Novel applications of nanobodies

Camelid derived single domain peptides are a novel form of antibody which maintain the same antigen binding properties but have greater stability and smaller size than traditional antibody. These molecules can be conjugate with several chromophores, for instance with green fluorescent protein for cellular imaging applications.
A group from the University of Munich (German) applied its deepen knowledge about GFP modifications to nanobodies, this is the name of camelid peptides. The result was an improvement in GFP brightness modulation. By performing a phage display screening, they found out seven different molecules able recognize GFP and enhance or minimize its fluorescent signal. To validate the system, they produced a GFP- tagged oestrogen receptor and a nuclear binding enhancer nanobody, in presence of hormone the receptor moved into the nucleus and GFP signal increased five- fold its brightness. Several applications can be thought for this tool. For instance, nanobodies for each cellular compartment can be useful to determine how the protein of interest tagged with GFP change their position inside the cells in response to specific stimuli. Alternatively, protein- protein interaction can be studied because nanobody can bind one protein and after interaction with the second protein fluorescent signal can be modulated, as well as it should be possible to evaluate the duration of interaction self. A novel and flexible tool is now available for biologists.

Monoclonal antibodies

Monoclonal antibodies are important tool in molecular biology, diagnostics and clinical studies. These protein are produced by a single cells isolated from immunized animals. Current protocol requires an immunization of an animal host, for instance rabbit or mouse; then spleen cells are selected in vitro and B cells are isolated. B cells from spleen are fused with tumoral mouse cells of mieloma in order to stabilize and make possible the B lymphocyte culture. Indeed, B cell culture is difficult to set up and maintain.
The hybridoma technology allows overcoming these difficulties because of genetic transformation of mieloma cells. Finally, hybridomas are serially diluted and the antibodies are obtained from cell hybridomas cultures derived from a single cell. Which are the advantages of monoclonal antibody in respect with polyclonal ones? Monoclonal antibodies are codified by the same gene and none point mutations are present to generate some difference into antibody population. Thus, the whole population is identical and specifically recognizes one antigen. Cross reactivity is reduced with monoclonal antibodies and the interaction between antigen and antibody is usually more stable. Furthermore, this technique is also really flexible because it’s virtually possible to create antibody versus each antigen, when it’s possible to immunize the host animal. Which are the applications for monoclonal antibody? Monoclonal antibodies are currently used in molecular biology and biochemistry laboratories for imaging, western blotting, immunoprecipitation and so on. A lot of protocols are based on antibody use. In diagnostics, monoclonal antibodies are used in ELISA dosage or in flow cytometric analyses, as well as infection detection or pregnancy diagnosis.

Clinical applications of monoclonal antibody are prevalently in oncology. In 1997, the first monoclonal antibody was approved for non- Hodgkin lymphoma treatment. Since this year, several antibodies have been optimized against breast cancer, leukaemia, colon cancer and recently lung cancer. Each antibody recognizes a tumoral antigen and specifically kills only the cells (tumoral) that present that molecule. Thus, adverse effects associated with the use of monoclonal antibodies are reduced if compared with traditional drugs. Based on their specificity, antibodies can be used to carry other useful drugs to cells. For instance, an antibody can be conjugated to radioactive compounds to be addressed to cancer cells. Furthermore, other drugs can be carried into the brain, giving the capability of monoclonal antibody to overcome the blood- brain barrier. Parkinson’s disease can be treated with this approach. Improvement of biochemical characteristics of monoclonal antibodies is one challenge for scientists for the next future. Indeed, it’s important to improve the delivery of monoclonal antibody into all districts of human body. The specificity will be a must if clinical or diagnostic applications are planned for the monoclonal antibody. Furthermore, cheaper technology must be optimized to allow large scale production. Research development in this field is really promising.

New challenges for synthetic biology

Synthetic biology is a modern discipline that tries to manipulate cells and program them to perform some activities useful for us. Different products or services may be provided by cells, for instance production of biofuel or toxin identification into a body as well as controlled insulin release. Synthetic biology requires a strict control of the system, but many parts involved into the process have not been known yet.
For instance, DNA sequence in the promoter region is not always well characterized and this is a crucial point to increase cell productivity. So, the first point to be clarified to obtain results from synthetic biology is the knowledge of expression system. Furthermore, it’s important to understand how all parts can work together. This is another challenge for synthetic biology because of the complexity of regulation mechanism into the cells. Circuits can work in an unpredictable manner and results are often few understandable. Finally, even if the circuit seems to work very well, the system could not be reliable in all situations and it could fall because of genetic mutations which could arise in any time. In summary, synthetic biology is an important challenge for scientists for the next future. Biofuel, toxin detection and insulin release are only three of different purposes for which this discipline must be applied.

Immune system involvement in TB

Tuberculosis is one of the diseases eliminated from occidental countries, even if few cases are reported every year. Granuloma formation into lung is the fundamental characteristics of this disease and the involvement of our immune system in this process is argued since long time. In advance of publication number of Cellular and Molecular Immunology an important paper illustrates how our immune system has a consistent role in granuloma formation and which kind of T cell are involved.

The comparison between blood samples derived from Tuberculosis patients or healthy donors demonstrated that the presence of a special group of T cells – namely the IL 17 producing γδ T cells- in peripheral blood was significantly higher in patients than in healthy donors. In vitro re-stimulation with tuberculosis mycobacterium antigen generated an increase in IFNγ producing T cells ratio in patients than in healthy donors, while the ration of IL17 producing cells was similar within two groups. These data were consistent with other results obtained in mouse model and confirmed the role of IL17 in granuloma formation. The identification of γδ T cells as major IL17 producer may arise some advantages for possible pharmaceutical treatment, but further elucidations are required to better understand all the mechanism at the basis of this complex disease.

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.

Mitochondria- ER tethering

Mitochondria are important organelle that produce ATP in cells. Even if a lot of information are available about the function and evolutionary biology related to this cellular district, few is understood about the communication between mitochondria and the rest of cells in terms of molecular transport. In particular, some proteins have been recognized to form junctions between mitochondria and endoplasmic reticulum (ER) that is the compartment responsible of lipids synthesis and export into vesicles, but still be mysterious the nature of the connection.

Science published an important paper about this topic: authors identified a method to genetically screen and recognize proteins involved in the connection between mitochondria and ER in yeast. They designed a synthetic mitochondria- ER tether, called ChiMERA, consisting of an N-terminal mitochondrial signal sequence and a transmembrane domain from protein Tom70, connected through the central GFP domain to C-terminal ER tail anchor from protein Ubc6. The expression of ChiMERA in yeast strain that showed lethal mutations in mdm12 gene, important for mitochondria morphology and functionality, rescued the growth suggesting that the correct interaction between mitochondria and ER was achieved. In summary, this work acquires a pivotal role in research of other native organelle tethers and, maybe in far future to replace this cellular function with artificial proteins, if needed.

Nanog and pluripotency

In August 2009 Cell journal published an important article about the role of Nanog in pluripotency. Silva and colleagues showed that Nanog played a crucial role in development of pluripotency in mice. Indeed, they generated a KO mouse for nanog gene and they observed a block in reprogramming cells to achieve the full pluripotency; Nanog re-expression allowed the complete restoration of the reprogramming process indicating the central role of this protein in the acquisition of pluripotency.

Researchers were not able to obtain embryonic stem cell culture from nanog null mice because the embryos didn’t form the inner cell mass and so the pluripotent compartment. Molecular explanation of the role of Nanog is probably related to the signalling pathway that involves also Oct4, it seems that Nanog is important to sustain that pathway by inducing a self maintenance of the system. Nanog is also important for the reactivation of chromosome X, another crucial step to reach the pluripotency. This work increases our knowledge to obtain the induction of pluripotency in vitro: this goal is crucial for instance for many applications of regenerative medicine. Defining the factors that control, start and maintain pluripotency will help us to obtain safer and better induced pluripotent stem cell cultures.

Microinjection of yeast

Yeast is a commonly used genetic model, budding and fission have been largely studied, but few methods to introduce DNA, protein or other compounds, have been described until now. Indeed, yeast presents the rigid cell wall that avoids injection and all techniques currently used to manipulate cells. It has been described that piezo-impact micromanipulator worked well to overcome biological barriers; thus, this tool was employed also for yeast injection with good results.

The procedure, reported in the last volume of Nature Methods, allows to introduce material during fission yeast. At the microscope it’s possible to immobilize and buckle one cell, through mechanical stress, while sorbitol in the surrounding medium avoids cell disruption. Material that has to be injected enter into the cell is released from pipette and passed through the hole into the wall. The major difference between usual microinjection technique is that the pipette doesn’t touch cell surface. Why is this method so innovative? For the first time yeast has been manipulated and now inhibitors, RNA and proteins could be introduced in. Thus, new experiments could be thought and performed, keeping in mind that yeast is really diffused, as a tool for genetic studies and recombinant protein expression and other applications could be now imaged.