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.

Manipulation of bacterial genome in yeast

Even if manipulation of bacterial genome is often difficult and challenging, engineering allows to better understand bacterial biology and genetics. Researchers from C. Venter Institute improve a protocol to clone bacterial genome in yeast, manipulate it and boot it up in bacteria self. To do this they chose an “easy” model, Mycoplasma, because this organism doesn’t have bacterial wall, its genome is small and A-T rich, so is more properly replicated in yeast than ones rich in G-C. Furthermore Mycoplasma has non-standard genetic code that can not be translated in yeast, preventing the synthesis of bacterial proteins toxic for yeast.

What did scientists perform to achieve this important result? They cloned Mycoplasma genome into yeast artificial chromosomes (YACs), genetically manipulated it and then transplanted it into the final organism receiver. Two concerns could prevent this goal: one was the possibility that restriction endonucleases recognised foreign sequences and degraded them and the second one was that yeast modified bacterial genome. Fortunately this last event didn’t occur, while to limit endonucleasic activity, scientists hypermethylated donor genome and eliminated endonucleases from receiver organism. This protocol could be improved in order to become a conventional technique for bacterial manipulation in order to have another tool to solve human needs in medicine and environmental preservation.

Challenging in food safety

Food safety is one of most important challenge in our countries. Indeed, adulteration and poising food are often described in newspapers. FDA and ESFA are the organisations issued to guarantee food safety in U.S and Europe, respectively. Which are the most common problems that determine food adulteration. The first cause of this kind of problem is the bacterial contamination: indeed bacteria can provoke food spoilage as well as they are pathogen in some cases.

To decrease the possibility of food spoilage, numerous testing must be performed in food processing company. The safety ideal “from farm to fork” created by FDA can be reached only through an attentive analysis of raw materials, processing and packing procedures in order to assure maximal sterility in each step. Furthermore, some laboratories are specialised in bacterial, toxin and sterility testing: the common goal is to provide consumers with safe and wholesome food at least for all shelf-life of food self. These laboratories usually offer a broad range of services and usually communicate with their clients through LIMS system. LIMS systems, indeed, are able to collect large number of data analysis, reports and general forms and rapidly make them available to clients.

Ig Nobel prize

On October 1st at Harvard’s Sanders Theatre, Ig Nobel prizes were awarded. Ig Nobel prize is awarded to improbable researches that first make scientists laugh and then make them think. As correspondent Nobel prizes, awards for different application field are assigned: from medicine to economy, from peace to literature, the most improbable researches are highlighted.

In 2009 Medicine Ig prize was awarded to dr. Unger who has studied for 60 years the role of cracking knuckles in the genesis of finger arthritis: to do this, he has cracked the knuckles of his left hand without touching his right hand for 60 years. Public Health Ig Nobel prize was awarded to an innovative kind of brasserie that can be converted in more protective face masks, as necessary. This model is U.S patent since 2007. Japanese scientists won the Ig Nobel prize for Biology: they showed how a thermophilic bacteria isolated for faeces of giant panda could reduce kitchen refuses more than 90% in mass. Last but not least, Peace prize was awarded for an experimental study about the opportunity to be smashed over head by an empty or full beer bottle: in both cases energy to break beer bottle is higher than those required to fracture human skull, so in both cases beer bottles are dangerous instruments in physical disputes. We hope to make you laugh but then you should think!

Reference: http://improbable.com/

Direct RNA sequencing

Recent paper published on Nature describes an innovative method to directly sequence RNA. mRNA is usually isolated from total cellular lysate and converted in cDNA by reverse transcriptase; then, cDNA is sequenced with protocols currently used for DNA sequencing. The use of an enzyme for retro-transcription could insert mistakes into cDNA sequence with quite high frequency: an advantage of direct RNA sequencing is to bypass this step.

How does RNA sequencing occur? Polyadenylated RNAs are bound onto solid support; DNA polymerase able to use RNA as a template binds one fluorescent nucleotide complementary to the first basis of RNA. This binding generates fluorescent signal, that is read. After reading, fluorescent probe is cleaved and the following basis on RNA can be recognized and bound to another fluorescent nucleotide. In this way, after a fixed number of cycle, all RNA molecule is sequenced. Even if this technique is innovative and extremely useful, it needs to be further improved and tested because some problems due to RNA stability could arise. Moreover, it has to be ascertained the proficiency of DNA polymerase that must perfectly work. However, we hope that this method could enter as soon as possible in clinical practice.

Telomeres and Nobel Prize

Last month (10 September 2009) in this blog, we talked about telomeres and their significance in cell biology. Telomeres are chromosome portion positioned at the extremity of chromosome self; they protect DNA from fusion or other genetic abnormalities that may occur during DNA replication. Telomeres are progressively consumed for the lack of activity of telomerase, provoking aging and cellular death in regulated way. Giving the importance of this biological structure, in October 5, 2009, Carol Greider, Elizabeth Blackburn and Jack Szostak won Nobel Prize for physiology and medicine.

Since 1980, they worked in three different Institution in USA, at the Johns Hopkins Medical School in Baltimore, Maryland, the University of California, San Francisco and Harvard Medical School in Boston, Massachusetts, respectively. Nobel prize history is always interesting and curious: for instance C. Greider saw the telomerase activity for the first time during Christmas day in 1984: she had a really important present for Christmas, but also she was working in this special day. So, Nobel Prize is the result of a lot of works, sacrifices and also serendipity. Someone criticizes this award, because prof. Olovnikov in 1973, so at least 10 years before Noble prize winners, highlighted the importance of the ends of chromosome during replication in his theory of marginotomy. Is an old question that Noble prize is often related to policy and politic aspect. In all case, the scientific value of this discovery is out of discussion. Telomere and telomerase enzyme have a crucial role in cancer, indeed in cancer cells telomerases continue to construct telomere after multiple replication cycle –normally after a defined number of replication cells die- and cells still alive, accumulate mutations and become malignant and maybe metastatic. Studies about telomerase inhibitors represent a new therapeutic modality to treat cancer and an hope for patients: some clinical trials have been started, directly acting versus telomerase enzymatic activity.

However, there is still a lot of information missing about telomere and telomerase and further basic studies must be performed in order to well understand this complex mechanism: indeed, it is not completely understood how telomerase activity is regulated in each telomere and how telomerase block DNA repair enzymes that can recognize break in the double stranded helix and re-stitch the torn end and again, how cancer cells can maintain the activity of telomerase at so high levels. All these questions will be solved in future research. Finally, this is the first time that two women are awarded together for Noble Prize and, in general, only 10 women have been awarded for this important prize. We hope that academic and science world will offer more career opportunities to women that want to follow this way than that are available now: Nobel Prize Award 2009 could represent a good starting point.

New method for prions identification

In 2001 Protein misfolding cyclical amplification (PMCA) technique was described: ultrasound waves catalysed a process in which misfolded prions leaded normal prions to misfolding in tube test. In this way it has been demonstrated that misfolded prions replicate without the presence of DNA and translation machinery. Researchers from the University of Texas Medical School in Houston, continue this work in order to simulate in vitro and in few hours what happens in human brain during prions generation lasting several decades.

In particular, PMCA technique was modified and further ultrasound waves cycles were added. They used homogenate from brain of hamster, mouse and human and applied their technique: they observed misfolded prions in hamster and mouse but not in human, probably because human prions have a lower probability to change their folding and the process is also slower. This technique has to be improved to allow to study sporadic origin of prions. By contrast, previous PMCA technique can be used as diagnostic tool to recognize the presence of misfolded – so dangerous- prions in patients that show neurodegenerative problems. Indeed, spontaneous prions are often associated in human to neurons disruption and neurodegenerative disease and early identification of their presence can allow the choice of better therapeutical approach.

Kinase activity profiling

In previous posts we have already discussed about kinases and phosphorylation detection. Today, we would like to focus our attention on one new method to profile kinase activity during cell cycle, pharmacological inhibition, cancer, signalling pathway activation. In June 2009, PNAS journal reported this approach: researchers of the Department of Cell Biology in the Harvard Medical School monitored the activation state of kinases in cell lysate by analysing the phosphorylation of 90 synthetic peptides, known as substrates, through mass spectrometry.

As an internal standard for quantification, they used heavy isotope-labelled peptides. The assay is quite easy to do: they used 96 format and plated total lysate and ATP, then they added the substrate to phosphorylation reaction and measured with liquid chromatography coupled with mass spectrometry. In this way they produced a fingerprint of kinomes of several cell lines of breast cancer, exactly showing which pathways were activated. In the paper they presented a novel Src family site in vivo, but this approach could have other important applications. For instance, it’s possible to compare the activation state of pathway in normal and tumoral cells, indeed in cancer alterations in kinase activity are often reported. Furthermore, the quantification of phosphorylation is important to check inhibitory properties of small molecule kinase inhibitors.

Cryopreservation

In scientific laboratories cryopreservation is usually used to store biological materials, such as cells and tissue. These kind of samples are frozen till -196°C in liquid nitrogen and maintained in special racks in nitrogen tanks. What does happen during freezing process? At so low temperature all biochemical cellular processes are blocked and also cell death is avoided, thus cells can be conserved and stored. Several protocols are reported in literature about cryopreservation; indeed, this process presents also some potential risks.

Firstly, it will possible to observe some solution effects if different solutes freeze at different temperatures: in this case some problem could arise if solutes were toxic at high concentration. Secondarily, extracellular ice could be generated and cause cellular damage by mechanical crushing. Anyway, much more dangerous than extracellular ice is the intracellular one that is fatal for cells. In order to avoid intracellular ice formation, it’s possible to freeze cells with cryoprotectant agents that lower the freezing temperature and increase the viscosity: this process is named vitrification and instead of crystallizing, amorphous ice are formed. Several molecules can determine both these effects but larger molecules are preferred because mostly contribute to increase the viscosity. Dimethyl sulfoxide is the common cryoprotectant used in cellular biology: DMSO is inexpensive and easy to use, but at high concentration is really toxic for cells. Cells cannot stay for long time in DMSO solution before and after freezing because it permeates through the membranes. So, one common procedure to freeze cells is to prepare freezing medium, resuspend cells in it and immediately put vials at -180°C. This passage at -180°C is useful to control rate and slow freeze. Alternatively, cells must be rapidly thawed and DMSO removed or diluted, cells must be plated at higher density than usual in order to increase the likelihood to obtain a vital population. All tissues, cells, blood samples, semen, oocytes and embryos can be frozen.

An important issue in laboratory is to manage these frozen samples. Indeed, it is preferable that nitrogen tanks would be rarely open in order to avoid temperature alteration and nitrogen evaporation. For this reason, position, type, date of freezing of a sample must be annotated in a special lab book in order to easily recognize the right sample. Better yet, it could be useful to us a special software to manage a large number of samples. That kind of software, like FreezerPro from RURO is commercially available and guarantee the perfect identification of samples without loosing time or taking the risk of choose the wrong ones. Furthermore, in quality control regime this kind of IT tool are often mandatory

Improving fluorescent probes

Cell imaging is one important tool to verify protein expression, localization and interaction in living cells. Several probes are now available to specifically colour cells, but intracellular retention is a problem for an high number of them. It’s great challenge to improve intracellular retention and also a great deal because improving intracellular retention means also improve the sensitivity of fluorescent detection.

Calcein, a fluorescein derivative, contains two imino- diacetic acid groups which are responsible for the optimal intracellular retention. Researchers of the University of Tokyo synthesized membrane- permeant derivatives of fluorescein containing either one or two imino- diacetic acid group and decided that two groups, as are present in calcein, are optimal for improving intracellular retention.

They designed novel fluorescent probes for imaging highly reactive oxygen species and nitric oxide. With these new probes they visualized low levels of target proteins in living cells over a relatively long period of time, which it’s not possible to monitor with traditional probes. This chemical approach to prevent cellular leakage should be broadly applied for all probes fluorescein- based as well as on other kind of scaffolds and could be considered a general strategy to increase the sensitivity in living cells.