miRNAs as tool to reprogram somatic cells

In the mouse, pluripotent sem cells can be induced by using certain miRNAs. A recent work published on Nature Biotechnology demonstrated that the addition of miRNAs to a list of factors usually used to reprogram somatic cells to pluripotency increases the efficiency of the process. In particular, scientists from the University of California identified in mouse embryonic stem cells that have a special cell cycle (lack of checkpoint at the G1-S transition) and, for this reason, proliferate rapidly a pool of miRNAs ( embryonic stem cell specific cell cycle regulating miRNA, ESCC) that regulate this so particular cell cycle.

Considering that cell cycle and differentiation are strictly linked, when embryonic stem cell like-cycle is reproduced in a differentiated cell, it could be possible to promote de-differentiation. Indeed, miR 290 cluster was added to somatic cells (mouse embryonic fibroblast) with other factors such as Oct4, Sox2 and Klf4, and increased the efficiency which these cells were reprogrammed. The miRNA system is redundant, and several molecules have the same seed sequence and are interchangeable in their functions. It’s not clear if miRNA could totally substitute all transcription factors in reprogramming somatic cells. Other work has to be done in this field to define the final cocktail that could be use for this purpose.

Identification of miRNA target genes

microRNA are short RNAs that are important in gene regulation in all organism, form Drosophila to human. Indeed, in mammals they induce the RNA-induced silencing complex to target sites usually located at 3’untranslated regions (UTR) of mRNAs, determining translation repression or RNA degradation. To identify putative genes targets, computational approach is generally used, while experimental validation is more difficult.

Four algorithms, miRanda, TargetScan, RNA22 and PITA, have been developed to identify targets; comparison between these algorithms allows to reduce the number of predicted genes, usually thousand of genes. So, scientists run their search with at least two algorithms and consider only the overlapping targets. Another important factor in mRNA target search is to consider the 3’ UTR: different databases of miRNA target genes propose different 3’UTR, thus to compare the result from two algorithms is crucial to start with the same database of 3’UTR. Furthermore, one miRNA could target many genes, the identification of group of genes targeted by one miRNA could give some information about the localization and the biological function of proteins, codified by these genes. Functional profiling of miRNA target can be performed through the Gene Ontology website. miRNA is a complex but really interesting tool that cells use to tune the expression of certain proteins, specially for instance during embryonic development.

Comparison between Baculovirus and E. Coli, as expression system

Baculovirus expression system is commonly used for protein expression, in particular it is used for eukaryotic proteins that cannot be correctly folded in E. Coli. This virus, not infectious for human, infect insect cells, such as Sf9, Sf21 or Hi5, and are isolated to performed high amount of recombinant proteins. Indeed, Baculovirus genome could be easily modified with current molecular biology techniques –molecular cloning, following by recombination and transposition- and propagated in normal cellular biology lab, not P2 or P3 chambers are necessary to use this virus. It’s possible to insert long fragment of exogenous DNA into virus genome, this aspect is important and represent a good advantages in respect of E. Coli because eukaryotic proteins are usually codified by long genes.

Another significant advantage is the capability to introduce post-translational modifications, such as phosphorylation, glycosilation that are necessary to obtain the complete functionality of the recombinant protein. Thus, proteins produced with this system could be employed for instance in biochemical assays, after purification, and in screening of pharmacological compounds. By contrast, the main disadvantages associated with Baculovirus are the cost and the yield: insect cells culture is more expensive than E. Coli culture, because of the media, sera and molecular reagents necessary to make the transfection. Furthermore, the yield is lower and if 1 mg of recombinant protein can be produced form 1 litre of E. Coli culture, much more litres of insect cells are necessary to produce the same amount of protein.
What are the parameters important to choose one type of expression system in respect of another one? Firstly, it has to be known very well the protein that has to be produced: literature review gives interesting suggestions and the most common expression system is usually the best one. Secondarily, the aim of the study is important, indeed if a functional study has to be performed, it’s crucial to have an active protein also in a little amount, by contrast for crystallization study is important to consider that milligrams of protein have to be purified. Finally, both Baculovirus and E. Coli are tool easy to use, but in the case of Baculovirus some experience in cellular biology rather than biochemistry is important. The timing has also to be considered because Baculovirus-insect cell system requires at least one month, after cloning, to perform first infections; by contrast, E. Coli needs only cloning procedures to express the protein of interest. Both recombinant viruses and bacteria can be frozen, in order to have reagents ready to use, it’s important keep sterility and a great classification if numerous proteins are studied at the same time in the lab: special software are commercially available for this purpose. In conclusion, both techniques are useful in biochemistry, structural biology and molecular biology and in respect of the aim of the study is crucial to choose the more convenient one.

Challenging on drug combination

Drug combination represents an important field of research for a long series of reason. Firstly drug combination could have a therapeutic advantage if it generates synergy, so when the activity of two drug given in combination is higher than the sum of the activity of each drug given alone.

Unfortunately, synergy in the activity often determines synergy also in side effects. In literature it’s reported that synergistic combination could arise in more specific and particular cellular context than single agent activities. A simulation of bacterial metabolism allowed to perform multi-dose experiments relevant to several diseases and outlined the combinations that gave selective synergy in respect of drug target activity. For instance, an anti-inflammatory drug showed an increasing selectivity through different expression level of the target of another drug. The specificity of synergistic combinations generates new opportunities for therapeutic approach to improve the control of cellular system, so complex and interesting. Moreover, synergy could lead to decrease effective doses of the drug and, maybe, reduced collateral effects. Finally, formulation of couple of drugs in one pill could improve also the patient compliance. Much work has to be done to identify new combinations specially for drugs whose patent is terminated.

Minicells to target tumour cells

A difficult challenge in cancer therapeutics is to develop drugs that can overcome the heterogeneity and resistance of cancer cells. A tumor usually consist on several type of cells that contribute to tumor growth, tissue invasion and metastasis in different way. Furthermore, during a treatment drugs become ineffective because of survival of cells that escape death induced by drug.

Small minicells derived form bacteria was used to targeted delivery of drug into cancerous cells. Antibodies can target minicells to tumor cell surface receptors and release drug at the specific site. A recent publication demonstrated how it was possible to subsequently administrate short hairpin RNA and cytotoxic drug in order to firstly knock down a multidrug resistance protein, then kill the cells made vulnerable. This strategy was tested on xenograft tumor in mice model and gave the opportunity to tune the dosage of cytotoxic drug, diminishing adverse effects. Minicells were not toxic for animals and didn’t compromise their survival: this approach seems promising to specifically treat resistant tumor and the use of shRNA allows to personalize the treatment. Other important challenges in next future will be the discovery of other route to target minicells, because resistance mechanism will be early developed to decrease receptor expression on cell surface.

Risk of skin cancer during sun exposure

During summertime, we spend part of our day to bronze our self, but as reported in scientific literature, UV rays can induce double strand breaks into DNA molecule, causing serious problems. Skin tumour is the most common cancer and between skin cancers non-melanomas are the most diffused.

Basal and squamous cells carcinoma are not lethal and usually don’t spread in metastases; surgical intervention or local chemotherapy are successfully used to care these cancers. By contrast, melanoma is more dangerous and, in some cases, causes death of patients when is diagnosed too late and is already spread in other part of the body. Sunburns is the principal risk factor for all skin cancers that normally appear in region of body that are not often exposed to sun, for instance bell, and can slowly start melanine production and, so, the natural protective process; red or fair hair with light eyes are characteristics of people that more frequently is affected by these tumours. Prevention is essential to reduce risk, in particular it’s essential to limit sun exposure during childhood and, in general, during hottest hours of day from 10AM to 4 PM, always wear protective clothes or sunscreens that block UV rays.

Challenges in pluripotency

Pluripotency is a special feature of stem cells to renew and generate part of the body. Totipotency is characteristic of embryonic stem cells that can generate each organ or tissue, while pluripotency is more limited and only few tissue can be product by stem cells. Four factors seem to control pluripotency: Oct4, Klf4, Myc, Sox2.

All these genes are crucial during embryonal development and in some cases mutations could lead to serious diseases, such as cancer for Myc gene, or impaired development for Oct4 and Sox2. A recent work published on Nature journal demonstrated that, by using these four factors (previously used to reprogram fibroblast), it could be possible to generate human pluripotent stem cells from fetal, neonatal and adult primary cells. Human induced pluripotent cells showed similar features of embryonic stem cells in terms of morphology and gene expression and were able to form teratomas when injected in immuno-deficient mice. This work opens new perspectives in stem cells research: for instance in next future, it could be possible to culture primary cells directly form patients and induce pluripotent cells in order to repair tissue, make transplantation or care degenerative diseases without troubles of rejection.

Challenging on data management

Data management is a crucial aspect of scientific work, not only to correctly collect and retrieve information, but also to constantly have a complete vision of the work. In quality control system, data are classified by a code, defined by scientists and common for all laboratory members. For instance, files can be named with date, operator, project and kind of experiment: in this way it’s possible to identify one file without loosing time to check all folders and open every document.

Otherwise, it’s possible to use special software that is commercially available to manage your data. In lab research different kind of data can be generated: firstly raw data, such as numbers, pictures or texts (for example patient interview in clinical studies). Raw data have to be conserved, retrievable and in some cases protected by password, especially in clinical studies when sensible information about patient’s health are collected. These data can derive from several instruments that work with their specific program: they have to be converted in a unique format in order to be useful for elaboration. Raw data are usually resumed in graphs or tables that can immediately and communicate the result from complex experiments. At this point, it’s crucial to choose the best way to communicate results and valorize your work. Indeed, few graphs and table in scientific article have to present a long work made by several scientists in some cases. A correct data management system is useful during article writing to retrieve all important information and it’s cost- effective because avoid to loose time in searching raw or elaborated data or in repeating experiments those data are missing.

Not only in laboratory research, but also in clinical trial is important to correctly manage data for different reasons: firstly in clinical trials data often derive from patients and are subjected to privacy law, secondarily a really large amount of data are usually produced, especially during latest phases of trial when thousand patients are enrolled, lastly clinical trials are expensive and correctly managing data decreases costs. Data management software is feature-rich and includes planning, preparation and performance rather than raw data collector. Moreover, scientist contact information, deadlines, milestones and progress report have to be available to allow a complete overview of the project. The software is usually used in different centers around the world because clinical trials involve several institutions and must be user-friendly and verified from external intrusion.

In conclusion, data management is a challenging aspect of modern science for many reasons: data generated from research or clinical studies are a huge amount and often derive from different institutions that collaborate: this means that coordination between centres in data collection is essential; moreover, data are conserved and protected in respect of quality control standard and privacy law, respectively. Modern and efficient software is available now, and in next future it’s necessary to further customize IT products in order to make data management easier and less expensive.

A wonderful DNA-pHmeter

In our opinion nanotechnology represents one branch crucial for the future of scientific progress: in this context DNA-pHmeter is a wonderful invention. Cellular compartments are characterized by different pH, optimal for enzymatic activity; checking the pH in each cellular compartment could be useful to better understand reaction mechanism and so on.

Scientists from National Center for Biological Science in Bangalore, India, developed a DNA based device in which structural changes in DNA shape are induced in relation to proton concentration. In presence of low pH, cytosines tend to pair with other cytosines in an unusual base-pairing and lead to the formation of an intercalating four-stranded DNA. If fluorescent tags are added at the end of each strand, it could be possible to measure changes induced in DNA shape by pH through FRET –fluorescence resonance energy transfer-. This approach is innovative and simple to use and can be employed without specific instrumentation, rather than spectrofluorimeter. Furthermore, I-switch, this is the name of DNA-pHmeter, works in less than one minute and shape change is reversible. Several modifications could be introduced in DNA molecule to perform specific study, for instance ligands that could target I-switch in a specific cellular compartment. Good work!

Advances in In vivo imaging

In vivo imaging is a technique currently used in laboratory to follow, for instance, tumour growth as well as drug distribution through the body. Why is this approach so informative? Firstly, small animals, like mice, are live when are analysed, this represents a great advantage because you can observe phenomena in real time into the body. Secondarily instrumentation available now is efficient and sensitive, and application fields of in vivo imaging are continuously increasing. Two techniques are diffused in research laboratory: PET, positron emission tomography and optical imaging approaches.

PET is largely used in hospital and is based on detection of gamma radiation released from radioactive compound or reporters to track processes taking place in the body. By contrast, optical techniques rely on the deepen knowledge on bioluminescence and fluorescence. Fluorescent dyes, now in picomole amount, are quantitatively measured: looking at the absorption of different wavelength it’s possible to determine where light source is located. A challenging problem associated to optical imaging is the strong dependence of signal to depth: modern instruments try to solve this issue by working around light scattering. In conclusion, giving the advances in this approach, in vivo imaging is a promising techniques to be used not only in clinical study, but also in pre-clinical research.