The path to personalized medicine

Advances in personalized medicine, or the use of an individual's molecular profile to direct the practice of medicine, have been greatly enabled through human genome research. This research is leading to the identification of a range of molecular markers for predisposition testing, disease screening and prognostic assessment, as well as markers used to predict and monitor drug response. Successful personalized medicine research programs will not only require strategies for developing and validating biomarkers, but also coordinating these efforts with drug discovery and clinical development.     

What Can Medicine Learn from the Human DNA Sequence?

The cooperation of biochemistry with clinical medicine consists of two overlapping temporal phases. Phase 1 of the cooperation, which still is not finished, is characterized by joint work on the pathogenesis and diagnostics of systemic metabolic diseases, whereas in phase 2 the cooperation on tissue and cell specific as well as on molecular diseases is prevailing. In view of the conceptual revolution and shift in paradigm, which biochemistry and medicine are presently experiencing, the content of cooperation between the two disciplines will profoundly change. It will become deeply influenced by the results of the research into the human genome and human proteome. Biochemistry will strongly be occupied to relate the thousands of protein coding genes to the structure and function of the encoded proteins, and medicine will be concerned in finding new protein markers for diagnostics, to identify novel drug targets, and to investigate, for example, the proteomes of the variety of tumors to aid tumor classification, to mention only a few areas of interest which medicine will have in the progress of human genome research. The review summarizes the recent achievements in sequencing the human DNA as published in February 2001 by the International Human Genome Sequencing Consortium and Celera Genomics and discusses their significance in respect to the further development of molecular, in particular genetic, medicine as an interdisciplinary field of the modern clinical sciences. Only biochemistry can provide the conceptual and experimental basis for the causal understanding of biological mechanisms as encoded in the genome of an organism.     

Proteomics meets blood banking: Identification of protein targets for the improvement of platelet quality

Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage — a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients.     

分子标记方法在生态学中的应用潜力和局限

近几十年来,分子标记方法在生态学中得到了广泛的应用,并显示出重要的应用潜力,着重表现在:①目前分子标记方法主要应用在种群生态学中,在今后的研究中,它将在进化生态学,保护生态学,恢复生态学及生态系统生态学中显示出巨大的应用空间;②现有的一些分子标记方法还有更大的应用空间;③对同一研究问题,结合不同的分子标记方法可以取得更好的研究成果;④新的分子标记方法的产生,将有利于生态学的研究工作的深化和获得新的发展。同时,由于一些分子标记研究费用较高,获得的基础数据重复性较低,研究过程中涉及到一些放射性的安全性问题等都可能影响它在生态学领域的应用。因此,针对生态学不同的要求选择和优化分子标记手段是十分重要的。     

抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。     

Sequence-based linkage analysis

The rapid decrease in the cost of DNA sequencing will enable its use for novel applications. Here, we investigate the use of DNA sequencing for simultaneous discovery and genotyping of polymorphisms in family linkage studies. In the proposed approach, short contiguous segments of genomic DNA, regularly spaced across the genome, are resequenced in each pedigree member, and all sequence polymorphisms discovered within a pedigree are used as genetic markers. We use computer simulations consistent with observed human sequence diversity to show that segments of 500-1,000 base pairs, spaced at intervals of 1-2 Mb across the genome, provide linkage information that equals or exceeds that of traditional marker-based approaches. We validate these results experimentally by implementing the sequence-based linkage approach for chromosome 19 in CEPH pedigrees.     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.     

Personalised Medicine in 2012: Editorial to the Special Issue of New Biotechnology on "MOLECULAR DIAGNOSTICS & PERSONALISED MEDICINE"

This special issue of New Biotechnology is focused on molecular diagnostics and personalised medicine and appears at an epochal moment in the development of the field. The practice of medicine is taking a significant and irrevocable turn towards personalisation, due to the great progress in areas such as genomics, pharmacogenomics and molecular diagnosis. It becomes increasingly apparent that to deliver the promise of personalised treatments, more and more novel medicines discovered today will be presented together with innovative companion diagnostics. The contributions to this volume touch on many disciplines, ranging from cell biology to genetics, immunology, molecular diagnostics, pharmaceutics and economic issues. The contributions of clinicians and basic scientists are synergistically presented to underline better the wide spectrum of studies that can contribute to the new field of personalised medicine. The promising perspectives of individualised treatments are related not only to higher effectiveness, but also to increased efficiency. This is relevant not only for the individual patient, but even more so for the general public, within a wider economical perspective where resources are limited and it becomes more and more mandatory to close the gap between social costs and benefits. This approach follows the steps of a stratified and individualised medicine and finds its final goal in an individualised healthcare.     

Medicinal mushrooms and cancer therapy: translating a traditional practice into Western medicine

Modern medical practice relies heavily on the use of highly purified pharmaceutical compounds whose purity can be easily assessed and whose pharmaceutical activity and toxicity show clear structure-function relationships. In contrast, many herbal medicines contain mixtures of natural compounds that have not undergone detailed chemical analyses and whose mechanism of action is not known. Traditional folk medicine and ethno-pharmacology coupled to bioprospecting have been an important source of many anticancer agents as well as other medicines. With the current decline in the number of new molecular entities from the pharmaceutical industry, novel anticancer agents are being sought from traditional medicine. As the example of medicinal mushrooms demonstrates, however, translating traditional Eastern practices into acceptable evidence-based Western therapies is difficult. Different manufacturing standards, criteria of purity, and under-powered clinical trials make assessment of efficacy and toxicity by Western standards of clinical evidence difficult. Purified bioactive compounds derived from medicinal mushrooms are a potentially important new source of anticancer agents; their assimilation into Western drug discovery programs and clinical trials also provides a framework for the study and use of other traditional medicines.     

Proteome signatures—how are they obtained and what do they teach us?

The dawn of a new Proteomics era, just over a decade ago, allowed for large-scale protein profiling studies that have been applied in the identification of distinctive molecular cell signatures. Proteomics provides a powerful approach for identifying and studying these multiple molecular markers in a vast array of biological systems, whether focusing on basic biological research, diagnosis, therapeutics, or systems biology. This is a continuously expanding field that relies on the combination of different methodologies and current advances, both technological and analytical, which have led to an explosion of protein signatures and biomarker candidates. But how are these biological markers obtained? And, most importantly, what can we learn from them? Herein, we briefly overview the currently available approaches for obtaining relevant information at the proteome level, while noting the current and future roles of both traditional and modern proteomics. Moreover, we provide some considerations on how the development of powerful and robust bioinformatics tools will greatly benefit high-throughput proteomics. Such strategies are of the utmost importance in the rapidly emerging field of immunoproteomics, which may play a key role in the identification of antigens with diagnostic and/or therapeutic potential and in the development of new vaccines. Finally, we consider the present limitations in the discovery of new signatures and biomarkers and speculate on how such hurdles may be overcome, while also offering a prospect for the next few years in what could be one of the most significant strategies in translational medicine research.     

Global genetic analysis

The introduction of molecular markers in genetic analysis has revolutionized medicine. These molecular markers are genetic variations associated with a predisposition to common diseases and individual variations in drug responses. Identification and genotyping a vast number of genetic polymorphisms in large populations are increasingly important for disease gene identification, pharmacogenetics and population-based studies. Among variations being analyzed, single nucleotide polymorphisms seem to be most useful in large-scale genetic analysis. This review discusses approaches for genetic analysis, use of different markers, and emerging technologies for large-scale genetic analysis where millions of genotyping need to be performed.     

Metabolomics through the lens of precision cardiovascular medicine

Metabolomics, which targets at the extensive characterization and quantitation of global metabolites from both endogenous and exogenous sources, has emerged as a novel technological avenue to advance the field of precision medicine principally driven by genomics-oriented approaches. In particular,metabolomics has revealed the cardinal roles that the environment exerts in driving the progression of major diseases threatening public health. Herein, the existent and potential applications of metabolomics in two key areas of precision cardiovascular medicine will be critically discussed: 1) the use of metabolomics in unveiling novel disease biomarkers and pathological pathways; 2) the contribution of metabolomics in cardiovascular drug development. Major issues concerning the statistical handling of big data generated by metabolomics, as well as its interpretation, will be briefly addressed. Finally, the need for integration of various omics branches and adopting a multi-omics approach to precision medicine will be discussed.     

Vaccinomics and a new paradigm for the development of preventive vaccines against viral infections

In this article we define vaccinomics as the integration of immunogenetics and immunogenomics with systems biology and immune profiling. Vaccinomics is based on the use of cutting edge, high-dimensional (so called "omics") assays and novel bioinformatics approaches to the development of next-generation vaccines and the expansion of our capabilities in individualized medicine. Vaccinomics will allow us to move beyond the empiric "isolate, inactivate, and inject" approach characterizing past vaccine development efforts, and toward a more detailed molecular and systemic understanding of the carefully choreographed series of biological processes involved in developing viral vaccine-induced "immunity." This enhanced understanding will then be applied to overcome the obstacles to the creation of effective vaccines to protect against pathogens, particularly hypervariable viruses, with the greatest current impact on public health. Here we provide an overview of how vaccinomics will inform vaccine science, the development of new vaccines and/or clinically relevant biomarkers or surrogates of protection, vaccine response heterogeneity, and our understanding of immunosenescence.     

Regression-based quantitative trait loci mapping: robust, efficient and effective

Regression has always been an important tool for quantitative geneticists. The use of maximum likelihood (ML) has been advocated for the detection of quantitative trait loci (QTL) through linkage with molecular markers, and this approach can be very effective. However, linear regression models have also been proposed which perform similarly to ML, while retaining the many beneficial features of regression and, hence, can be more tractable and versatile than ML in some circumstances. Here, the use of linear regression to detect QTL in structured outbred populations is reviewed and its perceived shortfalls are revisited. It is argued that the approach is valuable now and will remain so in the future.     

Molecular Genetics and Developmental Physiology: Implications for Designing Better Forest Crops

Current tree biology related to tree genetics and breeding has two important developments that have not well been integrated in the literature. The first is the physiological and biochemical dissection of plant yield, whereas the second is the genetic mapping based on molecular markers, such as RFLPs, RAPDs, AFLPs, and microsatellites. Genetic mapping has revolutionized traditional quantitative genetic analysis by which the genetic variation of a character is described in terms of its mean and (co)variance without the knowledge of the underlying genes. By integrating physiological and developmental studies of yield traits, genetic mapping can provide a unique means for detecting key QTL that play important roles in affecting tree growth and metabolism. The incorporation of these QTL into commercial populations through gene transformation or marker-assisted selection will move current breeding programs strictly based on an empirism to an approach that is mechanistically oriented. In this review, we discuss how plant physiology and development are merged with genetic mapping to formulate the strategy of molecular breeding in which superior forest crops are selected at the gene level. It is anticipated that this novel breeding strategy can potentially provide major breakthroughs for tree breeding.     

Isolation and PCR amplification of genomic DNA from dried capsules of cardamon (Elettaria cardamomum M.)

Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage tissue such as cardamom capsules. The yield of DNA was 6–7 μg g⁻¹ tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence Characterised Amplified Regions (SCAR) markers for profiling cardamoms.     

Immunomodulation confers herbicide resistance in plants

In order to create a novel mechanism for herbicide resistance in plants, we expressed a single-chain antibody fragment (scFv) in tobacco with specific affinity to the auxinic herbicide picloram. Transgenic tobacco plants and seedlings expressing this scFv against picloram were protected from its effect in a dose-dependent manner. This is the first successful use of an antibody to confer in vivo resistance to a low molecular weight xenobiotic (i.e. < 1000 Da). Our results suggest the possibility for a generic antibody-based approach to create crops resistant to low molecular weight xenobiotics for subsequent use in the bioremediation of contaminated soils, crop protection and as novel selectable markers.     

A rapid method to map mutations in Drosophila

Background

Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map.

Results

Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb.

Conclusion

The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens.     

Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature,lymphatics, and leukocyte-endothelial interactions

The ability to determine various functions of genes in an intact host will be an important advance in the postgenomic era. Intravital imaging of gene regulation and the physiological effect of the gene products can play a powerful role in this pursuit. Intravital epifluorescence microscopy has provided powerful insight into gene expression, tissue pH, tissue pO2, angiogenesis, blood vessel permeability, leukocyte-endothelial (L-E) interaction, molecular diffusion, convection and binding, and barriers to the delivery of molecular and cellular medicine. Multiphoton laser scanning microscopy (MPLSM) has recently been applied in vivo to overcome three drawbacks associated with traditional epifluorescence microscopy: (i) limited depth of imaging due to scattering of excitation and emission light; (ii) projection of three-dimensional structures onto a two-dimensional plane; and (iii) phototoxicity. Here, we use MPLSM for the first time to obtain high-resolution images of deep tissue lymphatic vessels and show an increased accuracy in quantifying lymphatic size. We also demonstrate the use of MPLSM to perform accurate calculations of the volume density of angiogenic vessels and discuss how this technique may be used to assess the potential of antiangiogenic treatments. Finally, high-speed MPLSM, applied for the first time in vivo, is used to compare L-E interactions in normal tissue and a rhabdomyosarcoma tumor. Our work demonstrates the potential of MPLSM to noninvasively monitor physiology and pathophysiology both at the tissue and cellular level. Future applications will include the use of MPLSM in combination with fluorescent reporters to give novel insight into the regulation and function of genes.