rapid progress could be achieved if greater emphasis was placed on true experimental work. Furthermore, we need to begin evaluating experimental data in the context of an integrated Al stress response if we are to achieve a full understanding of the cellular basis of Al resistance.     

Variable expression of some "housekeeping" genes during human keratinocyte differentiation

We investigated the expression levels of four cellular "housekeeping" genes during epithelial differentiation. Differentiation is a dynamic process and various cellular RNAs have been targeted for use as internal controls during differentiation of human keratinocytes, but the consistent expression of such standards has not been previously validated. We used the organotypic (raft) culture system to grow stratified and differentiated epithelium in vitro. We compared cellular RNAs from epithelial tissues of both normal human keratinocytes and keratinocytes whose differentiation scheme is altered by the replication of human papillomavirus. Using ribonuclease protection assays to quantify RNA expression levels, we found that beta-actin and glyceraldehyde-3-phosphate dehydrogenase levels fluctuated during epithelial differentiation, whereas cyclophilin RNA and 28S-ribosomal RNA were the most consistently expressed during epithelial differentiation. These stably expressed cellular RNAs can be targeted as controls to permit quantitative expression analyses of cellular and pathogen RNAs during epithelial differentiation under various experimental conditions.     

Introducing "Frontiers in Zoology"

As a biological discipline, zoology has one of the longest histories. Today it occasionally appears as though, due to the rapid expansion of life sciences, zoology has been replaced by more or less independent sub-disciplines amongst which exchange is often sparse. However, the recent advance of molecular methodology into "classical" fields of biology, and the development of theories that can explain phenomena on different levels of organisation, has led to a re-integration of zoological disciplines promoting a broader than usual approach to zoological questions. Zoology has re-emerged as an integrative discipline encompassing the most diverse aspects of animal life, from the level of the gene to the level of the ecosystem.The new journal Frontiers in Zoology is the first Open Access journal focussing on zoology as a whole. It aims to represent and re-unite the various disciplines that look at animal life from different perspectives and at providing the basis for a comprehensive understanding of zoological phenomena on all levels of analysis. Frontiers in Zoology provides a unique opportunity to publish high quality research and reviews on zoological issues that will be internationally accessible to any reader at no cost.     

Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig

Domestic pig （Sus scrofa domestica） is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags （ESTs） have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different nonnormalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account.     

Aging mechanism as the "down side" of adaptation: a network approach

Many diverse hypotheses on aging are in play. All from "aging genes" over decreasing telomere length to increased level of gene mutations has been suggested to determine an organism's lifespan, but no unifying theory exists. As part of a growing interest toward more integrative approaches in the field we propose a simplistic model based on the "use-it-or-lose-it" concept: we hypothesize that biological aging is a systemic property and the down side of adaptation in complex biological networks at various levels of organization: from brain over the immune system to specialized tissues or organs. The simple dynamical model undergoes three phases during its lifetime: (1) general plasticity (childhood), (2) optimization/adaptation to given conditions (youth and adolescence) and (3) steady state associated with high rigidity (aging). Furthermore, our model mimics recent data on the dynamics of the immune system during aging and, although simplistic, thus captures essential characteristics of the aging process. Finally, we discuss the abstract model in relation to current knowledge on aging and propose experimental setups for testing some of the theoretical predictions.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Cytogerontology at the beginning of the third millenium: from "correlative" to "gist" models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the "Hayflick's model". More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some "gerontological" parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the "Hayflick's limit", why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cells in vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop "gist" models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our "stationary phase aging" model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of "senile" damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the "genuine" aging of cells (i.e., increasing probability of their death with "age") and the aging of multicellular organisms.     

"4D Biology for health and disease" workshop report

The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine.     

Characterization of FSH-regulated genes isolated by mRNA differential display from pig ovarian granulosa cells

The present authors have isolated FSH-regulated genes from primary granulosa cell cultures with or without Follicle Stimulating Hormone (FSH) treatment using mRNA differential display. mRNA differential display consists of amplification of partial sequences of cDNAs (150–400 bp) corresponding to 3' ends of cellular messenger RNAs, and thus, generates 3' expressed sequence tags (3' ESTs). Five thousand cDNA bands were examined, among which the present authors have isolated and sequenced 16 different FSH-regulated products. These sequences were compared with those available in databases. Three of the sequences showed similarity to identified genes from other species (bovine NADH dehydrogenase subunit 4, Xenopus chromosome sequence-associated polypeptide E and transformation-sensitive protein IEF SSP) and four others with human ESTs. Regulation of the corresponding genes has been checked by RT-PCR since most of these are expressed at a low level. FSH-regulation was confirmed for 12 mRNAs (four down- and eight up-regulated). The present authors have also mapped 12 of these ESTs on porcine chromosomes regions using a somatic cell hybrid panel.     

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

Research options for controlling zoonotic disease in India, 2010-2015

Background

Zoonotic infections pose a significant public health challenge for low- and middle-income countries and have traditionally been a neglected area of research. The Roadmap to Combat Zoonoses in India (RCZI) initiative conducted an exercise to systematically identify and prioritize research options needed to control zoonoses in India.

Methods and Findings

Priority setting methods developed by the Child Health and Nutrition Research Initiative were adapted for the diversity of sectors, disciplines, diseases and populations relevant for zoonoses in India. A multidisciplinary group of experts identified priority zoonotic diseases and knowledge gaps and proposed research options to address key knowledge gaps within the next five years. Each option was scored using predefined criteria by another group of experts. The scores were weighted using relative ranks among the criteria based upon the feedback of a larger reference group. We categorized each research option by type of research, disease targeted, factorials, and level of collaboration required. We analysed the research options by tabulating them along these categories. Seventeen experts generated four universal research themes and 103 specific research options, the majority of which required a high to medium level of collaboration across sectors. Research options designated as pertaining to ‘social, political and economic’ factorials predominated and scored higher than options focussing on ecological, genetic and biological, or environmental factors. Research options related to ‘health policy and systems’ scored highest while those related to ‘research for development of new interventions’ scored the lowest.

Conclusions

We methodically identified research themes and specific research options incorporating perspectives of a diverse group of stakeholders. These outputs reflect the diverse nature of challenges posed by zoonoses and should be acceptable across diseases, disciplines, and sectors. The identified research options capture the need for ‘actionable research’ for advancing the prevention and control of zoonoses in India.