The MSAT Scientific Advisory Panel met in February 2020 to discuss applications for research grants and 4 projects were accepted for funding. We are indebted to our lay advisor on the Panel, John Telford who has kindly shared the summaries on each of the grants.

John has also written a summary blog that helps to explain how each of the research projects link together to support the continued growth and development of our understanding about Multiple System Atrophy (MSA). You can read the blog series here.

Project 1

Don’t ‘Diss’ the Dustman …

This theme keeps recurring: that the key to MSA – and to Parkinson’s – could be incompetent rubbish collection. Getting to the bottom of this – at the cellular level – is what this project is all about.

Dr Maria Xilouri of the Biomedical Research Foundation of the Academy of Athens is the lead researcher for this project. She has been working for many years on the role of aberrant forms of the protein alpha-synuclein in neurological conditions such as MSA and Parkinson’s.

Background to the research – taking out the rubbish

These ‘bad’ forms of alpha-synuclein are part of the ‘rubbish’ we are talking about. The functions of proteins, which are long molecules made up of amino acids, depend on how they are folded. Sometimes they become misfolded and in this state can be toxic. Clearly this toxic rubbish needs to be removed.

MSA and Parkinson’s result from certain cells in the brain dying off. In Parkinson’s it is nerve cells that are directly targeted whereas in MSA the cells that are affected are those cells – called glial cells – which nourish and physically support the nerve cells. And it is alpha-synuclein, misfolded in different ways, which apparently is the culprit. In both diseases deposits of molecules of alpha synuclein in aggregated form are found in brain cells and it is thought that these are the result of the cells trying but failing to clear up and dispose of these misfolded proteins. It is like putting your rubbish into black bags but not having the dustman come round to collect them.

One of the main rubbish disposal systems in the cells of our body is known as the Autophagy-lysosome pathway (or ALP for short). For some reason we put the accent on the ‘o’ in the first word and the ‘y’ in the second: AutOphagy, LYsosome. When the system is behaving normally, any bits of rubbish in a cell, such as old proteins that have served their purpose or fragments of other material that are the result of cellular processes or have come in from outside, are packaged into microscopic containers called lysosomes. This rubbish is then worked on by enzymes inside the lysosomes to break them down into smaller molecules that can be more easily and safely recycled.

Two broad aims

The first broad aim of this project is to understand better the details of what is going on in the case of MSA. It does appear that the ALP is not working properly. But why not? Early results suggest that misfolded alpha-synuclein (along with another rogue protein named p25alpha) interferes with autophagy if it can get a toe-hold. So it looks like there is a vicious circle operating: The ALP is meant to clear away these rogue proteins. But, once these proteins are present, they can compromise an ALP that is already functioning below par. But is it a pre-existing vulnerability of the ALP or the ingress of a particular strain of misfolded protein that kicks it all off? Or does it need both?

The second broad aim is to see whether the ALP can be given a helping hand to restore the rubbish-collection function to such an extent that the alphasynuclein and p25alpha are effectively removed and the glial cells remain healthy and can continue to play their vital role.

Tools and techniques – the nitty gritty

To achieve these objectives the team will use a selection of genetically engineered and non- non-engineered mice and examine their brain cells for the levels of expression of a whole range of sub-cellular entities and substances that are involved in the ALP. In some cases mice genetically engineered to produce aberrant alphasynuclein in their brain cells will be used and in other cases non-engineered mice will be treated with alphasynuclein fibrils which are able to stimulate the production of ‘bad’ alphasunuclein in brain cells. It is too complicated to describe here but the investigations will involve measuring the relative levels of the different substances produced in the brains of mice which have been bred, or have been induced, to have conditions resembling MSA and Parkinson’s. Correlations between levels of aberrant alphasynuclein and p25alpha on the one hand and substances involved in the Autophagy Lysosome Pathway on the other will be looked for in order to puzzle out the precise disease mechanism. A good understanding of disease mechanism can give pointers to where interventions may be effectively made.

A comprehensive biochemical analysis of post-mortem brains will also be done to corroborate the findings in real human cases.

They will then go on to gain further insights by seeing what happens when the functioning of the ALP is deliberately depressed.

To achieve the second broad aim, the ALP will be activated and enhanced to see how well this rescues the brain cells – the nerve cells and glial cells – in the mice which have been induced or engineered to have MSA-like and Parkinson’s-like conditions. There are a number of well-tried methods for experimentally boosting the ALP. This will be done with cell cultures of both neurons and glial cells. Again the details of these investigations are too complex to describe here suffice it to say that laboratories like that of Maria Xilouri are wonderfully equipped to carry out the observations, measurements and assays that are required.

The project in context

The Scientific Advisory Panel was impressed by the design of this project and the proven competence and experience of the team proposing it. This is not a stand-alone project but taps into the years of research from around the world that have yielded the current state of knowledge and understanding. Research tools, techniques and resources – such as the genetically engineered mouse models – have painstakingly been developed so that an enormously sophisticated platform is in place from which to make further advances in this important field.

If you want to learn more about autophagy and lysosomes you could start with this Wikipedia article: https://en.wikipedia.org/wiki/Autophagy . You will see that it is a bit more complex than the above account!

Project 2

Disrupted genetic instructions …

The lead researcher for this project is Dr Conceição Bettencourt who is a Postdoctoral Research Associate at the UCL Institute of Neurology working with Prof Janice Holton’s lab. She has been working in the field of DNA methylation for several years because it has been found that this process is significantly and deleteriously affected in MSA. What is going on? And could it be put right?

So what is DNA methylation and why is it important?

DNA – which is organised into 46 chromosomes – is the instruction manual for making protein molecules which are the agents for making a cell do what it is supposed to do. The DNA is identical in every cell in the body so theoretically every cell can produce all 20,000 plus proteins. But clearly a further set of instructions is needed so that only the appropriate genes for a particular type of cell – a heart cell or a nerve cell, say – get switched on to make just the right set of proteins for that context. A major way in which this is done is through methylation. This is the attachment of the biochemical methyl group to appropriate places along the DNA chain, determining which genes are active and to what extent.

In MSA and other diseases the pattern of methylation becomes changed so that some proteins are produced when they shouldn’t be, or they are produced in the wrong amounts or not produced at all when they should be.

Why is this important for MSA?

Much more needs to be known about the molecular changes involved in MSA pathology – the details of what happens, what precedes what and what causes what. Changes in DNA methylation mirror the pathological chain of events. In some cases a methylation change could be the cause of other events and in other cases the consequence. There are also differences in these chains of events between different but related diseases, for instance between MSA, Parkinson’s and PSP. There are also methylation differences between the different areas of the brain that are affected in MSA. MSA pathology is highly complex and needs further teasing out. Studying methylation differences has already yielded a lot of preliminary information and this project is aimed at making important advances from here.

A fuller understanding of the pathological pathways are likely to beneficial in two main ways. First, discerning different patterns for methylation in different diseases – and in the different types of MSA – could provide a more accurate way of diagnosing that someone had MSA rather than Parkinson’s, for instance. i.e. It could provide a so-called diagnostic biomarker.

Second, and more importantly for the MSA patient, a good understanding of the disease pathology could point to targets for drug intervention for treating the disease, slowing its progress or reducing its symptoms. Because methylation is a reversible process, harmful protein imbalances caused by it maybe could be corrected. Also it could be that faulty methylation is what makes certain brain cells more vulnerable to the build-up of aberrant forms of the protein alpha-synuclein – the condition that is characteristic of both MSA and Parkinson’s. Correcting the faulty methylation could provide a defence against further alpha-synuclein build-up.

How does the team intend to explore methylation in practical terms?

The project is based on analysing post-mortem brain tissue from samples held in the Queen Square Brain Bank. Much of the sampling has already been done and a mass of data has been accumulated which the project will analyse alongside new data. The patterns or signatures of methylation encompassing thousands of genetic sites will be determined and comparisons will be made between the samples from MSA, Parkinson’s and PSP patients and non-diseased persons. This will also take account of the severity and stage of disease reached so that a detailed picture of the progression of the changes can be built up.

The members of the team and their collaborators cover a huge range of expertise including epigenetics, transcriptomics, bioinformatics, neuropathology, biochemistry and neurology making them well-placed to successfully deliver the objectives of the project.

Project 3

Tampered Genetic Code

The Lead Researcher is Dr Christos Proukakis of UCL who works at the Queen Square Institute of Neurology where the Brain Bank is housed. Dr Proukakis has worked for many years on somatic mutations in Parkinson’s and other neurological diseases which puts him in a very good position to explore it further in MSA.

Every cell in the body of an individual ultimately comes from the single cell produced at conception. So they all contain identical copies of the ‘original’ DNA don’t they? Well, no. Sometimes a genetic mutation occurs in an individual cell, or in a group of individual cells during a person’s lifetime. This is often the case in cancer. The mutation can then cause the cells to misbehave. It as if a hacker has got in and tampered with the instructions that the cells use to carry out their function. Such mutations are called ‘somatic’.

There is now a lot of evidence that somatic mutations can occur in brain cells. The problem is that if a mutation is confined to the brain, it is not detectable in parts of the body outside the brain. This is why the research will involve looking in detail at post-mortem samples held in the Brain Bank to explore different parts of healthy and MSA-affected brain. This is the third project which highlights the importance of donations to the Brain Bank.

Copy number variation

In earlier work it has been discovered that indeed a few cells in MSA-affected brain do have a particular somatic mutation which leads to extra copies of a gene for the protein alpha-synuclein. This is significant because of a parallel situation in Parkinson’s; in an inherited form more alpha-synuclein than normal is produced and this in turn tips certain brain cells into being susceptible to Parkinson’s. MSA is similar to Parkinson’s in that misbehaving alpha-synuclein is implicated although in different cell types.

The project is aimed at firming up the hypothesis that this so-called copy number variation mutation, when it is present in MSA brain, occurs in the same places where the cells are most affected by the MSA pathology. This would confirm a causal link. In MSA these places are the various regions where oligodendrocytes (the cells that support and nourish the nerve cells) contain inclusions of aggregated alpha-synuclein. The project will focus on four main brain regions and look for differences in relation to the two main MSA types.

The investigations will go beyond broad correlation, looking at:

(a) how the degree of copy number variation affects age of on-set,

(b) how much it occurs not only in oligodendrocytes but also in affected neurons and in the precursors of the oligodendrocytes as well, and

(c) whether an alpha-synuclein inclusion in a cell is always or generally associated with a copy number variation mutation in the same cell – which would indicate a causative relationship.

It is hard to answer in a satisfying way how much closer this project will bring us to a cure. But it is clear that if, as suspected, it allows us to see what precisely is initiating the pathology, it will enable the precise targeting of effective interventions to treat it.

Project 4

PROSPECT-M Study

MSA is a difficult disease. It affects areas – several of them – which are deep inside the brain and can hardly be examined. Signs and symptoms are similar, in the early stages, to other diseases and it is comparatively rare. Getting adequate information about it is difficult. The PROSPECT study was set up to address this.

The basic idea is to collect biological samples, brain scans and other objective information and clinical assessments from as many patients as possible who are believed to have MSA. All this material and information is stored permanently to provide a solid resource – a Bio Bank – that can be freely accessed by MSA researchers. Regular updating of samples and information for all patients will provide a consistent record of disease progression – a massive asset that will augment the existing brain bank of post-mortem tissue.

Blood samples, Cerebrospinal fluid (CSF) and skin samples all contain loads of biological substances that reflect the body’s metabolism and there is growing success in finding the right signals to show that a person definitely has MSA rather than something else. Determining these biomarkers is vital because diagnosis at present is so difficult.

The new funding

The funding agreed this year will support the longitudinal aspect of the study. A cross-sectional study is when one set of samples and measurements per patient is taken at one point in time. A longitudinal study is when a series of sets of samples and measurements are taken regularly over time. The benefit of the latter is that changes can be seen that are associated with the progression of the disease.

So what? If the research has already provided tests to show that you have MSA for sure, why would you want to know how far you are down the line if nothing can be done about it?

Here is where there is some tentative good news. Research into finding the causes of MSA and to look for clues for how to slow or stop its progress has been going on for decades. This has begun to yield fruit so that right now at least three treatment approaches have got as far as clinical trials.

The reliability of clinical trials depends on knowing firstly that each patient actually has the disease and not something masquerading as it. The biomarker aspect of the PROSPECT study has made much progress in providing this. Secondly, you need a way of knowing with certainty whether the treatment is having a measurable effect on modifying the progress. The forthcoming clinical trials will use assessment in the clinic to measure whether the treatments are having a tangible effect in slowing the disease down. But this is a blunt instrument – we all know that symptoms can vary so much from day to day. So this longitudinal study is timely – although not quite soon enough to provide the definitive method for measuring disease progression in the trial participants.

Much of the funding will support recruiting 100 patients from four centres in the UK a “large, biomarker-confirmed, trial-ready cohort” for rapid enrolment into MSA trials. This is no trivial task because of the comparatively low numbers of patients with probable MSA. Biosamples will be taken from these recruits on a yearly basis along with brain scans and clinical assessments.

The collection of samples, the establishment of the joint biobank and the determination of useful biomarkers is being done in collaboration with other research centres in France, Spain, Germany and Russia.

The story continues…..

Neurofilament Light Chain (NfL)

Various biomarkers are actively being established which, when taken together, can provide a profile for indicating a high probability of MSA. Another marker: the measured level of neurofilament light chain (NfL) in blood and CSF though not specific to MSA, seems to correlate well with the severity of this condition. If other markers indicate that a person does have MSA, measuring the changes in NfL can give a good indication of how the MSA is progressing. The project will work on confirming the reliability of this measure.

The trials provide an opportunity to demonstrate how good NfL is as a measure of disease progression. Measured changes in the levels of NfL in patients given the treatments and those not given them, will be compared with the normal estimations of disease progression through clinical assessment and brain scans. This could establish NfL measurement as an important tool for the routine assessment of disease severity and progression. NfL measurement could be used to back up conclusions about whether treatments actually work.

The three types of trial

Exenatide – This is a drug already in use for the treatment of diabetes. There are indications that it can slow Parkinson’s and possibly MSA.

Alpha-synuclein antibodies – As several neurodegenerative diseases result in inclusions of aggregated alpha-synulein in cells, much work has been going on to see whether anti-bodies can be used to remove this protein so that its propensity to aggregate harmfully is eliminated.

Anti-sense Oligonucleotide Therapy (ASO) is a way of interfering with the molecular mechanism of producing a protein and so could be used to suppress the production of alpha-synuclein which in a misfolded, aggregated form is believed to be a major toxin that causes the cells to degenerate in MSA.

As trials of these potential treatments get under way, it is expected that PROSPECT participants can be offered for enrolment because they are collectively on the point of becoming the “large, biomarker-confirmed, trial-ready cohort” envisaged.

The MSAT Scientific Advisory Panel awarded funding for 4 research projects. We are indebted to our lay advisor on the Panel, John Telford who has kindly shared the summaries on each of the grants.

Project 1:

Detailed assessment of the role of somatic genomic copy number variants (CNVs) in multiple system atrophy

Lead Applicant: Prof Christos Proukakis, UCL Queen Square Institute of Neurology

The Wrong Number

In an inherited form of Parkinson’s disease there is a genetic mutation in which there are more copies than usual of the gene that produces the protein alpha-synuclein. The result is that more of this protein is produced in brain cells and gets laid down as damaging inclusions in the cells.

MSA, on the other hand, is largely not inherited. So why am I even mentioning this? Well, a comparatively recent discovery by Prof Chris Proukakis is that such mutations can arise not from inheritance from one’s parents but later on during the body’s development or through ageing. He and his team have discovered that sets of mutated cells with duplications of the alpha-synuclein gene (known as SNCA) are often found in those areas of the brain which atrophy in MSA. How and why these so-called ‘somatic’ mutations are caused are mysteries that remain to be revealed further down the line. MSA is not the only neurological disease where this phenomenon appears to exist.

Right now there is a need to confirm the close association of Copy Number Variations (CNVs) – as these mutations involving extra copies of the genes are called – with the presence of inclusions in the same cells. They also need to prove that the CNVs actually cause the excess alpha-synuclein through the normal transcription processes in the cell and therefore are drivers of the disease.

Along the way some important anomalies need to be cleared up such as why sometimes inclusions occur without CNVs and why sometimes CNVs don’t give rise to inclusions. There could be other MSA-promoting conditions and other somatic mutations that cause the same thing. And it could also be that an element of the experimental technique used to reveal protein inclusions could be partly failing and need changing. Such factors need to be explored to get the accurate picture.

All this is an illustration of how complex biology is, particularly at the cellular level. At the risk of delving into philosophy, this is an example of why researchers must be painstaking and take care not to jump to quick conclusions following what appears to be a significant insight. Without thoroughly investigating and understanding what and how something is going wrong, it is not possible to come up with a tailored remedy that stands a chance of working. Good research is not cheap but we are fortunate in the field of neurology to have an abundance of excellent and dedicated researchers.

Project 2:

ClearSpeechTogether versus standard NHS speech intervention: A single, mixed method, rater-blinded pilot randomised controlled trial for people living with MSA-C

Lead Applicant: Anja Lowit, Strathclyde University, Glasgow.

Speech and communication

The word ‘Research’ might make you think of labs, white coats and test tubes and as such rather alien to your everyday life and the struggles that MSA brings. But the MSA Trust has decided to focus a good proportion of its funding effort on clinical research – research whose relevance to you, the MSA patient or carer, is more immediately obvious.

A notable example from this year’s grant awards is a research project to do with speech. As you probably know only too well, MSA often adversely affects speech. Volume drops, articulation suffers and you find your intelligibility declines. As communication is such a vital aspect of everyday life, this can be hard to take.

What can be done? Speech and Language Therapists (SLTs) are key. For those people with speech problems who are referred to the NHS, a standard Speech and Language Therapy course is likely initially to provide you with six weekly one hour one-to-one sessions with a therapist in which speech and communication difficulties are systematically addressed. The patient is taken through a number of different exercises and strategies aimed at having an enduring effect. This can be very beneficial but, of course, is quite expensive in terms of the use of the therapist’s time. It also begs the question of what further therapy might be needed for a progressive condition like MSA once the initial course is over.

What if a good proportion of the therapy was conducted in a group setting? The team running this project has already explored a programme, called ClearSpeech Together, with people who have other sorts of ataxia in which initial individual sessions with an SLT are followed up with frequent sessions in groups where the members follow a programme of exercises together. The SLT is still actively engaged but their time is used interacting with more people at once. Other important advantages are that patients are involved in mutual delivery of the therapy and are drawn into active participation with the others in the group thereby reinforcing the communication skills and strategies that easily get lost with conditions like ataxia and MSA. They feel better supported socially and psychologically than with individual SLT sessions. The work so far has shown that not only is this approach effective and a good use of therapists’ time but is also accepted well by the patients. The groups have often continued after the formally prescribed courses have finished, becoming valued social as well as therapeutically-targetted gatherings.

This particular project to be funded is aimed at testing out the feasibility and effectiveness of this approach amongst people with MSA-C over two years. If it has a positive outcome, a larger project will be designed with a view to making it standard within the NHS.

Project 3:

Is Multiple System Atrophy a distinct strain of alpha-synucleinopathy?

Lead Applicant: Professor Steve Gentleman, Department of Brain Sciences, Imperial College London, Burlington Danes Building,

Hammersmith Hospital Campus London

Antibodies take the strain

MSA, Parkinson’s and Dementia with Lewy Bodies are called synucleinopathies because all involve the mis-folding and laying down of inclusions in brain cells of a protein called alpha-synuclein. It seems that molecules of this protein, which usually go around in the cells singly, mis-fold and then begin to join together first in small numbers (when the groups of molecules are called oligomers) and then in greater numbers to form fibrils. It is a bit like ‘spoons inside a drawer’. Moreover it seems that once this process begins, unfolded molecules are induced to join in by the initial oligomers. Prion diseases progress this way.

Now in Parkinson’s these aggregates of misfolded alpha-synuclein are laid down in nerve cells (neurons) in particular parts of the brain whereas in MSA they are laid down in a different type of brain cell called glial cells in different parts of the brain and are called Glial Cytoplasmic Inclusions (GCIs). Why the difference? Evidence over the last few years has suggested that alpha-synuclein can misfold in more than one way. This means that there can be various so-called ‘strains’ of this misfolded and aggregated protein. And as you have guessed, a particular strain is believed to have properties that incline it to affect neurons and another has properties that incline it to affect glial cells in other parts of the brain. Glial cells are the sort of cell that supports and nourishes neurons and when they are affected and stop working, neurons also die and the various symptoms of MSA develop.

This project is centred on perfecting the ability to detect the different strains of alpha-synuclein using specially generated anti-bodies. We usually know antibodies as proteins used by the immune system to identify and neutralise foreign objects such as pathogenic bacteria and viruses. But they can be created to recognise and stick like labels to other entities such as our misfolded protein. In this case there has been success in producing antibodies that can bind to particular strains with a good deal of specificity. This project will continue this work and use various novel techniques to detect and make visible in post-mortem brain tissue when this occurs. These new techniques, it is hoped, will enable the researchers to explore and verify the pathological processes involved in MSA at the cellular level especially regarding the role of the different strains of the aberrant protein.

They will then go on to find out whether they can detect the binding of the antibodies to the MSA-specific strain of alpha-synucelin in cerebrospinal fluid (CSF). If so, this would provide a way of confirming, while they were still alive, and ideally at an early stage, that a person had MSA and not, say, Parkinson’s.

Furthermore, once there is a way of firmly diagnosing MSA and tracking its progress, the effect of potential new treatments could be more precisely measured.

In addition the great hope is that specific antibodies could be used to attach to and remove deposited alpha-synuclein – on the assumption that this is a root cause of the disease.

Project 4:

An MSA Clinical Pathways Platform and Patient Registry – Monitoring disease, quality of care and developing evidence-based care algorithms, starting with a Dysphagia Clinical Care Pathway.

Lead Applicant: Dr Viorica Chelban, Queen Square Institute of Neurology University College London

Hard to Swallow

Impaired swallowing (known as dysphagia) is a troubling and distressing feature of MSA which develops during the disease. This is clear if you look at the MSA section of the HealthUnlocked online Forum. (https://healthunlocked.com/msa-trust/posts) On average dysphagia is moderately serious after 5 years or so. Aspiration pneumonia – food and liquids going into the lungs instead of the stomach – can occur as the problem get worse. I know; my wife died of it. And that is why in serious cases patients may undergo a procedure known as a PEG – Percutaneous Endoscopic Gastrostomy. A tube is fitted which passes through the abdomen straight into the stomach through which food and liquids can be passed. Without it patients can become malnourished and dehydrated. Gastrostomies may be called for in a number of other neurological and non-neurological conditions including Parkinson’s and Motor Neurone Disease. But in MSA a body of evidence for when and how PEGs should be employed and what the outcomes are likely to be in terms of effective disease management and quality of life has not yet been built up. This project intends to address that.

The meat of the project is to create a national registry of MSA patients focussing on swallowing difficulties. The data for each patient will look back at when difficulties appeared, how they progressed and what treatment was given – including whether a gastrostomy was recommended and, if carried out, how effective, acceptable and beneficial it was. More data will be added as patients are monitored and their condition progresses. As we know, the symptoms and progress of MSA manifest themselves in such a variety of ways in separate individuals that the only way to get a meaningful overall picture is to collect and compare a lot of real data. This is what this register will enable and has not been done comprehensively before. But more than that, patients, carers and clinical specialists will all be actively involved in contributing their detailed experiences and insights into swallowing impairments and their treatments. Online questionnaires and surveys and other methods such as working groups, conferences and consultations will be used to structure the register, acquire the case data, analyse the information and draw out conclusions that are important for clinical practice and for the best management of dysphagia in MSA.

Of course, that will not be the end of the story. The ultimate aim is actually to formulate a Clinical Care Pathway for the management of swallowing problems in MSA. The detailed patterns that are discerned from the richness of the registry data will provide an evidence base for what treatments are most effective in different varieties of cases – including in what circumstances certain treatments should be avoided. The Care Pathway as elaborated would then be an invaluable and specific tool, universally promoted, for improving and optimising clinical practice in this area.

2023-24 Grant Call

Multiple System Atrophy Trust are pleased to announce their 2023-24 Grant call for innovative and novel research projects, both scientific and clinical.

We would be delighted to receive pre-proposals in the following areas:

Scientific research into MSA, with priority given to Biomarker studies in MSA and Studies of validation or other aspects of prodromal MSA

and

Research focused on improving the clinical care of people living with MSA.

Grants of up to £150,000 are available to researchers and up to three projects will be awarded in this grant round.

We are holding a pre-proposal stage to support researchers, which should be a short preview of the research you wish to undertake.

The MSA Trust Scientific Advisory Panel will assess the pre-proposals and will contact those who they wish to invite to proceed to full application stage.

The cut-off date for receiving pre-proposals is 8^th November 2023 at 5pm GMT.

Please download the grant application form below.