Wnt pathway and its involvement in oncogenesis

   Wnt proteins are cysteine-rich secreted ligands, involved in development control in organisms ranging from worm to mammals. 19 Wnt members are expressed in mammals and are classified in two distinct groups based upon their ability to induce transformation in mouse mammary epithelial cell line C57MG. The highly transforming group includes Wnt1, Wnt3, Wnt7a, while the intermediately or non transforming members are Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt11. The two classes of Wnts signal through different pathways and lead to different developmental outcomes. In the “canonical pathway” activation of beta-catenin mediates signal transduction.

   Briefly, in absence of Wnt ligands, cytoplasmatic beta-catenin is phosphorylated by a destruction complex composed of Axin, Adenomatous polyposis coli protein (APC) and glycogen synthase kinase 3B. Phosphorylated beta catenin is also ubiquitinated and degraded in proteosoma. In contrast, when Wnt interacts with Frizzled serpentine receptors and their co-receptors LRP5 and/or 6, the destruction complex is inhibited. Indeed, axin is relocated to the membrane where is inactive. Thus, beta-catenin accumulates in the cytoplasm and after its translocation into the nucleus forms a complex with TCF/LEF family proteins. This interaction allows the specific activation of gene expression. Some Wnt proteins can activate a beta-catenin independent signalling, namely the “non canonical pathway”. This activation occurs via three different mechanisms. Specific Wnts and Fz receptors can increase the intracellular calcium concentration through trimeric GTP-binding proteins and activate calcium/calmodulin-dependent protein kinase II and protein kinase C (PKC). Otherwise, Wnt-Fz complexes act through heterotrimeric G proteins to activate phospholipase C and phosphodiesterase. Lastly, Fz mediates the planar cell polarity pathway which activates small G proteins, including Rac and Rho, c-Jun N-terminal kinase (JNK) and Rho-associated kinase. Even if two classes of Wnt proteins have been proposed, is not totally clear which pathways are activated by each Wnt. It seems that the receptor context determines Wnt signalling output, suggesting a major complexity in the Wnt cascade control with relevant implications in development and disease, such as cancer. Indeed, Wnts are, for instance, genetically altered in MMTV-induced tumour or overexpressed in many breast cancer cell lines.

   Over-expression of WNT3a, WNT4, WNT6, WNT8b, WNT9a, WNT10b that signal through the canonical pathway correlates with a poor prognosis. Colon cancer seems also affected and caused by alteration of Wnt canonical pathway. Several studies are publishing on this topic because it’s very interesting and important and recently, the evidence that cancer stem cells use Wnt pathway to maintain their stemness has made this pathway much more crucial. Further works will clarify the role of each Wnt pathway component in order to figure out how this pathway works. Moreover, Wnt pathway will be a possible therapeutic target and each protein will be validated and inhibited. The opportunity to attack cancer against multiple sides is largely accepted by the scientific community. In this vision, the inhibition of Wnt pathway could give a significant contribution.

The Baculovirus expression system

The Baculovirus expression system provides a versatile and reliable system for the production of recombinant proteins in insect cells. Although many different viruses belong to the Baculovirus family, the most widely used is Autographa Californica multiple capsid nucleopolyhedrovirus (AcMNPV) which can be propagated in Spodoptera Frugiperda (Sf), Trichoplusia Ni (Tn) insect cell lines.Virus genes are expressed in early, late and very late phases of infection: the early and late genes are largely related to the production of viral particles which then bud from the infected cell thus spreading infection to other cells. The very late genes that encode for polyhedrin and p10 are required for the production of the occlusion bodies containing the virus particle in the nucleus of the host cell. Both of these genes are under the control of strong promoter, but can be deleted from the virus genome without interfering with the production of infectious virus particles. As consequence, foreign genes can substitute the polyhedrin and p10 sequences to derive expression vector. This system has been largely used to express mammalian proteins because it guarantees, even if with some limitation, to obtain a correct folding. This capability is extremely important when active enzymes must be synthesized. Further advances in this techniques allow the production of all challenging proteins.

Novel HIV antibodies identification

The identification of effective HIV antibodies is a scientific and medical challenge that has not solved yet. Last week, it has been published in Science journal an article about the isolation of selective antibodies against more than 90% strains of HIV virus that could be used for vaccine design. Scientists at the Vaccine Research Center generated a probe to selectively identify and pick out only the antibodies that act against the part of the virus’s protein envelope that interacts with CD4+ cells. Replacing them with sequences from other viruses masked other portions of the envelope. In this way, the odds of fishing out wrong antibodies were significantly reduced. Thus, scientists screened more than 25 million of B cells derived from 15 HIV patients and isolated only three antibodies able to exactly mimic the binding between CD4+ cells and viruses. The next step is to better characterize these antibodies to learn about the maturation process that makes possible recognizing the HIV virus. Another aspect that should be kept in mind is to evaluate how many HIV patients are able to produce similar antibodies in order to be sure that the vaccine will be not too specific or restricted.

Docking application on aspirin derivatives development

Docking analysis is usually used to improve drugable molecules or develop new derivatives from old drugs. Based on this application of docking analysis, scientists from the Department of Bioinformatics at the Bharathiar University in India, generated and validated a series of aspirin analogues that act on prostaglandin H1 enzyme. Aspirin is a common drug used to treat pain during inflammation. Its properties on platelet aggregation allow its use on thrombosis prevention. Through the analysis of interactions between the small molecule to the active site of the enzyme, they identified the pentanoate group as a possible good binder Thus, they proposed this group as a new type of non- steroidal anti- inflammatory drugs with higher precision on target and fewer side effects. The work has been published on Nature Precedings and it seems really promising. Indeed, COX-1 inhibitors are largely used and their side effects still are quite dangerous. Thus, it should be important to develop safer small molecules. TO this purpose, the molecular docking is a method well validated and it has been previously used to develop several other drugs or improve the existing ones.

Data sharing in neuroscience

Data sharing is one of most important challenge in several scientific fields and it has already given good results in genomic and proteomic analysis. Also neuroscience should benefit from data sharing, in particular from functional magnetic resonance imaging (fMRI) data sharing. This technique is used to map functional region of brain that are monitored and recorded in resting or working state. The entire map of functionally tuned regions in the brain constitutes the connectome and it may help scientists to better understand brain physiology and functioning. Michael Milham and his group of psychiatry at the New York University propose to other scientists worldwide to share their images of functional magnetic resonance in order to dispose more samples to analyze and compare. Indeed, even if these images have been generated in uncoordinated manner and with different purposes, data sharing could be extremely useful. Thus, they proposed the 1000 Functional Connectomes Project in which they wanted to collect data from fMRI. More than 35 centers decided to have part in this project, shared the images of more than 1400 volunteers, and deposited them into an open access database. Applying analytic and computational methods to evaluate and aggregate shared data, Milham and coworkers demonstrated a universal architecture of functional connections in the resting human brain. Furthermore, they highlighted the consistent loci of variability between individuals and centers and the brain region for which age and gender emerged as significant determinants. This study was published on PNAS Journal in 2010. Other follow-up studies will be necessary to validate the results described here. For instance, the information collected in the Functional Connectomes database could be used to build normative maps of functional system in the brain, as well as to interpret laboratory test results, thus using this method for a clinical application. The Functional Connectomes project is continuously growing and their supporters and creators encourage all groups involved in neuroscience imaging to share their data. This collaborative effort will give important scientific result because of the synergistic effect of data sharing: indeed, data that are brought together can be much more than the sum of the parts. Based on that, data sharing could be crucial also in other medical disciplines where imaging may explain biological processes. Of course, specific software will be necessary to effectively manage the huge amount of data derived from data sharing and the computational biologist will be fundamental in the laboratory. In conclusion, sharing data and computational analysis have become milestones in several field of science, and we are sure that this collaboration will give a significant contribution in scientific advances.

Morphoproteomics

Morphoproteomics is a novel medical discipline that combines histopathology, molecular and cellular biology and protein biochemistry to define the protein circuitry in diseased cells. In this way it should be possible to identify specific target for customized therapeutic intervention. One preclinical study on prostate cancer overlaps data from phosphorylation analysis with phospho-specific probes and morphological evaluation of cellular compartmentalization. The identification of peculiar localization of certain phospho-analytes demonstrated the efficacy of this kind of personalized studies and their clinical utility. Another study demonstrated the feasibility of this approach in neck and head squamous carcinoma, that often has poor prognosis and high mortality rate. In this case, mTOR pathway was analyzed in terms of potential clinical significance of its activated components. Rapamycin is an inhibitor of this pathway. The inhibitory effects on cellular growth were directly correlated with mTOR activation, and a paint of the block was obtained by immuno-hystochemistry. On conclusion, morphoproteomics gives details about the signal transduction by using the combination of several techniques. This approach has been positively evaluated in certain disease, like cancer where an alteration in protein circuitry often causes the disease. Other pathological model would be available soon to validate this approach also in other disease.

Similarities between pandemic influenza viruses

Scientists from the Mount Sinai School Medicine (New York) recently publish an interesting paper about the similarities between two viruses, namely the Spanish influenza virus and H1N1. The Spanish influenza virus caused a incredible number of death in 1918 during the first world war, worldwide. This virus is formerly extinct and for this reason it could be used as a bioterrorist agent. The comparison between the Spanish influenza virus and H1N1 demonstrates an homology of antigenic sites of haemagglutinin A. This observation induced scientists to investigate whether mice and humans previously vaccinated against H1N1 were also immunized against the Spanish influenza virus. Human sera containing antibodies anti-HA were collected and passively transferred into mice infected with the Spanish influenza virus and protected them from lethality. Thus, immunization against H1N1 protects also from the Spanish influenza virus infection. In case of bioterrorist attack or accidental virus escape from scientific laboratories the emergency could be easily managed. This study is pionieristic and other researches could be done to identify similarities between viruses. Such as approach, it could be useful to validate current therapies against other viral target, diminishing the cost of clinical trial required to market new anti-viral drugs.

Challenging on transcriptome

mRNA translation is one of crucial process in cellular metabolism. Several approaches have been developed by cells to perfectly regulate the translation. microRNAs bind mRNA and determine its half life, while numerous proteins directly bind the mRNA and favor or avoid the interaction with ribosomes. Studying the interaction between proteins and mRNAs is important to well understand whether alteration in these binding sites have any contribution to genetic disease that are caused by a loss of an RNA- binding protein such as fragile X mental retardation or familial amyotrophic lateral sclerosis. Scientists form the Rockefeller University described a method to isolate complexes protein- RNA. They used the UV radiation that are known to form covalent bonds between protein and mRNA. So, live cells are exposed to UV radiation, and an immunoprecipitation against the protein binder allows to identify the sequence involved in the binding. This technique called CLIP (PDF), cross linking and immunoprecipitation, can be applied also to high throughput experiments. To do this some improvement are necessary in order to separate the signal from background. The use of photo- activable ribonucleotides introduces into the RNA some non-toxic and efficient linkers. Indeed, a specific base change during reverse transcription, scoring for thymidine to cytidine in the sequenced cDNA allows to precisely map the interaction site. This is an important step to unravelling the gene regulation.

Is science a good job?

It has been recently published on Nature Journal a sort of survey about the satisfaction level of scientists in different countries. By using fixed values ranged between 0 and 1 the average mark was 0.861 that keeps in consideration all the parameters evaluated in this survey. Scientists were asked to judge their salary, vacation plan and retirement plan, some financial aspect of the work that are essential to appreciate the job self. Other parameters described in this study are the interest in the project, the possibility to work autonomously, the usefulness of relationship with boss and colleagues. Chinese, Italian and Japanese scientists seem the most unsatisfied of their job because of difficulties from a financial point of view; by contrast USA and UK scientists are happiest because they think to have a good financial profile and good autonomy. The general opinion is quite positive, in all countries scientists appreciated the creativity of the job that makes interesting science. Thus, around the world being scientist represent the best job to do! Of course, an improvement is also necessary specially in those countries where this job is not financially considered enough or where the scientific autonomy still be far.

3D cellular culture

Several trials have been already done to produce a three dimensional support to grow cells. Collagen matrix, gelatin, nanofibers are some examples and each of them has some problems or limitations that have not allowed to be selected as a method of choice for three dimensional cell culture up to date. The advantage of three dimensional cell culture is to restore the normal environment where one cell is located into the tissue or in organism. The monolayer on a plastic surface or a suspension avoids the interaction with matrix or with other cells and, as a consequence, it affects cellular behavior. Furthermore, the plastic surface cannot be deformed in a similar way like the matrix and cells partially loss their plasticity. Matrigel and gelatin or collagen layer may help to maintain the three dimensional conformation of cells, but at the same time they are not totally inert surface and may activate some signaling pathways usually silenced. A possible alternative to matrigel or collagen layer is paper. Yes, the normal chromatographic paper has been proposed as a possible support to grow three dimensional cell culture. A recent paper published in PNAS journal describes this protocol. Scientists soaked the chromatography paper in hydrogel and cells and papers were layered at the bottom of tissue dish. In this way, cells were able to grow in contact with other cells cultured in the upper paper. To analyze one layer it was possible to peel the paper from the stack and work on it. The major advantage of the method, rather the low cost, is to sample a living tissue without fixing it or making thin sections. You can split the tissue and put inside the paper layer and when you want to analyze it, you just have to open it. In contrast, the main limitations are the lack of vascularization that is normally present in the natural tissue and the impairment of fluid flow. Thus, further work has to be done to make possible the local fluid flow that is really important in certain studies to study, for instance, the pharmacodynamic of drugs. Of course, even if this model is really interesting, as well as other three dimensional supports, it cannot substitute the use of animal model in science for many reasons. In particular, animals have vasculature, time dependent physiological and pathological remodeling, inflammation and immune system that are not present in any three dimensional in vitro model. All these elements are fundamental to understand the biological mechanism of pathways and characterize new drugs. In conclusion, paper support seems a very interesting model to study preclinical development of drug or to deep cell cell interactions and further applications will be published soon.