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.

Mutational analysis of promoter region

Promoter region is the regulatory portion of genes that controls transcription. Even if this portion is crucial for finely tuning protein expression, and is known that epigenetic alterations are dangerous for this control, poor knowledge are available about the role of point mutations. Indeed, several point mutations are identified in promoter region, their biological consequence is poorly understood and studied. Scientists of the University of Washington set up an array to screen the effect of mutation on promoter functionality. They analysed all possible point mutations into a core promoter: the construct presented the native sequence, followed by mutated promoter and the transcription fragment. Each construct was codified by a barcode; thus, after transcription, a quantitative measure of transcriptional activity was obtained. In this way, all mutations were screened and quantitatively characterized. A surprising result was that one mutation generated down-regulation of the expression, while two mutations didn’t alter so much the transcriptional activity. A possible explanation was that the interaction between DNA and polymerase was stabilized in presence of one mutation, reducing the capability to move onto DNA molecule. This experiment was performed in vitro with cellular extract. Scientists knew that the best way to accomplish this screening is to use transfected cell lines, and this will be the possible next step.

The value of negative results

Negative results are similarly important than positive results for science advances. Indeed, a large number if hypothesis could be excluded if we considered negative results from other studies. Unfortunately, negative results are not so easily accepted and published in peer reviewed journal, thus are not available for science community.
Scientists from the German Research Centre for Environmental Health and from the University of Munich spent their time to collect negative results from protein-protein interaction studies and built a database, called “negatome”. They used data from literature search and from structural information retrieved into Protein Data Bank and identified almost two thousand of not interacting pairs. The novelty is that the not-interaction is experimentally demonstrated and published, while in previous work not- interaction was determined from not co-localization: if two proteins are differentially located, they will not interact. This sentence could be true, but is not predictive of interaction, just co-localization. The Negatome can contain some false negative pairs, but in general it can be considered a useful tool to compare and confirm results from immunoprecipitation or two hybrid system or other techniques that usually generate some false positive. We hope that this example should be followed by other databases for negative results.

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.

Statistics in science

A common English quote says that there are three kinds of lies: lies, damned lies and statistics. This sentence was wrongly attributed to Disraeli, whereas it was firstly published by Marc Twain. Even if it could seem true, statistics is the only tool available up to date to manage a large amount of data and to give their a mean. Several functions are usually used in statistical analysis; the most common are the average, standard deviation, frequency and also the number of observation is reported in scientific publications.
All these data are defined as descriptive statistics because of their direct characterization of the population of interest. In other cases more complex calculations must be done to find significance from the study. Linear or logistic regressions are models to identify the trend that better explain and summarize the collected data. From linear or logistic regression is possible to interpolate or extrapolate data in order to predict results into the studied range or outside the studied range, respectively. Furthermore, from statistical analyses prevalence, incidence, absolute risk, odd ratio and relative risk are determined. All of these parameters are so important that must be determined during clinical study and are subsequently used to compare different protocols, drugs and so on. Clinical studies are classified as experimental studies or observational studies. The main difference between these two classes is the presence of a treatment given to patients in the experimental studies, whereas in the observational studies none treatment is followed but the selected population is just observed to determine the parameters of interest. In both cases, given the high number of patients enrolled in order to obtain significant results, statistics is useful to compare treated cohort and placebo cohort, for example, as well as to evaluate the role of specific component of the study, namely called covariates.
The major part of software that help to collect and manage data from a clinical study have also some algorithms to calculate standard statistical parameters during the data collection self. Revision and further elaboration must be done by professionals in order to correctly consider study results. Statistics is important not only in clinical trials, but also in all experiments performed in science. Indeed, to be sure to have obtained a result as a consequence of certain conditions and not due to serendipity, all experiments are usually repeated three or more times. Data presentation normally comprises average and standard deviation or confidence interval and significance is determined by a series of tests that should be described in material and methods section of the publication. In conclusion scientists must have some basis of statistics because this discipline confers value to their experiments and make them shareable and comparable with scientific community. Even if statistics is considered as a lie in some cases, it will be useful for science advances.

The pediatric knowledge base

The pediatric knowledge base (PKB) is an IT system developed at the Pediatric Pharmacology Unit of the Children’s Philadelphia Hospital. This project involved professionals from several disciplines, such as medicine, pharmacology, statistics, bioinformatics. They built a model in which clinical data from patient were combined with electronic medical records and drug profiling, in order to obtain an IT tool able to continuously learn information about the patients self.
PKB helps physicians to choose the better pharmacological therapy in terms of dose, agent performance, collateral effects and drug combination. Based on the profile of three common drugs, such as vancomycin, methotrexate and tacrolimus considered as prototype, PKB monitors the drug use and helps to predict the outcomes in children patients that are often more difficult to treat. PKB is updated by numerous hospital members, in this way deep knowledge about clinical adverse events or clinical advantages as well as innovative protocol can be shortly acquired with great benefits for patients. Furthermore, PKB is web based and it could be shared between hospitals of different countries. A consortium is planning to be created to coordinate PKB implementation across the world. Using PKB as a model, it could be possible to set up similar dashboard for other class of patients in the next future.

IT system protection

The broad diffusion of technologies such as computers, databases, mobile phones and so on, has determined a considerable change in our life. We usually use this kind of instruments at the work or school, at home and in other situations of everyday life.
It’s really important that the global IT system would be safe and protected from hacking attacks. Last year, an important mobile phone manufacturer company found vulnerability in its system and phone calls could be listen by everyone knew this failure. This first episode started an intensive research on security system in order to preserve IT and our privacy. Scientists were called to identify flaws into algorithms, acting like legal hackers, and were invited to share their skills and experiences with the community. So, manufactures changed their habits to reject the possibility to reveal flaws in their systems and choose an ethic approach to improve the worldwide knowledge. Also manufacturers that produce IT systems for pharmaceutical companies must consider the opportunity to look for new devices to protect all sensible data regarding the clinical study. Indeed, personal details of patients contained in clinical study are usually shared between centres involved in the trial and become more sensitive of possible attacks. Thus, it’s ethic that these data should be preserved.

RFID to easily identify frozen samples

RFID means radio-frequency identification and is used to define tagged object that can be recognized through radio-waves. This system is composed by two part: the first one is an integrated circuit in which is possible to store and process information and modulate and de-modulated radio frequency; the second part is usually an antenna required for receiving and transmitting the signal. The first use of RFID was during the II World War when allies used this system to distinguish their planes from those of enemy; then in 1973 it was US patented by Mario Cardullo with a business plan showing uses in transportation, banking, security and medical. Now, RFID technology is currently used for several application: from passport to animal identification, from race timing to transportations payments.

In healthcare RFID are undergone to severe regulation to defence the privacy of consumers. In 2006, Food and Drug Administration highlighted the importance to do not include information about consumers, health practitioners or other uses of the product out of label. The RFID tag may be covered with a seal containing a logo, a message unrelated to product and an unique serial number and, mostly important, the tag will not substitute the traditional labelling process. This point wasn’t observed by GSK when they used RFID tag to control HIV treatment capsules and for this reason a violation was signalled. Another important use in scientific world is the tracking of nuclear substances during both storage and transportation. Indeed, scientists from the Department of Energy Argonne National Laboratory set up a system in order to gain information about the nuclear materials in real time, allowing the constant monitoring of the materials self. Moreover, some RFID devices have been added to notebooks to streamline information management and retrieval. This tool could be useful in big pharma, or big institutions in order to correctly manage all information without loosing time.

Sample identification is another important application in the scientific laboratory; for instance, it could be really useful to recognize frozen samples –cells, proteins and RNAs- without opening the box. The great advantage of this system is the perfect preservation of biological materials, indeed everyone knows that continuous and repeated extraction from liquid nitrogen seriously damage biological samples. Furthermore, RFID allows to immediately control the box status and find if vials are missing. In quality control system this system is strongly recommended because it makes transparent audit process. Modern RFID technology is miniaturized and cheap, so it could be applied on each cryovials through self-adhesive cryo safe labels. Using RFID tags has a lot of advantages specially if the freezing service is centralized or common for several laboratories: not only box contents are always well described, but also operators’ mistakes can be immediately repaired without loosing precious materials. Also in small scale laboratories this system could have some applications to manage samples during years specially if there is a quick turnover of workers, as usually happens in academic labs. RFID system is a modern tool to facilitate scientific job.

Databases

Two main databases are now available: the EMBL-EBI and the NCBI for Europe and US, respectively. These two databases are connected and all the information present are available in both systems. Another database is provided by a Japanese laboratory and is online at genome.jp.

Main databases contain information about DNA sequence, two examples are EMBL datalibrary and GenBank; all other databases regarding RNA, proteins and polymorphisms or rare diseases are connected to these two ones. Databases are usually checked by operators or software: the difference between these two control systems could be observed in the redundancy because manual control is usually more systematic than these performed by software. About proteins, three secondary databases are currently used: Swiss-Prot, TrEMBL and PIR. In these websites several bioinformatic tools are available to align sequences, predict primary and secondary structure of proteins or determine the isoelectric point, all these information are important especially at the beginning of the study. Other databases like PDB or Modbase offer three-dimensional structures of proteins and prediction of three-dimensional structures, respectively. As well as PROSITE collects information about protein motifs, functional domains and so on. Last but not least, Genome.jp is preparing a new tool, KEGG pathway, useful to retrieve information about enzymes and metabolic pathway. Good work!