Gone FISHing: new applications for an old technique

The use of fluorescent in situ hybridisation (FISH) has allowed for the advancement of molecular cytogenetics and kept research involving this technique at the cutting edge of science. However, FISH has a number of shortcomings, including a low resolution in its imaging, and restrictions in the sizes of probes that can be used. Extensions on this technique have therefore been developed in an attempt to improve upon the use of this process. An example of one such extension is the advent of comparative genomic hybridisation (CGH), a technique which utilises fluorescent probes to identify an increase or decrease in gene copy number that may be present within the genome in question. This development, along with a number of others, have potential to be powerful tools for several levels of investigation.

In a recent seminar given by Dr Miguel Pita, the use of fluorescent in situ hybridisation (FISH) was called into question. The primary focus of this talk was the application of fluorescent probes when using comparative genomic hybridisation (CGH). This is an important new technique within diagnostic medicine, with numerous other real world applications. CGH relies on the labelling of entire genomes to determine any amplification in gene copy number within an individual’s karyotype. This is a powerful tool, and has a number of future beneficial applications, which will be explored in more detail within this article.

Whilst completing my masters research project I was heavily reliant on the process of FISH to gain a greater understanding of the karyotype and any changes within the karyotype of Brassica napus (oilseed rape), as well as its parents Brassica oleracea and Brassica rapa. Whilst this was a crucial tool within this project, it was important for me to understand the limitations of this technique, which were highlighted within this seminar.

The birth of hybridisation techniques

The identification of tissues and the distribution of their chemical constituents was a key aspect of many early discoveries, and began by using various stains targeted (non-specifically) towards cellular structures. However, the lack in specificity of these stains limited their usefulness, with many moving away from these kind of techniques.

With no new major advances in the cytogenetic aspect of this field, molecular genetics became the main calling point for the majority of researchers. It took several decades for the advent of the fluorescent nucleic acid probes that we now commonly associate with molecular cytogenetics. However, probes of this kind were still incredibly primitive, relying heavily on radiolabelled probes, which came with the inherent drawbacks of using such chemicals. It therefore took a number of further advances before molecular cytogenetics was able to step into the limelight once again.

This began with the refinement of these in situ techniques, including a much greater increase in their sensitivity and resolution. Today, the majority of these cytogenetic approaches use fluorescently labelled sequences of DNA to detect areas with which they share homology, which is now commonly known as fluorescent in situ hybridisation (FISH)¹.

FISH is now used for a number of different purposes, including genetic counselling, within medicine and for general research purposes.

The procedure usually involved in the production of FISH probes

The process of FISH begins with the development of a probe. The sequence from which this probe is designed can be obtained in a number of ways, including PCR and micro-dissection of desired chromosomal sections. Once this sequence has been obtained, it is necessary to fluorescently label. These can be labelled either directly or indirectly, with the most common techniques used being nick translation or through the use of PCR utilising tagged nucleotides.

Once these sequences have been labelled, they are ready to be applied to chromosome preparations that have been suitably fixed upon a substrate. These spreads, and the effect that the labelled probes have upon them, can then be visualised and examined through the use of a fluorescent microscope¹.

The use of probes in this way has been an incredibly powerful tool within medicine and research, and has allowed for molecular cytogenetics to once again emerge as a leading investigative tool. The process of FISH is further explained in figure 1.

More examples of how FISH has been used

FISH is often used in medicine as a diagnostic tool. One such example of a disease that is used in this way is within downs syndrome. It is in this way that FISH probes are designed for sequences common only to chromosome 21. The detection of three fluorescent signals therefore confirms the presence of three copies of chromosome 21 (trisomy 21), therefore confirming a disease phenotype. This identification is not only limited to trisomy 21, with other trisomys also easily identified^{2, 3}.

FISH is also used to detect the presence of deletions that cause disease phenotypes such as prada-willi/angleman syndrome, as well as whole chromosome probes to detect the presence of translocations, including within certain forms of cancer^4,5.

However, whilst FISH is a very powerful tool, there is a still a limit on the resolution of this technique, with the size of the probe limited to above 100,000bp. There have therefore been a number of developments and expansions within this technique to extend the scope of its applications, either through increasing its resolution, or by applying this technique differently to which it was originally intended to extend its practical applications.

Expansions on the FISH technique

One such example where the scope of FISH has been extended by increasing the resolution that can be achieved from this kind of technique is illustrated by the use of tyramides. This works by adding proteins that are targeted for the required sequence (which can be much smaller than those required within standard FISH), followed by the addition of tyramides. These tyramides then hybridise around these small probes, thus amplifying the signals that can be produced. This process is illustrated further in figure 2.   Whilst this kind of hybridisation is much less accurate than the FISH technique, it does provide a more general idea of gene location, and at a much higher resolution. This kind of technique therefore has strong potential for preliminary diagnostics, as well as in paving the way for new methods of developing FISH with increasingly higher levels of resolution⁶.

Comparative genomic hybridisation (CGH)

Another example where the scope of FISH has been extended is with the development of comparative genomic hybridization (CGH). CGH is capable of revealing duplications and deletions within a patient genome when compared to a control genome. Within this technique, genomic DNA is extracted from the patient you wish to examine, followed be extraction of genomic DNA from a control patient. These two samples are then labelled with their own independently coloured label and mixed together. These two samples are then applied to a control metaphase spread (which is not derived from the patient that is being treated)⁷.

Visualisation in this way can see which coloured hybridisation is present in higher or lower quantities than others, as well as areas that are present in the same quantities (visualised by a mix of the two colours). This indicates any change in gene copy number variation within a patients, including whole chromosome arm translocations, smaller translocations, and duplications in much smaller numbers.

Whilst much recent research has been geared towards the use of bioinformatics in order to determine genetic abnormalities, this would be an unrealistic approach towards diagnosis in humans. This is mainly due to the fact that diagnosing in this way would require the sequencing of whole individual organisms, which would be time consuming and expensive. The use of CGH is a much easier and more powerful tool for initial visual identification of a disease phenotype, therefore reducing diagnosis times within patients and allow for much faster treatment; greatly benefitting suffers of a number of diagnosable treatments.

CGH is also emerging as a powerful tool for the genetic screening of embryos to detect abnormalities in their ploidy level. Changes in chromosome number lead to a large number of miscarriages, as well as conditions such as trisomy 21, causing downs syndrome, as mentioned previously. Prior to the advent of CGH, detection of chromosomal abnormalities was a much longer and more arduous process, therefore highlighting one particular area where the application of CGH has eased investigation and diagnosis⁸.

CGH also has applications beyond those involved purely in healthcare. Two examples that were explicitly mentioned within this seminar are as follows.

The first example involved comparing the karyotypes between the prave vole, which is monogamous in nature, and the meadow vole, which is non-monogamous in nature. In this case, CGH was used to determine whether chromosomes may have recently fused to try and provide some explanation behind this difference in behaviour. Being able to further understand this kind of genetic control could have important implications regarding the understanding of these animals as a species.

The second example given was also a comparison between two different animal species. In this case, C. parallelus erythroptic and C. parallelus. These two species exist in two distinct geographical areas, with the existence of a hybrid zone between the two species. The resultant progeny produced between these two species within the hybrid zone are infertile. It is thought this infertility is due to an increase in the number of bacteria within that can survive and thrive within these hybrids. CGH has therefore been employed in an attempt to determine any chromosomal insertions that may occur between these hybrids to determine why it is that bacteria can thrive better within this hybrid environment. This is not only important to further understand this species, but could also give us a greater understanding over the types of genes that are important to fight off bacterial infections, which could have human implications with further research.

The use of CGH is therefore an important step in advancing the field of cytogenetics. However, it is also crucially important that other techniques of this kind are also developed and enhanced upon in order to ensure that quick and easy diagnostic medicine can maintain its momentum, reduce the time where a patient is unsure of their diagnosis, and meet the needs of all those involved in the administering of treatment. The specific use of CGH and other more classical techniques in tandem also have optimistic applications in understanding oncogenic phenotypes, taking us one step closer to personalised treatment in response to this disease^9,10.

If techniques like this one, and others mentioned within this article, continue to develop and be expanded upon, cytogenetics can remain an important tool for a wide range of diagnostic purposes. It is important that cytogenetics is not left behind by the advent of more molecular based tools, as it is clear that cytogenetics has not given all that it is able to give.

What are your thoughts on this seminar topic? Have you learnt about a new technique that maybe you hadn't heard of before? Comment below with any questions and don't forget to +1 if you enjoyed this article. Thanks for reading!

References:

1.      Levsky, M.J. Singer, R.H. (2003) Fluorescence in situ hybridisation: past present and future. J Cell Sci. 116. P2833-2838.  

2.      Bishop, R. (2010) Applications of fluorescence in sity hybridisation (FISH) in detecting genetic aberrations of medical significance. Bioscience Horizons. 3 (1): p85-95.

3.      Pinkel, D. Landegent, J. Collins, C. Fuscoe, J. Segraves, E. Luxas, J. Gray, J. (1988) Fluorescence in situ hybridisation with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. PNAS. 85 (23) p9138-9142,

4.      Fox, J.L. Hsu, P.H. Legator, M.S, Morrison, L.E, Seelig, S.A. (1995) Fluorescence in situ hybridisation: powerful molecular tool for cancer prognosis. Clin Chem. 41 (11): p1544-9.

5.      Kearney, L. Shipley, J. (2012) Fluorescence in situ hybridisation for cancer-related studies. Methods Mol Biol. 878: 149-74.

6.      Van Gijlswijk, R.P. Wiegant, J. Raap, A.K. Tanke, H.J. (1996) Improved localization of fluorescent tyramids for fluorescent in situ hybridisation using dextran sulphate and polyvinyl alcohol. J Histochem Cytochem. 44 (4) p389-92.

7.      Pinkel, D. Albertson, D.G. (2005) Comparative genomic hybridisation. Genomics and Human Genetics. 6. P331-354.

8.      Frenny, S. Harsh, S. Kiumarri, P. Stuti, T. Manisha, D. Patel, B. Jayesh, S. (2014) 1 (1) p 3-9.

9.      Cremer, T. Cremer, C. Lichter, P. (2014) Recollections of a scientific journey published in human genetics: from chromosome territories to interphase cytogenetics and comparative genome hybridisation. Hum Genet. 133: 403-416.

10.  Lall, M. Saviour, P. Puri, R. Paliwal, P. Mahajan, S. Verma, I. (2014) Molecular cytogenetics. 7: p9-13.

 

U1 isn't just interested in pre-mRNAs cleavage.

The spliceosome is crucially important for the proper processing of pre-mRNA into fully functioning mature mRNA. The spliceosome is a structure composed of proteins and a number of snRNPs. However, the concentrations of each snRNP are not the same, with a drastically higher level of the U1 snRNP present within cells. This paper therefore investigated the potential role of this snRNP, and found that its role in splicing is not its sole function, with evidence suggesting that it is also involved in the protection of pre-mRNA, thus preventing its premature processing. This research could have implications on the human disease spinal muscular atrophy, which is known to be caused by changing concentrations of snRNPs.

Ribonucleic acid (RNA) is a class of biological molecules which are crucially important for a vast number of functions, including the proper regulation and expression of genes. RNA is further divided into further categories, one of which includes messenger RNA (mRNA), which is used in cellular organisms to convey genetic information and allow the synthesis of the specific proteins required for an organism’s survival. However, prior to the production of mature mRNA, primary transcripts (pre-mRNAs) must undergo post-transcriptional processing such as splicing, which is carried out by the spliceosome.

The spliceosome is predominantly assembled from snRNPs as well as a number of other protein complexes, and functions by removing introns from the pre-mRNA transcript. The U2 spliceosome is composed of the U1, U2, U4, U6 and U5 snRNPs. It is these snRNPs which recognize the sequences which define the classes of introns, giving them a crucial process during splicing reactions. 

Whilst snRNPs are crucially important for the proper assembly of the spliceosome, each snRNP is not present in equal concentrations. This is highlighted in the case of the U1 snRNP in which there are thought to be 10 million molecules within each human cell, a much higher concentration than is observed in other snRNPs. 

The full implications on cellular functions that changes in the level of snRNPs could have is not currently known. However, previous research has found that perturbing the biogenesis of snRNPs induces a high level of splicing abnormalities. A greater understanding of the role of snRNP abundance is important, with implications in a number of human diseases, including spinal muscular atrophy which is caused by a reduction in the level of functional SMN protein. 

However, there is often difficultly in researching the varying levels of snRNPs and the effect that they may have, due to the high disparity that occurs between different cells depending on their function. Recent research by Kaida et al (2010) therefore wished to further expand knowledge in this area though investigating the effect of a reduction in the levels of snRNPs individually. This was tested through the use of antisense morpholino oligonucleotide (AMO) technology, which uses sequence of oligonucleotide designed to be complimentary to specific sequences which are known to be transcribed into mRNA, which stops it from performing the cellular role it is normally associated with.

AMO was used to mask the 5’ end of the U1 snRNA, which is the region associated with the identification of splicing sites, thus preventing its normal function. The action of AMO was seen to be dose dependent, with a concentration of 7.5µM U1 AMO finally used, as this level deemed the 5’ site completely inaccessible. Treatment with the general splicing inhibitor SSA was also used as a reference to understand the U1 snRNPs full effect. 

The results from this treatment suggested a role for U1 snRNP unrelated to its normal role in splicing. It was found that, when the U1 snRNP function was knocked out a much higher concentration of prematurely cleaved and polyadenylated pre-mRNAs could be observed. This suggests that this snRNP is capable of protecting pre-mRNA transcripts from premature cleavage and polyadenylation (PCPA). Whilst the mechanism through which this type of protection is provided is not currently known, several models have been proposed (explained in fig 1). One method suggests it is believed that U1 prevents this activity through masking sites with potential polyadenylation signals (PAS), making them unavailable for their function of pre-mRNA processing. This dual action could also go some way to explaining why such high levels can be observed within cells, owing to the fact that they may be needed in separate locations along a genome simultaneously.

However, what can be conferred with a high level of certainty is that U1 must be used to modulate the level of gene expression within a cell, by defining the numbers of pre-mRNA transcripts which can reach maturity and affect the protein production of the cell.

It may therefore be an important step in the future research of this work to fully understand the mechanism through which U1 confers protection, therefore potentially allowing us to artificially control its function both in normal patients, as well as those suffering from diseases where gene expression induces a negative phenotype.

Do 40S and 60S come together sooner than we thought?

The ribosomal subunits 40S and 60S are produced and assembled whilst they are still within the nucleus. However, there seem to be several different levels of control which prevent their formation into a fully functioning 80S ribosome, which is only relieved once they are exported into the cytoplasm. This has often led researchers to believe that 80S ribosomes can be found only within the cytoplasm. However, recent research has brought this theory into question. 

The synthesis of proteins is required for the viability of all living cells, with the code for these proteins contained within chromosomes as a sequence of bases on DNA. For this sequence to be developed into functioning proteins, a number of complex processes must first take place, chief amongst which is the transcription of DNA into messenger RNA (mRNA). This mRNA can then be used to assemble simple amino acids into complex proteinacious structures. However, the process of transcription is not possible without the large molecular machine called the ribosome, which acts as the primary site of protein synthesis.

During the production of a ribosome, the subunits 40S and 60S must join together to form a fully mature and transcribing ribosome (80S), the formation of which acts as a main indicator that transcription is occurring within a cell.  Much previous research has suggested that the 60S and 40S subunits are synthesised in the nucleus through various complex mechanisms, with the 40S and 60S subunits being incapable of associating with mRNA, preventing their proper functioning until they are exported into the cytoplasm. Once in the cytoplasm they are able to form the fully functioning 80S ribosome, which is then capable of transcribing DNA and producing functionally mature mRNA .

There are several levels of control which are capable of controlling each of these components. Studies in saccharomyces cerevisiae indicate that this level of repression can be controlled by nonribosomal assembly factors (AFs) which bind to pre-40S and pre-60S subunits preventing their activation and assembly into the 80S ribosome until they are exported into the cytoplasm. In addition to this, it is also believed that other proteins are crucial for the translocation of these ribosomal subunits through the nuclear pore, the control of which may also be involved in preventing the assembly of 80S before they are required. There are also several lines of evidence which suggest that the 40S ribosome is not processed properly whilst it still resides within the nucleus, further preventing it from forming a mature ribosome, unless exported into the cytoplasm.

However, with a recent study indicating that immature 40S subunits can actually initiate translation whilst still residing in the nucleus, as well as numerous other studies confirming this fact, it is now clear that 40S is capable of interacting with 60S and forming an 80S-like structure, which is able to produce a low level of inefficient translation.

With this information in mind it was the goal of a recent paper by Al-Jubran et al (2013) to fully determine the cellular location of the functional 80S ribosome within Drosophila. To do this a number of ribosomal proteins (RPs) were identified, with their molecular position determined both when in their 40 and 60S subunits, as well as when contributing to the mature 80S structure. If it was found that these RPs are situated closely to each other in the 80S ribosome, then they were tagged in such a way as to make them fluoresce when they are within close proximity to each other. This would give a clear indication of exactly where 80S ribosomes were situated within a cell.

The results from this visualisation indicated that the majority of ribosomes were in fact located primarily in the cytoplasm of the cell. However, there were also a number of signals which suggest that a properly functioning ribosome can be found within the nucleus, with higher levels of intensity found at the nuclear periphery and the nucleolus.

However, these results had to be further confirmed to ensure their authenticity. One such confirmation was gained through treatment with puromycin. Usually, when a cell is treated with puromycin the 80S ribosomes become inactive and non-translating. When these non-translating ribosomes were tagged in the same way, no signal was produced. This indicates that the 80S ribosomes that were visualised without the puromycin treatment must have been actively transcribing, giving further weight to the idea that transcribing ribosomes can be found within the nucleus.

Therefore, this research confirms findings by previous research which indicates the presence of 80S ribosomes within the nucleus, as well as cementing a strong technique that is capable of visualising these ribosomes, in a simple and clear cut way. This research could be further developed through quantifying the level of transcription that is being produced by these nuclear 80S ribosomes. This would make it much clearer whether the transcription of mRNA before cytoplasmic export is essential for the proper functioning of the cell, or whether this process simply occurs at this stage to kick start transcription of all genes. 

The role of centromeres in the bouquet formation of Tetrahymena thermophilia.

Bouquet formation, homologous pairing and crossing over in early meiosis are all processes that are strongly dependent on the centromere.  

When meiosis is initiated one of the first structures that is seen to form in almost all organisms is the chromosome bouquet. The chromosome bouquet is an arrangement where telomeres bunch together in a confined area of the nuclear periphery with centromeres at a polar position to them, which as its name suggests resembles a bouquet of flowers. This structure allows for the the pairing of homolgous chromosomes within the cell. 

Tetrahymena thermophilia is a unicellular ciliated protist who’s micronuclei elongate and stretch dramatically during its meiotic prophase, which is the point at which Tetrahymenas exaggerated bouquet forms. Loidl, Lukaszewicz, Howard-Till and Koestler at the University of Vienna have released a paper that may help to explain what mechanisms are taking place during this process in the unicellular protist. 

This paper investigates the importance of Double stranded breaks (DSB’s) and centromere function in Tetrahymena’s bouquet formation, suggesting that centromeres have essential functions in recombination and chromosome pairing.

To begin their investigation Loidl et al attempted to understand the function of centromeres during Tetrahymenas nuclear elongation. They did this by constructing strains where the H3 histone Cna1p, was disabled through RNAi depletion. Under wild type conditions where telomeres and centromeres segregate during the formation of the bouquet, centromeres cluster at the periphery of the nuclei.  However with this RNAi mutant immunostaining detected only background staining, with no clear organisation of centromeres.

This paper also discusses how the bouquet arrangement of centromeres and telomeres at opposite poles of the nucleus is highly dependent on in the interaction of microtubules with the kinetochore. It was found that whilst microtubule interaction is the main contributor to nuclear elongation and that centromeres play no role in the elongation of the cell. Microtubules have two known functions in Tetrahymena; to elongate the nucleus and to hold the centromeres at a fixed position of the nucleus. 

It was already known that DSB’s were needed for the bouquet to form, as it is an ATR-dependent response. ATR being an enzyme that is activated in the persistent presence of single stranded DNA, which is a common intermediate for most DNA damage repair pathways.  Loidl et al set out to confirm that the bouquet was actually necessary for DSB repair by adding nocodazole- a microtubule inhibitor – to prevent the formation of the bouquet within the Tetrahymenas nuclei. 

As an additional measure DSB-formation and repair were monitored using pulsed-field electrophoresis in both the control and the nocodazole treated cells. Both of these experiments showed that DSB’s were repaired independently of bouquet formation. Therefore this proves that whilst DBS’s are need for the initial formation of the bouquet, the bouquet itself is not needed for the repair of those DSB’s.

To try and further understand the role of DSB’s and how they regulate bouquet formation Loidl et al created a scenario where DSBs were continuously produced. To do this they treated meiotic phase cells with cisplatin – an inducer of DSBs.

Cells treated with cisplatin were no longer able to exit the bouquet stage, suggesting that the trigger for the cells release from the bouquet stage must be an intermediate stage in the DNA repair.

The bouquet structure is highly conserved amongst a vast number of different species, but it’s function varies

slightly from organism to organism. For example in Arabidopsis, like with Tetrahymena, telomeres are linked with the nuclear periphery. However, in contrast to Tetrahymena the centromeres do not cluster at a single point at the opposite pole to the telomeres, but are more dispersed randomly across the nucleus with no evidence to suggest that they are involved in homologous chromosome pairing or recombination. Instead chromosome pairing occurs in zygotene, when a structure loosely similar to the bouquet is formed.  In mammals and budding yeast chromosome pairing has been proved to be led by telomeres and dependent on the protein SUN1 which anchors the telomere to the nuclear membrane. A similar protein of which could not be found by Loidl et al for Tetrahymena.

The next step in the investigation of Tetrahymenas exaggerated bouquet is a more in depth look at the function of telomeres and telomere associated proteins. By doing this there will be either confirmation of the views that this paper has put forward or give us a better insight into the function of telomeres allowing us to appreciate their involvement in the bouquet forming process.

By understanding Tetrahymenas bouquet completely and fully we will be able to apply this knowledge to other organisms to help us determine the processes that govern their bouquet formation. This would be especially important is we could apply this new knowledge to ourselves and the processes that go on in our cells during meiosis. This would give us a much clearer insight into how diseases and disorders may form, and therefore give us clues on how to prevent these diseases. 

Pretty soon our food is going to run out – but what can we do about it?

The human population of the earth has been increasing exponentially over the past few hundred years, a trend which is showing no sign of stopping. But with all these extra mouths to feed, do we have the resources to actually keep feeding them?

One of the biggest problems that the human race faces at the moment is the rapid development of global poverty and global hunger, issues which will only be worsened if the population increases to nine billion, as predicted.  Whilst these are two separate problems, they can be simultaneously resolved in a long term way through the promotion of agricultural growth in areas where poverty is particularly rife. However, communities which experience particularly harsh levels of poverty usually occur in areas whose land is incapable of sustaining a large number of crops. These areas therefore require new types of crop species which can grow with a much lower level of nourishment, whilst still producing the same yield of food.

Therefore, research geared towards producing these new varieties of plants is of critical importance. The main method which could be used to produce these variants is through increasing a plants level of genetic recombination.

Recombination is a molecular process which occurs within the cells of plants during meiosis, a specialised round of cellular division which produces gametes, cells with half the usual number of chromosomes (haploids). During this time, chromosomes which are genetically very similar to each other called homologues pair together and form cytological structures called chiasmata. When these chromosomes then resolve during a stage called anaphase, the resultant chromosomes often contain pieces of genetic information from each of the chromosomes which originally pair (recombinants). This introduces a level of genetic variation within a population, which is often the driving force behind evolution, and the adaptation to different environmental influences.

This process occurs in all sexually reproducing animals. However, in certain plant species recombination is kept under incredibly strict control in an attempt to ensure the stability of their genome. Whilst this is a positive outcome for these plants in their natural environment, when attempting to produce variants with more resilient phenotypes this produces a difficult obstacle. It is therefore the aim of many researchers to further understand the mechanisms which govern recombination, as well as any techniques which could be adapted to try and artificially induce much higher levels of recombination.

It is this type of research which I am currently involved in whilst completing my masters at The University of Birmingham, UK. During my time working in this lab I will be attempting to determine whether okadaic acid, a phophase 2A inhibitor, is capable of inducing a much higher level of recombination in Brassica napus between chromosomes which normally don’t recombine at all.

This type of research is much different to previous research which has produced genetically modified (GM) crops, which usually involves placing foreign genes into an organism which would never have been present in the wild. This type of technique is often poorly favoured by the public at large, with many suggesting that the repercussions of manipulating nature in this way could never be fully understood.

However, the research that I am involved in is interested simply in inducing the expression of genes that were are already present within the genome, but were never allowed to surface and influence the phenotype. This is a much more environmentally safe method of producing high yielding plants, and one which would be a globally accepted resolution to current issues around poverty and food security.

Could this type of research be the answer to some of the big questions that we are going to have to face in the near future? Whilst it certainly has potential, we are still a long way away from being able to completely know the truth. But don’t worry, I’ll keep you up to date if I ever find out the answer to my tiny scope of research, and let’s just hope that the hundreds of other labs around the world do the hard work for us.

What do you think about this research? Comment below with any thoughts or questions. 

Volunteers required for whole genome sequencing – Will you participate?

Today the UK has launched a personal genome project, urging 100,000 people to contribute their genetic information and have their genome sequenced to be put on public record. Is this a big step in the advancement of sequencing huge sample sizes? Or is making our entire genome available  to anyone online a step too far?

Our DNA is what makes us who we are; a sequence of bases in each and every one of our cells which contains the code to making what we see in the mirror every morning. It is possible for someone to look at your DNA and determine almost everything about us; the colour of our hair, our eyes or any genetic diseases we may have or be prone to, without ever having actually laid their eyes on us.

Whilst it would have previously been near impossible for many people to gain access to our DNA, a new initiative has been set in place by The Personal Genome Project UK (PGP-UK) urging 100,000 volunteers to donate their genetic information for their genome to be sequenced. This has already been undertaken by other countries, including America in 2005. However, this is the first time a project like this has been attempted in the UK, and many sceptics have their doubts.

The aim of those conducting this project is to accelerate researchers understanding of genes, both normal and defective, as well as how different environmental influences can affect those genes. However, the majority of companies sponsoring this research (one of which being google) are hoping to use this data for commercial exploitation, to specifically target advertising for drugs that each of us may need, based on our genetic code.

This is one reason why this project in controversial, but many also have issues with the broad spectrum of individuals who will be able to view your genetic code. Whilst names and addresses will not be included on record, the research group involved has warned that the security of participants is not guaranteed, and they could potentially be identified.

Because of this, a number of tests are included in the application of those who are applying to be involved, in which only a score of 100% will be accepted, which is designed to ensure that all those involved fully understand the potential risks.

If you were decide to join the project, and then be accepted, you will receive a kit to take cheek swabs, and also asked to attend a clinic to provide more extensive samples, with your genetic information published within a month.

It is hoped that this sort of extensive record of so many peoples genomes will allow for a number of major diseases, which have a large effect on public health, to be linked to genes which have previously been unidentified. This would be crucial information for those researching therapies for these diseases, and could dramatically advance our understanding of them.

Do you think you’d be interested in taking part in this project? Or do you think that having this kind of information about yourself on public record is a step too far? I for one know I’m definitely going to be signing up. 

Money doesn't grow on trees - or does it?

A recent paper published in nature communications has given evidence that Eucalyptus trees may be capable of absorbing deposits of gold from within the earth. At a time when new gold discoveries have fallen dramatically, could this be an answer to finding them?

New discoveries of gold have fallen by 45% in the last 10 years. A large amount of gold may be found deeper within the earth beneath sediments, but locating these deposits has proven incredibly difficult. One new technique that is currently in development is called biogeochemistry, which is essentially the use of biological systems (for example plants) to determine which minerals are present in the soil they grow in, through observing the concentrations of minerals which can be found in the plants themselves.

However, there are a number of problems with using this type of data, chief amongst which is the fact that gold concentrations within plants is usually incredibly low, with no unequivocal evidence suggesting that the concentration of gold in plants has any correlation to the amount of gold in the soil in which they grow. It was therefore the aim of this research to provide more solid evidence for this theory.

To do this, researchers observed the activity of Eucalyptus trees which were known to be growing above a gold deposit, buried beneath a thick layer of other sedimentary minerals.

Whilst it has previously been shown that gold particles are present around the soil of Eucalyptus trees, this research, with the use of the Australian synchrotron (a machine which uses X-rays to view matter in vivid detail) was able to provide evidence for the presence of tiny amounts of gold in the leaves, twigs and bark too, proving that the trees were actually absorbing this material through their roots buried deep beneath the earth.

This could be an important discovery in the mining of precious minerals, and even those that aren’t so precious. Normally, to find a deposit of ore, extensive exploratory mining would have to take place which would usually result in a dead end. This is both expensive and invasive to the environment that sits on top of the ore. However, if this new method of detection could be developed more extensively, all that would be required to locate what we were looking for would be a sample of leaves or twigs from the vegetation in the area. These samples would then be able to tell us exactly what was within the soil, and whether more extensive mining should take place.

What do you think about this new discovery? Can you imagine being able to pop into your back garden one day and being able to tell exactly what was in the soil by looking at just one leaf?

Comment below with your thoughts and questions and don’t forget to +1 and reshare if you enjoyed this article. 

Is a tumours micro-environment a source of innate resistance to anticancer drugs?

The RAF-MEK-ERK pathway is often mutated in a number of cancers, causing signals for cell proliferation and survival to be relentlessly activated. Various inhibitors of certain components in this pathway have been developed, but with little efficiency. It is believed that the micro-environment of a tumour can confer a level of resistance to some cancer therapies through the secretion of HGF, a growth factor produced by stromal cells.

Each new development of a treatment against cancer is met with difficulties. This study by Straussman et al focuses on the RAF-MEK-ERK pathway. This is a pathway through which extracellular signals are transduced into intracellular signals, through interaction with extracellular receptors. These signals cause the expression of transcription factors which regulate the synthesis of genes required for cell survival and proliferation, key genes when considering the formation of a cancer.

Previous research has targeted the RAS protein, a component of the RAF-MEK-ERK pathway, with unsuccessful results. This has led to research directed at the kinases downstream from RAS. It is one of these downstream kinases investigated by Straussman et al, in the form of RAF and its potential inhibitors.

RAF inhibitors (RAFis) work by interfering with the RAF protein in the RAF-MEK-ERK pathway, preventing this pathway from transducing the signals for increased proliferation. It has previously been seen that inhibiting the mutated RAF reduces cancerous growth. However, these types of responses are almost always followed by a re-emergence of that tumour, brought on through the formation of resistance. Here it is suggested that the tumour microenvironment may be conferring that resistance through the secretion of soluble factors.

Whilst the role of the microenvironment in growth and metastasis is well documented, only recent research has suggested its function in drug resistance. In order to test the microenvironments role in tumour drug resistance, Straussman et al began by developing a co-culture system. In this co-culture system, GFP–labelled tumour cells were cultured alongside stromal cells to assess modulation of drug sensitivity. This was quantified by measuring how levels of GFP changed over a set period of time. This test resulted in the observation that, when this co-culture system was exposed to RAFis, those RAFis were frequently rendered ineffective when cultured alongside stromal cells.

Strausman et al then investigated the effect of one RAFi in particular (PLX4720). To do this they tested the ability of stromal cell lines to provide 7 mutant BRAF (V600E) melanoma cell lines with resistance to the anticancer drug. This resulted in six out of the seven developing resistance to PLX4720.

It was therefore concluded that stromal cells can render certain anticancer drugs ineffective.  Straussman et al confirmed that soluble factors secreted from stromal cells were responsible for the formation of resistant tumour cells. This conformation was important, as it allowed Straussman et al to identify the exact resistance causing factor. To do this they conducted an antibody-array based analysis of a large number of secreted factors. This allowed them to compare the conditioned medium obtained from the previous 6 stromal cell lines that developed resistance to PLX4720, with stromal cell lines that had not exhibited any sign of rescue activity.

From this, HGF, a growth factor that plays a role in activating the receptor tyrosine kinase MET, was identified as the source of rescue. HGF is capable of restarting this pathway through activating MEK, bypassing the RAF component of this pathway.  Straussman et al then confirmed the presence of HGF in a number of patients being treated with a RAFi, as well as confirming the phosphorylation, and therefore activation of MET (See Figure).

In these studies it is also predicted that the presence of stromal HGF in patients is a form of innate resistance, with patients capable of producing HGF showing a much poorer response to treatment than those unable to produce it.

However, further evidence was required to fully confirm that the presence of HGF was the cause of resistance. To collect this evidence, Strausmann et al tested whether recombinant HGF was capable of inducing resistance upon tumour cells, whilst simultaneously testing whether HGF-neutralising antibodies blocked resistance to PLX4720. This confirmed that HGF was indeed capable of producing the resistant phenotype.

Could these results have a clinical impact on the treatment of cancer?

These results are important clinically in defining why certain cancer treatments aren’t always effective, as well as identifying where research should be taken to combat this resistance. This paper can be compared to those investigating sorafenib, a molecular inhibitor of a number of protein kinases which has been approved in the treatment of kidney and liver cancer.

Future developments in this field should focus on whether the formation of resistance can be blocked through inhibition of HGF, as well as identifying a time scale on how long stromal cells take to confer resistance to this treatment. If this time scale can be determined, a treatment could be developed which involves combining therapies at specific times to amplify their effectiveness. It may also be important to investigate further whether other RAFis are deemed ineffective by HGF, which could make generating second generation inhibitors important. However, it may be more prudent to investigate whether the activation of MEK, ERK or MET can be inhibited. This would have the same effect, but because inhibition would be taking place further along the pathway, there is less likelihood that resistance will form.

As mentioned by the authors, further research should also take place into investigating whether this type of resistance has a role against other anti-cancer drugs, as this may give us crucial information in combating against them.

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