Metabolism and physical exercise

The important benefits of physical exercise are broadly known, but there was a gap between empyrical experience and biochemical metabolism. A recent study published on Science Translational Medicine journal helps to fill this gap. In particular, scientists from the Massachusetts General Hospital in Boston USA demonstrated by mass spectrometry the biochemical changes in runners metabolism. They analyzed the profile of 302 people and marathon runners before and after ten minutes run in terms of 210 metabolic compounds that may change their levels during physical activity. So, they gave greater importance to metabolic intermediates than to changes in protein expression levels. They identified changes in 21 compounds, not all previously related to physical activity. Plasma indicators of glycogenolysis, modulators of insulin sensitivity as well as regulator of lipid oxidation were identified. Furthermore, an increased expression of Nurr77, a transcriptional regulator of glucose metabolism and lipid homeostasis genes was evaluated. Thus, this work demonstrated the benefits of physical exercise from a molecular point of view. It could be interesting in further studies to understand what is the regulation mechanisms, whether epigenetic modifications or positive selection do occur and whether these alterations are genetically ereditable.

Ten years after the Human Genome Project

The Human Genome Project started ten years ago, with the challenging promise to sequence the whole genome and definitively understand all genetic secrets.
Two astonishing –but also scientifically interesting- surprises were presented to scientists of all countries: firstly only few genes are present in the genome, and this number -20000- is not so different from those of other species; secondarily, the major portion of DNA has regulatory functions rather than encoding significance. Thus, human genome sequencing has generated a lot of further questions about the mechanism of expression tuning. In 1960s Jacob and Monod demonstrated the presence of gene regulator in prokaryotic organisms, such as E. Coli; only few years ago we obtained the confirmation of this presence also in the human genome and numerous gaps have to be filled to reach a comprehensive understanding of molecular mechanism in cells. Epigenetic studies, microRNAs identification and gene expression analysis will help to gain a complete overview of human genome regulation, in addition to gene sequencing. Moreover, the Proteome Project will continue to clarify how proteins are involved in cellular life. Fortunately, a lot of open questions still be unsolved, and a lot of work has to be done by scientists worldwide.

Direct to consumer genetic services

Large expansion of direct to consumer genetic services is observed world-wide. This service offer single gene tests as well as screening of customer’s DNA at various loci to identify some polymorphism associated with certain disease. For instance, in women with familiar past history of breast cancer can be monitored for BRCA1 and BRCA2 genes, that are recognized as elevated risk factors. Furthermore, some genes involved in Alzheimer’s disease can be tested. A study was performed to evaluate the consequences of knowing the genetic makeup and the possible disease encountered in the future: it seems that knowledge about genetics doesn’t influence life, for instance by generating depression or anxiety. More concerns are about the scientific value of these tests, the modality to perform them and the clinical utility. Different approaches have been done by government to regulate this modern phenomenon: for instance, in Germany these tests are forbidden and only physicians can suggest genetic test carried out in appropriate institutions. In United Kingdom it has been proposed a self- policing by industry. In the US regulation is up to Federal states and varies largely. First implication of the large diffusion of this kind of test is the increase of analysis and examination to confirm genetic information. Medical intervention can not be the direct consequence of the test, but more deepened talks with physicians and genetic counsellors should be preferred.

Animal model for Parkinson’s disease

At the moment no animal models are available for human Parkinson’s disease. Everyone knows the importance to have right model to study a disease or test drug to care it; this lack must be rapidly overcome, giving the high number of people that suffers for this disease.

Michael J Fox Foundation and Sigma Aldrich set up a rat model in which it was possible to measure neurological damages and show symptomatically what is seen in Parkinson’s disease. Scientists knocked down five genes involved in the disease: leucine-rich repeat kinase 2 (LRRK2), α-synuclein (SNCA), DJ-1 (PARK7), Parkin (PARK2) and PTEN induced putative kinase 1 (PINK1). In this way only genes ownregulated in the disease can be studied, but this is the first step to build more accurate and useful models. They applied the Zinc Finger Nuclease to insert mutations in DNA of one day old rat embryo, then transplanted the embryos into pseudo-pregnant rat and obtain KO offspring. With ZFN technique is possible to eliminate the necessity to bred heterozygous chimeric mice to obtain KO animals. Genetically manipulated rats are now commercially available also for Parkinson’s disease and this opens new challenging opportunities to care this disease.

Gene therapy to restore sight

Leber’s congenital amaurosis is a pathology that affects 3000 people in USA and causes loss of sight from birth until reaching complete blindness at about 40 years. This disease interests a gene, RPE65 that helps in rhodopsin synthesis. If this gene is mutated, as occurs in the disease, photoreceptor cells into the retina will die.

During an important study carried out at the University of Pennsylvania, gene therapy was applied to blind dog to restore the activity of RPE65 gene and restore sight, as a consequence. Then, a small- scale study in human started in 2007, to ascertain the safety and efficacy of using virus to carry the RPE65 gene. First results showed that four of six young adults with Leber’s congenital amaurosis, had an improvement in their sight. Nevertheless, researchers knew that children with an intact retina –not already compromised by the disease- could have major benefits from this therapy. Thus, they performed the same study including four children for 8 to 11 years old and obtained great results in terms of increased light sensitivity. This work has been published in Lancet Journal in October, 2009. As all therapies that can improve the life quality of children, this work offers an hope for families that have so serious problems.

New technologies to identify gene function

After advances in DNA sequencing technology, the major task is to determine the functional role of proteins coded by these sequenced genes. Given the broad range of different functions carried out by proteins, it’s obvious that a multiplicity of techniques will be necessary, while DNA sequencing is achieved by few, easy and simply technique. A series of strategies based on generalization and systemization of genetics are emerging now as important tool to fill the gap between sequence and activity. One of these approaches is the analysis of the effect of perturbations of gene expression, by deletion, mutation or over-expression: after one of these modifications, we expect to observe a phenotypic change.

The challenge is to quantitatively measure phenotypes with enough accuracy and depth to define gene function. Two complementary approaches for determining complex phenotypes are currently used: in the first one many different parameters are simultaneously analysed, this is an high content screen; otherwise a single or limited number of aspects are observed, but the effect of perturbing each gene is followed in combination with a second perturbation, either another mutation or a chemical treatment. This genetic interaction profiling offers a high-resolution view of the function of each gene. Saccaromyces Cerevisiae is a model really useful for this kind of studies: a complete series of deletion strains of nonessential genes has been produced and has allowed to better understand the role of proteins important for yeast biochemistry and biology. Important results have been achieved also by using conditioning mutants that selectively grow in rich media: also in this case precious information has been retrieved. Rather than loss of function studies, methods for systematic gene over-expression have been optimized. Novel approaches in this field are interested in construction of untagged proteins in order to exclude that the presence of tag could interfere with the normal function of protein self.

The main goal of this systematic studies is to maximize the information flow, while minimally compromising the accuracy of phenotype detection. The introduction of large biomolecules into cells, such as DNA, RNA allows to directly analyse the role of one gene in the cellular life, and different kind of cells (mammals, primary cells, stem cells) can be used in this approach. Biomolecule is printed in an array onto glass slides, as done in conventional microarray. A monolayer of cells is deposited on top of the arrayed molecules and cells are transfected by taking up the material from glass. By using 96-well format plate, it’s possible to analyse the effects of a large number of biomolecules in a quantitative way. A plausible example of this method application is the effect of iRNAs on cellular proliferation: iRNA can be printed on bottom plate, cells are transfected (please note that is important to define the efficiency of transfection) and proliferation rate can be measured with normal treatment with MTT. In this way, genes important in proliferation could be identified.
Future efforts will be done to exploit a vast array of data that will emerge from large-scale genomic and proteomic projects to gain a deeper knowledge of the function of biological system.

Identification of miRNA target genes

microRNA are short RNAs that are important in gene regulation in all organism, form Drosophila to human. Indeed, in mammals they induce the RNA-induced silencing complex to target sites usually located at 3’untranslated regions (UTR) of mRNAs, determining translation repression or RNA degradation. To identify putative genes targets, computational approach is generally used, while experimental validation is more difficult.

Four algorithms, miRanda, TargetScan, RNA22 and PITA, have been developed to identify targets; comparison between these algorithms allows to reduce the number of predicted genes, usually thousand of genes. So, scientists run their search with at least two algorithms and consider only the overlapping targets. Another important factor in mRNA target search is to consider the 3’ UTR: different databases of miRNA target genes propose different 3’UTR, thus to compare the result from two algorithms is crucial to start with the same database of 3’UTR. Furthermore, one miRNA could target many genes, the identification of group of genes targeted by one miRNA could give some information about the localization and the biological function of proteins, codified by these genes. Functional profiling of miRNA target can be performed through the Gene Ontology website. miRNA is a complex but really interesting tool that cells use to tune the expression of certain proteins, specially for instance during embryonic development.

Challenges in pluripotency

Pluripotency is a special feature of stem cells to renew and generate part of the body. Totipotency is characteristic of embryonic stem cells that can generate each organ or tissue, while pluripotency is more limited and only few tissue can be product by stem cells. Four factors seem to control pluripotency: Oct4, Klf4, Myc, Sox2.

All these genes are crucial during embryonal development and in some cases mutations could lead to serious diseases, such as cancer for Myc gene, or impaired development for Oct4 and Sox2. A recent work published on Nature journal demonstrated that, by using these four factors (previously used to reprogram fibroblast), it could be possible to generate human pluripotent stem cells from fetal, neonatal and adult primary cells. Human induced pluripotent cells showed similar features of embryonic stem cells in terms of morphology and gene expression and were able to form teratomas when injected in immuno-deficient mice. This work opens new perspectives in stem cells research: for instance in next future, it could be possible to culture primary cells directly form patients and induce pluripotent cells in order to repair tissue, make transplantation or care degenerative diseases without troubles of rejection.

Genome-wide study on cardiovascular diseases

We have already talked about the importance of genome-wide analysis to study diseases and we have described some method for SNPs identification. In the current number of Nature Genetics an interesting study on cardiovascular diseases has been published. Co-authors belong to 95 institutions (hospitals, universities and research centers) in Europe and USA.

They analyzed the genotype of thousands patients and identified a correlation between high systolic pressure and mutations in limited region of the genome. In particular, they observed an association with the following eight genes: CYP17A1, CYP1A2, FGF5, SH2B3, MTHFR, c10orf107, ZNF652 and PLCD3.. This study has been confirmed by another work, always available in the same volume of Nature Genetics in which scientists identified four loci for systolic blood pressure (ATP2B1, CYP17A1, PLEKHA7, SH2B3), six for diastolic blood pressure (ATP2B1, CACNB2, CSK-ULK3, SH2B3, TBX3-TBX5, ULK4) and one for hypertension (ATP2B1). Why are so important these works? Firstly, these genes could be validated in further studies in order to understand if they could be considered as a target to prevent hypertension and develop new therapies for. Secondarily, this collaboration between so many institutions has to be a model for other studies about world-wide diseases.

References:
Nature Genetics 41, 667 – 676 (2009)
Nature Genetics 41, 677 – 687 (2009)

Signalling pathways as network of functional modules

Signalling pathways are usually described as a chain of consequential events, where proteins interact and transfer signal from external environment to nucleus. If some mutation occurs, all pathway is deregulated and it’s important to know where the mutation is in order to choose the correct approach to normalize it.

Is it possible to understand where is the mutation from genomic signature? It is! In Nature Reviews Genetics of June 2009 a new vision of signalling pathways is presented. Instead of a chain of reactions, functional modules are considered on basis of gene expression signature. From NCI-60 database authors identified genes related to the core protein of the pathway that showed the same variation of expression as this core gene.

For each pathway/ core protein several signatures have been identified and by relating signatures to mutants that selectively activate a specific downstream effector, it has been possible to assign each functional module (core protein and downstream effector) to genomic signature.

Which are the therapeutic implications of this new approach? For instance, it’s possible to define where is the mutation directly from genomic analysis, in this way a correct therapeutic approach can be chosen. Now, we have to increase our knowledge about the relationship between genomic signature and signalling pathway.