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.

Tetracycline derivative can modulate SMN expression

Survival of motor neuron centromeric (SMN) genes are involved in spinal muscular atrophy when mutated. This disease is an autosomal recessive disorder that progressively determines the loss of spinal alpha motor neurons, causing death during the childhood.

Deletion in SMN1 gene generates a truncated protein without functionality. In a similar situation another protein of the same family SMN2 is produced in larger amount, but this spliced form is unable to overcome the loss of SMN1 and restore its normal activity. SMN2 lacks an exon (7) important for protein function, this is the reason for which SMN2 cannot completely restore SMN1 activity. Researchers observed that tetracycline derivatives could interfere with splicing mechanism and promote the inclusion of exon 7 into SMN2 mRNA. They identified the less toxic ones in order to modulate SMN2 expression without collateral effects. Rather than the toxicity, the second problem that researchers had to solve was the blood brain barrier crossing because tetracycline derivatives cannot cross this barrier. Scientists proposed firstly an injection into brain, then the use of osmotic pump to internalize these useful drug in SMA patients. Giving the lethality of this disease, it’s important that all routes are checked and considered in order to find the right one.

iRNA in yeast

iRNA is a tool currently used in molecular biology to define the function of a gene: indeed scientists block mRNA transcription of specific gene and observe the cellular response. This technique is extremely potent and specific and allows to study one protein at time. In yeast, certain strains lack the normal RNA interference machinery and have alternative enzyme variants which can be transplanted into truly deficient species. iRNA is specially useful to study budding yeast, but this model lacks the Dicer enzyme, responsible for processing the double stranded RNAs into small interfering RNAs.
Scientists from the Whitehead Institute identified many proteins that show hallmarks of Dicer- mediated cleavage; they moved these proteins into new strains in order to reconstitute the iRNA machinery and they observed the complete silencing of all gene of interest. Thus, no limitations were found to study genes and proteins involved in yeast biology. Moreover, transposons are efficiently silenced without interfering with other genes. The system is greatly specific. Based on this positive result, scientists want to apply this finding to pathogenic yeast Candida Albicans in order to better understand the behaviour of this organism and identify some pharmacological target.

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.

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.