Regenerative medicine- Direct and indirect cell reprogramming

Regenerative medicine-Direct and indirect cell reprogramming
My last three blog posts commented on direct cell reprogramming. You may be wondering if there is a technology called indirect cell reprogramming. The answer is yes. It is formally called induced pluripotent stem cell (iPSC, cells that have the ability to become any cell in the body)-mediated reprogramming, and it was developed earlier than direct cell reprogramming. In this blog post, I give a brief introduction of the two technologies and the challenges faced by both technologies.

Both reprogramming technologies involve the conversion of one cell type into another, target, cell type, intended for therapeutic use. Usually, the conversion starts by using the most abundant cells in the body, such as human fibroblast cells, a type of cell found in connective tissue.

Indirect cell reprogramming
Indirect cell reprogramming is a technology that converts somatic cells to another type of cell via first transitioning through an intermediate state, induced pluripotent stem cells (iPSCs).¹ Therefore it is also called induced pluripotent stem cell (iPSC)-mediated reprogramming. The iPSCs have similar properties as embryonic stem cells in having unlimited differentiation capacity, while avoiding the ethical issues related to embryonic stem cells.² Moreover, as iPSCs are generated from patients, this eliminates the problem of tissue rejection following transplantation in patients. Once iPSCs are generated, the cells can then be directed for development and differentiation, to form desired cells.

Four main transcription factors (Oct3/4, Sox2, Klf4, c-Myc), called Yamanaka factors, that induce somatic cells into pluripotent states, were first identified by Japanese scientists, and the finding was published in 2006.¹ These transcription factors are highly expressed in embryonic stem cells, and are able to induce pluripotency in both mouse and human somatic cells.¹

Since the generation of the first induced pluripotent cells, some progress has been made to improve the quality of the iPSCs generated and the efficiency of the technology. Methods include:
1) replacing of c-Myc, which is oncogenic and may cause tumour formation, in many protocols of pluripotent cell induction;^3-6
2) using of viral delivery systems, such as an adenovirus, which does not integrate into the recipient’s DNA.^7,8 This choice of virus is because a retrovirus, which is used for delivery of the four factors in the original protocol, would integrate into host’s DNA and cause insertional mutagenesis, which increases the risk of tumorigenicity;
3) replacing the original Yamanaka factors by small molecules and chemical compounds. This could also avoid the use of retrovirus;⁹
4) expressing Yamanaka factors for a shorter time for induction. This saves the time required to generate, expand and differentiate pluripotent cells, and avoids the creation of teratomas, a rare type of tumor that can contain fully-developed tissues and organs.^10-12

Direct cell reprogramming
Direct cell reprogramming, also called transdifferentiation, is the conversion of a fully functional cell type into cell type of other lineage. The process bypasses a state of pluripotency. Transdifferentiation between some cell types can occur naturally in response to injury, and can be induced experimentally. I have made a brief introduction to transdifferentiation technology in a previous blog post.

A major limitation of direct cell reprogramming is the identification of reprogramming factors that promote conversion to a specific target cell type. The conventional strategy, which screens through a list of transcription factors that are important for the development of the target cells, is a time-consuming and laborious process. There are a few predictive software applications developed to facilitate the process. These include MOGRIFY¹³ and epiMOGRIFY¹⁴ which we mentioned in the last three blog posts, CellNet,^15,16 and the one developed by Massachusetts Institute of Technology (MIT).¹⁷

Challenges faced by the two technologies
As you can see from the general description of the two technologies, direct cell reprogramming is faster, cost-effective and more accessible than iPSC-based protocols. Direct cell reprogramming protocols do not use Yamanaka factors, which are licensed, to induce pluripotency. The technology does not involve ex vivo cell expansion and transplantation, as in iPSC-mediated reprogramming.

However, both technologies face the same problem of scalability, which could impede their therapeutic applications. For the direct cell conversion approach, most converted cells could either experience cell cycle arrest that limit the expansion of the cell populations, or they tend to revert back to what they were. This lowers the number of cells available for further use. Similarly, for the indirect cell reprogramming, forced differentiation of induced pluripotent stem cells to distinct lineages also leads to the problem of cell cycle arrest and inability to expand. Moreover, the efficiency of inducing mature cells into pluripotency is less than 4%, which is very low.¹⁸

Quality of induced pluripotent stem cells generated from the indirect cell reprogramming is also a concern. The iPSCs generated are highly tumorigenic.¹ This maybe due to the fact that the iPSCs are mostly derived from somatic cells of aged individuals. The risk that comes with this source of cells is the incidence of spontaneously occurring tumours, which commonly increases exponentially with aging. Concerns related to tumorigenicity and teratoma formation by these cells hamper their direct applicability.

Conclusion
Both direct and indirect cell reprogramming have been developed for more than a decade, and the technologies provide a feasible method to generate cells for biological studies, tissue engineering or transplantation. Diseases such as diabetes, heart failure, arthritis, and many more, which affect a high proportion of the world population, are hopefully to be treatable from the cell reprogramming technologies. Although no cell-based therapy has yet been officially approved in any country in the world, once the challenges mentioned above are overcome, the clinical application of this technology may not be far away.



References
1. K. Takahashi, and S. Yamanaka. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126 (4): 663–76.
2. B. Lo, and L.Parham. Ethical issues in stem cell research. Endocrine Reviews, Volume 30, Issue 3, 1 May 2009, Pages 204–213.
3. J. Han, P. Yuan, H. Yang, et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature, 2010;463:1096–100.
4. J. Chen, J. Liu, J. Yang, et al. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone. Cell Res., 2011;21:205–12.
5. H.Y. Li, Y. Chien, Y.J. Chen, et al. Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells. Biomaterials. 2011;32:5994–6005.
6. M. Maekawa, K. Yamaguchi, T. Nakamura, et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature, 2011;474:225–9.
7. N, Fusaki, H. Ban, A. Nishiyama, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser.B., 2009;85:348–62.
8. W. Zhou, and C.R. Freed. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 2009;27(11): 2667–74.
9.X. Li, J. Xu, and H. Deng. Small molecule-induced cellular fate reprogramming: promising road leading to Rome. Curr. Opin. Genet. Dev., 2018, 52, 29–35.
10. L. Kurian, I. Sancho-Martinez, E. Nivet, et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat. Methods, 2013; 10:77–83.
11. M. Thier, P. Wörsdörfer, Y.B. Lakes, et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 2012; 10:473–479.
12. A. Ocampo, P. Reddy, P. Martinez-Redondo, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell, 2016 December 15; 167(7): 1719–1733.e12.
13.O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
14. U.S. Kamaraj, J. Chen, K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 2020, 11, 509–522, November 18.
15. S.A. Morris, P. Cahan, H. Li, et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell, 2014, 158, 889–902.
16. P. Cahan, H.Li, S.A. Morris. et al. CellNet: network biology applied to stem cell engineering.Cell, 2014, 158, 903–915.
17. A.C. D’Alessio, Z.P. Fan, K.J. Wert, et al. A systematic approach to identify candidate transcription factors that control cell identity. Stem Cell Reports, 2015 Nov 10;5(5):763-775.
18. Rao MS, Malik N. Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012;113:3061–8.

Predictive bioinformatic platforms for direct cellular reprogramming-Mogrify Ltd (final part)

Predictive bioinformatic platforms for direct cellular reprogramming: Mogrify Ltd (final part)
Since its launch, Mogrify Ltd has started several projects on different therapeutic areas of regenerative medicine, which have made use of the prediction results from its computation platform Mogrify. In this blog post, I am going to present to you what I have found from the company’s website. (I am not sponsored by them, just looking.)

Application of the prediction from MOGRIFY
Through its wholly-owned subsidiary, Chondrogenix, Mogrify Ltd has stepped into the therapeutic area of musculoskeletal diseases such as osteoarthritis. The algorithm platform MOGRIFY identified a cocktail of transcription factors for the company, Chondrogenix, to convert, in a culture dish, different starting cell types from diseased patients, into functional chondrocytes that are capable of forming cartilage. Musculoskeletal diseases are usually caused by bone or cartilage defects. This technology paved the way for them to write an application request to the regulators to enhance the FDA-approved Autologous Chondrocyte Implantation (ACI) therapy, and to create additional reprogramming therapies using cells from donors (allogenic reprogramming therapy) or by direct cell conversion in living organisms (in vivo reprogramming therapy). Chondrogenix received two phases of funding from SBRI Healthcare for this project.^1,2

Mogrify Ltd is also using its computational platform to predict combinations of transcription factors to induce conversion of one cell type to another in order to produce proteins which are not produced sufficiently well by existing production systems. The resulting target cell types could provide researchers with improved access to important proteins found in human cell types that are difficult to obtain, and allow for more efficient antibody production methods for biologic drugs. This exploratory research is done with the collaboration of the MRC’s Laboratory of Molecular Biology (MRC-LMB) in Cambridge.³

In fact, Mogrify Ltd also collaborates with MRC-LMB to improve the MOGRIFY cell reprogramming platform itself. The enhanced version, MOGRIFY V2, incorporates data from next-generation sequencing and single-cell RNA sequencing into the algorithm to enhance the quality and accuracy of transcription factor predictions and cell conversion efficacy.⁴

Furthermore, Mogrify Ltd is also involved in allogeneic (using cells from a donor who is not the patient) T-cell immunotherapy for inflammatory and autoimmune diseases, by collaborating with Sangamo Therapeutics, a genomic medicine company in the US. Mogrify is responsible for the discovery and optimisation of the cell conversion technology from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) to regulatory T cells (Tregs). Sangamo expects to then use its technology and therapeutic development capabilities to transform these Tregs cells into ready to use allogeneic therapy candidates for the treatment of inflammatory and autoimmune diseases. The resulting collaboration will hopefully accelerate the development of scalable and accessible CAR-Treg cell therapies so that the treatments can be delivered more rapidly, in a more cost-effective way to a larger patient population.⁵

Financial Support
Since its launch in February 2019, Mogrify Ltd has attracted investments from strategic corporate investors to strengthen its business. According to a press release of the company in May this year, Mogrify Ltd has raised a total of US$33 million from seed funding and two rounds of Series A financing. The investors include Ahren Innovation Capital, 24Haymarket, Dr. Darrin M. Disley, OBE (CEO of Mogrify Ltd), Parkwalk Advisors, Astellas Venture Management, Dr Jonathan Milner (co-founder of Abcam PLC), and the University of Bristol Enterprise Fund III.⁶

In March 2019, Mogrify Ltd was awarded $555,000 (£420,000) from Innovate UK, a UK innovation agency, on a data-driven cell conversions project to produce cell therapies with potential applications in wound healing and oncology immunotherapy.⁷

Moreover, Chondrogenix (the subsidiary of Mogrify Ltd) received two rounds of funding from SBRI Healthcare, an NHS England initiative, championed by the Academic Health Science Networks (AHSNs). These funds were awarded for the purpose of generating a safe, efficient and scalable source of cartilage cells for the treatment of cartilage defects, osteoarthritis and other musculoskeletal conditions.^1,2

Conclusion
From the above information, it is obvious that Mogrify Ltd has ambition to develop and scale up reprogramming of cells for many autologous and allogeneic cell therapies, as well as to create in vivo reprogramming therapies (direct cell conversion in living organisms).^1-3,5 The two software platforms developed by the company no doubt speed up the process of direct cellular reprogramming, by using algorithms based on the big data already available in public repositories instead of performing trial and error experimentally. With the financial support it has got so far, I have no doubt that the company, in the near future, can meet its target of producing stable reprogrammed cells at scale for some areas of cell therapies. However, the company needs to overcome some challenges when it comes to clinically applying its reprogrammed cells.

The hurdles include the safety issues regarding the components suggested by the software platforms for cell conversion. There is a possibility that some biological factors such as cMYC, when overexpressed, can cause tumour growth.^10,11 The method to deliver biological factors safely is also a concern. Engineered viruses constructed to deliver transcription factors may integrate into a DNA region of target cells and cause a tumour later. In addition, the recipient’s immunity against the reprogrammed cells also needs to be addressed. These challenges, though, are also faced by other biotech companies focusing on regenerative medicine.

Besides the above-mentioned issues, I am eager to see the reports on the experiments, which apply the prediction from MOGRIFY⁸ and epiMOGRIFY⁹, to be published soon. I am also expecting Mogrify Ltd will merge the two software platforms into a single platform with synergistic power.



References
1. Mogrify subsidiary Chondrogenix secures funding from SBRI Healthcare to advance regenerative cartilage therapy to the clinic. Mogrify Ltd press release, 1st April, 2019. https://mogrify.co.uk/mogrify-subsidiary-chondrogenix-secures-funding-from-sbri-healthcare-to-advance-regenerative-cartilage-therapy-to-the-clinic/
2. Mogrify awarded $1.1M additional funding from SBRI Healthcare. Mogrify Ltd press release, 28th January, 2020. https://mogrify.co.uk/mogrify-awarded-1-1m-additional-funding-from-sbri-healthcare/
3. Mogrify enters research collaboration with the MRC Laboratory of Molecular Biology. Mogrify Ltd press release, 11th January, 2021. https://mogrify.co.uk/mogrify-enters-research-collaboration-with-the-mrc-laboratory-of-molecular-biology/
4. Mogrify solidifies IP position surrounding core technology and expands platform algorithm to enhance cell conversion. Mogrify Ltd press release, 17th December, 2020. https://mogrify.co.uk/mogrify-solidifies-ip-position-surrounding-core-technology-and-expands-platform-algorithm-to-enhance-cell-conversion/
5. Mogrify and Sangamo announce collaboration and exclusive license agreement for Mogrify’s iPSC- and ESC-derived regulatory T cells. Mogrify Ltd press release, 21st April, 2020. https://mogrify.co.uk/mogrify-and-sangamo-announce-collaboration-and-exclusive-license-agreement-for-mogrifys-ipsc-and-esc-derived-regulatory-t-cells/
6. Mogrify completes Series A financing totalling $33 million USD. Mogrify Ltd press release, 4th May, 2021. https://mogrify.co.uk/mogrify-completes-series-a-financing-totaling-33-million-usd/
7. Mogrify awarded $555,000 USD (£420,000 GBP) Innovate UK funding to accelerate regenerative cell therapies. Mogrify Ltd press release, 18th March, 2019. https://mogrify.co.uk/mogrify-awarded-555000-usd-420000-gbp-innovate-uk-funding-to-accelerate-regenerative-cell-therapies/
8. O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
9. U.S. Kamaraj, J. Chen, and K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 2020, 11, 509–522, November 18.
10. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006;126:663–676.
11. Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008;8:976–990

Predictive bioinformatic platforms for direct cellular reprogramming- Mogrify Ltd (cont'd)

Predictive bioinformatic platforms for direct cellular reprogramming: Mogrify Ltd (cont’d)
My previous blog post presented a brief understanding of recent progress in direct cellular reprogramming and the reason for the development of two software platforms by Mogrify Ltd. This blog post is about further details of the two platforms, MOGRIFY and epiMOGRIFY.

About MOGRIFY
MOGRIFY was first co-developed by two scientists, Julian Gough (Professor of Computer Science in the University of Bristol) and Owen Rackham (PhD student of Julian Gough in the University of Bristol), in 2011. They began the development of the platform by using the data from the FANTOM 5 consortium, an international consortium launched to provide gene expression data in virtually all cell types across the human body.¹ In 2014, they collaborated with Jose Polo from Monash University in Australia to test cell conversions in order to validate the prediction power of MOGRIFY. In 2015, based on the prediction results from MOGRIFY, the co-founders filed a patent application for the algorithm used by the computation platform, with over 30 direct cell conversions listed as example proof of what the algorithm can achieve.² The predictions and cell conversion results were then published in 2016.³

According to the paper, predictions from MOGRIFY have been applied on 173 human cell types and 134 tissues. In order to assess the predictive power of MOGRIFY, the performance of the algorithm was first compared against well-known, previously-published human cell conversions. MOGRIFY correctly predicted the transcription factors (enzyme proteins that bind to a particular DNA sequence and regulate the expression of genes) used in previously-published direct cell conversions from human fibroblasts (the most common type of cell found in connective tissue) into induced pluripotent stem cells,^4,5,6 and from B cells/ fibroblasts into macrophage-like cells.^7,8 In addition, MOGRIFY’s prediction list included four of the five transcription factors (or similar factors) used in the conversion of human dermal fibroblasts into cardiomyocytes (cells responsible for generating contractile force in the intact heart).⁹

The predictive capabilities of MOGRIFY® were also experimentally examined using human cells. Two cell conversions, human fibroblasts to keratinocyte cells (cell within the epidermis) and adult human keratinocyte cells to micro-vascular endothelial cells (cells isolated from small vessels within skin tissue), were performed using prediction results from MOGRIFY. By examining the morphological and molecular characterization of the reprogrammed cells, the conversions were found to be successful.³

The results showed that MOGRIFY provides a practical and efficient mechanism to facilitate the reprogramming of human cells.

However, since the publication of the results in 2016, no further reports of cell conversions using predictions from MOGRIFY, have been published, or at least I was not able to find any after extensive searches.

About EpiMOGRIFY
Once the cells are converted from other cell types, it is important to maintain the reprogrammed cells’ identity, as the converted cells tend to revert back to what they were before. Difficulty maintaining the differentiated cells makes a large-scale production of these differentiated cells hard to achieve. This can be a hurdle for the development of cell therapies.

EpiMOGRIFY was developed with the aim of identifying optimal culture conditions (with suggested small molecules and chemical compounds) required to maintain cell identity or induce cell conversion in chemically-defined media. The computation algorithm incorporated gene-regulatory information data and cell epigenetic landscapes for more than 100 human cells and tissues, from the ENCODE and Epigenome Roadmap consortia, to define culture conditions that can maintain the phenotype and function of the converted cell, or that can be used to induce cell conversion.¹⁰

The EpiMOGRIFY platform was co-developed by Duke-NUS Medical School (Professors Enrico Pettreto and Owen Rackham), Monash University (Professor Jose Polo) and Mogrify Ltd. Owen Rackham and Jose Polo were the co-developers of MOGRIFY.¹¹

Experiments validating the computational prediction in cell culture conditions for cell maintenance and differentiation have recently been published.¹² Using the suggestion factors predicted from EpiMOGRIFY, astrocytes (cells commonly found in the brain) and cardiomyocytes (cells that make up heart muscle) were able to grow as effectively, in terms of cell numbers and proliferation rate, as they are in the culture conditions that have long been used. Moreover, these cells appeared to better maintain their morphology, in terms of the biological structure and the presence of key markers for the cell types, when growing in the culture conditions with biological factors suggested from EpiMOGRIFY. These results provide a fundamental proof for EpiMOGRIFY’s predictive power to suggest culture conditions that are able to maintain a stable phenotype.¹²

Furthermore, using the cell conversion conditions predicted from EpiMOGRIFY, neural progenitors and embryonic stem cells were able to differentiate to astrocyte cells and cardiomyocyte cells, respectively. The differentiation efficiency was as good as that of existing differentiation protocols.¹² These results indicate that EpiMOGRIFY is highly accurate in identifying biological factors for cell conversion as well.

To be continued.

References
1. Our history. Mogrify Ltd website. https://mogrify.co.uk/about-us/
2. https://patents.google.com/patent/WO2017106932A1/en
3. O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
4. K. Takahashi, K. Tanabe, M. Ohnuki, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131, 861–872.
5. J. Yu, M.A. Vodyanik, K. Smuga-Otto. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007, 318, 1917–1920. 6. D. Huangfu, K. Osafune, R. Maehr, et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol., 2008, 26, 1269–1275.
7. H. Xie, M. Ye, R. Feng, & T. Graf. Stepwise reprogramming of B cells into macrophages. Cell, 2004, 117, 663–676.
8. F. Rapino, E.F. Robles, J.A. Richter-Larrea, et al. C/EBPα induces highly efficient macrophage transdifferentiation of B lymphoma and leukemia cell lines and impairs their tumorigenicity. Cell Reports, 2013, 3, 1153–1163.
9. J. D. Fu, N.R. Stone, L. Liu, et al. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Reports, 2013, 1, 235–247.
10. epiMOGRIFY PLATFORM. Systematically identify the epigenetically-predicted factors required to drive and maintain cell identity. Mogrify website. https://mogrify.co.uk/science/epimogrify/
11. Our history. Mogrify Ltd website. https://mogrify.co.uk/about-us/
12. U.S. Kamaraj, J. Chen, K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 11, 509–522, November 18, 2020.

Predictive bioinformatic platforms for direct cellular reprogramming- Mogrify Ltd

Predictive bioinformatic platforms for direct cellular reprogramming: Mogrify Ltd
Have you ever imagined one day we could have our liver, kidney or heart regenerated from our own skin cells, to replace our failed or damaged organs, without the need to wait for transplants from a suitable donor? Research studies in cellular reprogramming, targeting this prospect, have become a hot topic in recent decades. A new bioinformatic start-up, Mogrify Ltd, aiming to transform the development of cell therapies and to develop scalable off-the-shelf cell therapies, seems worth our attention.

Mogrify Ltd is situated in the Bio-Innovation Centre in Cambridge Science Park in the UK. According to the press release of the company on this March, the company has a headcount of over 60 scientific, operational, and commercial staff.¹ The main technology products of the company are two computation prediction platforms, MOGRIFY and EpiMOGRIFY, which are useful to facilitate the development of cell therapies.^2,3 The company was incorporated in 2016 and was launched in February 2019. However, the development of its first computational prediction platform, MOGRIFY version 1, was started in 8 years previously in 2011. The second platform, EpiMOGRIFY, was launched in 2020.⁴ Both platforms were validated and the results were published in peer reviewed journals.^5,6

Why did Mogrify Ltd develop the two computational prediction platforms? Why are the two platforms developed by Mogrify Ltd useful tools to facilitate the development of cell therapies? Let us briefly explore the development of direct cellular reprogramming, an important area in regenerative medicine.

Background of direct cellular reprogramming
As early as 1958, an experimental finding suggested that terminally-differentiated cells have a certain degree of plasticity and can be reprogrammed to alter cell fate.⁷ Nearly 30 years later, a single transcription factor (an enzyme protein that binds to a particular DNA sequence and regulates the expression of genes), MyoD, was shown to convert fibroblasts (the most common type of cell found in connective tissue) directly to myoblasts (embryonic progenitor cells that differentiate to form muscle cells).⁸ However, later findings suggested that a single factor is not sufficient to drive cellular reprogramming for most tissues. Since then, different combinations of key transcription factors and microRNAs, which are developmental regulators of the target cell lineage, have been identified to convert fibroblasts to various cell types with therapeutic purposes.^9,10,11

Traditionally, “minus-one” strategy is used to identify the combination of key transcription factors and microRNAs to convert mature cells to another mature cell type. In brief, scientists start with combinatorial screening using a pool of transcription factors and microRNAs, which are chosen according to the domain knowledge of the scientist on the biological networks controlling cell differentiation, to convert one cell type to another differentiated (mature, fully functional) cell type. Once the pool of the biological factors are experimentally proved to be efficient for the cell conversion, then a “minus-one” strategy is used to identify essential factors by removing one factor at a time from the pool, aiming at minimal combination required for cell conversion.

However, this method can be very time-consuming. Take for example an experiment which converted dermal or cardiac fibroblasts to induced cardiomyocyte (heart cells for contraction)-like cells: it was originally started with a pool of nearly 20 transcription factors and a similar number of miRNAs. “Minus-one” strategy was used to finally identify 3 key transcription factors for the cell conversion. You can imagine that it was a long, exhaustive task of trial and error.¹⁰ Moreover, the mechanism for direct reprogramming is cell type specific and still largely unknown for many cell types.

In addition, most of the converted cells cannot stay where they are after adding key biological factors for conversion. They either end up converted back to what they were, or rapidly exit the cell cycle and don’t proliferate. This is because cell fate is actually controlled by the epigenetic state, the regulation mechanism that happens on the DNA level, of each cell. Only the change in DNA level can bring permanent change in the fate of cells. Chemicals, microRNAs, and transcription factors with appropriate culture conditions which change the epigenetic state of cells could permanently change the cell fate. The addition of transcription factors and micro RNA which do not affect the change in DNA level can bring only a temporary conversion of the cells.

In general, traditional direct cell conversion method is not time efficient, and it is hard to produce stable, scalable converted cells for clinical applications. This presents a big challenge to the cell therapies which rely on converted cells using traditional direct cell conversion methods.

The two predictive computational platforms, MOGRIFY and epiMOGRIFY, were developed by Mogrify Ltd. to address the problems mentioned above. MOGRIFY® predicts transcription factors which promote any cell conversion from any source cell types.² EpiMOGRIFY identifies culture conditions with optimal combinations of transcription factors or growth factors required to maintain cell identity and epigenetically support the reprogramming of cells in chemically-defined media. These two platforms could help speed up the direct cell conversion process, and help to develop stable and scalable resulting cells that can meet clinical needs.

Data sources such as FANTOM5 and ENCODE, which were being incorporated to develop MOGRIFY® and epiMOGRIFY, have emerged thanks to the advances in developmental biology research and discovery, over the last two decades, of gene networks that drive cell fate.

To be continued.


References
1. Mogrify wins Hewitsons Award for Innovation in Business and Price Bailey Award for Business of the Year at CambridgeshireLive Business Excellence Awards 2020. Mogrify press release, 26 March, 2021. https://mogrify.co.uk/mogrify-wins-hewitsons-award-for-innovation-in-business-and-price-bailey-award-for-business-of-the-year-at-cambridgeshirelive-business-excellence-awards-2020/
2. MOGRIFY® PLATFORM. Systematically predict the transcriptomic switches required to produce any target cell type from any source cell type. Mogrify website. https://mogrify.co.uk/science/mogrify/
3. EpiMOGRIFY PLATFORM. Systematically identify the epigenetically-predicted factors required to drive and maintain cell identity. Mogrify website. https://mogrify.co.uk/science/epimogrify/
4. Our history. Mogrify Ltd website. https://mogrify.co.uk/about-us/
5. O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
6. U.S. Kamaraj, J. Chen, K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 11, 509–522, November 18, 2020.
7. J.B. Gurdon, T.R. Elsdale, and M. Fischberg. Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature, 1958, 182, 64–65.
8. R.L. Davis, H. Weintraub, and A.B. Lassar. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell, 1987, 51, 987–1000.
9. K. Batta, M. Florkowska, and V. Kouskoff, et al. Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Rep., 2014, 9:1871–1884.
10. M., Ieda, J.D. Fu, and P. Delgado-Olguin, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010, 142:375–386.
11. T. Vierbuchen, A. Ostermeier, and Z.P. Pang, et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 2010, 463:1035–1041.

A story of false data

A story of false data
In fiction, cancer research is about trying to find a cure for cancer. A single cure, that fixes everybody's cancer. While this certainly makes for good stories, and would indeed be wonderful if anyone ever managed to come up with it, most real-life cancer researchers settle for a lesser goal that is still an achievement: find drugs that will slow down (or, in some cases, remove) a particular type of cancer in a particular type of person. For example, they might come up with research that only works on nose cancer in Asians, and then only works some of the time. Nevertheless this can still add years of life to some people and is therefore worth checking.

In 2013-2014 I was involved in therapeutic aspects of cancer research, in further investigation of the interplay between the PRDM1 gene and the NF-κB protein that leads to different susceptibility rates of NK/T cell lymphoma (NKTCL) cancer patients towards chemotherapeutic agents. The research team of my supervisor had a leading study using a 26S proteasome inhibitor drug, bortezomib (Velcade), to act against NK/T cell lymphoma.¹ With the assumption that IL2 is an important factor in sustaining NK/T cell lymphoma, we investigated the combination treatment that can reduce bortezomib dosage by using either of the two fusion anti-IL2 receptor antibodies, basiliximab (Simulect®) and denileukin diftitox (ONTAK®). We found that IC50 (median inhibitory concentration) of NKTCL cells being treated by bortezomib can be decreased by ~53% to ~90% when combined with a low level of basiliximab. This was a promising result, but it still needed testing "in vivo" (in a live setting). As the lab I was in did not have enough live sample (and my supervisor was retiring and the grants were running out), we came up the idea of asking another lab (one in mainland China with whom we had long been in collaboration) to run the live tests for us.

I visited China, trained the people there to perform a couple of in vivo tests, and finished the job. Several months later I married and moved to the UK, and the lab in China kept informing us about the progress of their study by sending us results in pictures. Those results were coherent with the in vitro test results we had previously found in our lab. Maybe we really did have a new, more effective chemotherapy drug on our hands for people with that particular cancer type? I had to fly back to Hong Kong and check things out (we tried working remotely but the lab really wanted me there). Things seemed to be playing out like a film script, aeroplanes and all.

But sadly, our further analysis showed that the data from the China lab was very likely to be fake. I found two pictures sent at two different times, supposedly representing different samples, were actually identical. By reflecting and rotating one of the pictures later sent to me, I could not see any difference with the one that had been sent earlier. We decided not to publish it after all.

So, does our combination work? It has not been disproven. But science does not work on not disproving things, it works on positive proof. It might still be possible for other labs to re-check our combination, and we hope they will do so from our partial publications, but we were not able to show it ourselves. And we ended up being a little annoyed with that lab for faking their data (perhaps because they couldn't be bothered to run the real tests?) and causing all that excitement for nothing. But these things happen.

The quest to improve the lives of people with cancer continues, one small uncertain arduous step at a time.



References
1. L. Shen, W.Y. Au, T. Guo, et al. Proteasome inhibitor bortezomib-induced apoptosis in natural killer (NK)-cell leukemia and lymphoma: an in vitro and in vivo preclinical evaluation. Blood, 2007 Jul 1;110(1):469-70.

Cambridge Open Exascale Lab

Cambridge Open Exascale Lab
Recently I have been occupyed writing coronavirus-related blog posts, but with the first round of the COVID-19 vaccination programme almost at its final stage in the UK, and the recovery of business activities, I plan to start writing more about spin-offs and other initiatives from Cambridge University that involve with latest innovative technologies.

In this blog post, I would like to introduce you a world-class supercomputer laboratory, Cambridge Open Exascale Lab, the University of Cambridge. It is dedicated to designing fast supercomputers to bring the UK’s science, health and industry into the levels of complexity and performance that previously were out of reach.

Cambridge Open Exascale Lab is part of the Cambridge Research Computing Services (RCS), formerly called the High Performance Computing (HPC) Facility when it was founded in 1996. Cambridge Research Computing Services was established with the aim of providing high performance computing services to leading scientists, medics and engineers across the whole of the UK.

Cambridge Research Computing Services currently runs two supercomputers, called Peta4 and Wilkes (named after Cambridge computing pioneer Sir Maurice Wilkes, 1913-2010). These are running at rates of peta (10¹⁵) floating-point operations per second (FLOPS, a measure of supercomputer performance when totalled across thousands of CPU cores, useful when handling scientific tasks that are highly parallelisable between many CPUs). The newly established Cambridge Open Exascale Lab aims to develop supercomputing systems running at the speed of exascale (10¹⁸) FLOPS, some 1,000 times the scale of the current systems.

What exascale supercomputing systems could bring
Countries from the United States to Europe to Japan are in the race to produce the world’s first exascale computing system. The United States Department of Energy and Intel announced that the first exaFLOPS supercomputer, Aurora, would be operational at Argonne National Laboratory in Lemont, Ilinois, by the end of 2021. The European Union has a range of exascale programmes in the works under its European High-Performance Computing Joint Undertaking. Japan is aiming for the exascale version of its Fugaku supercomputer to be available to users within a couple of years.¹ You may wonder why exascale computing systems are so urgently being developed. Let’s explore.

Take the example of my field of expertise, biomedical science. In the last 10 years, a huge amount of biological data, such as genomic and proteomic data, has been generated, mainly due to the fall in price of sequencing. Combining this biological data with clinical data could help develop personalized medicine (also called precision medicine). Analysing an amalgamation of all this data tends to require computing systems with high processing capacity (and also high storage space, although this tends to be only a secondary consideration for supercomputers, because managing the processor interconnects is the most difficult task, whereas adding more storage is easy by comparison). With the invention of the exascale computing system, analysis of data from different data sources, which currently takes weeks or even months, could be shortened to hours or days, and thus allow more calculations to be explored within a research project’s time-frame. Personalized medicine could therefore be developed in a much faster pace.

In addition, exascale supercomputers will enable simulations that are more complex and of higher resolution. This allows researchers to explore the molecular interactions of viruses and their hosts, which aids in the design of vaccines.¹ Imagine how it might have helped if we could have had a vaccine against SARS-CoV-2 with more than 95% efficacy designed in a few days, or even in a few hours, during the initial stages of the COVID-19 pandemic.

The exascale power will allow climate forecasters to swiftly run thousands of simulations, introducing tiny variations in the initial conditions, to provide better insight into the potentially disastrous effects of climate change.¹

Besides life sciences, exascale computing is expected to benefit chemical design, pattern modelling, high-energy physics, materials science, oil exploration, and transportation.¹

In view of the benefits of exascale computing systems, the opening of Cambridge Open Exascale Lab could help the UK remain at the forefront of different fields of science.

Goals of Cambridge Open Exascale Lab
An exascale supercomputer will contain some 135,000 GPUs and 50,000 CPUs, each one being a multi-core chip with many individual processing units. This immediately creates the problems of huge power consumption, and a potentially difficult method of programming to enable almost a billion instructions being executed simultaneously. Furthermore, if the system is upgraded, researchers may need to re-examine millions of lines of code and optimize them to make use of the extra hardware, so that the programs can reach as close to the theoretical maximum processing power as possible. In addition, the ability to access memory (RAM or long-term storage) and retrieve data quickly is an issue in highly interconnected supercomputers, as evidenced by Cambridge startup Ellexus (recently sold to Altair), which focused on profiling the bottlenecks of I/O (input and output) to the storage devices in supercomputers, and found this was frequently more of an issue than the software designers had realised.

The Cambridge Open Exascale Lab’s plans include: analyse supercomputer power consumption with a view to how to reduce it; provide a method of giving supercomputer time more quickly to scientists that need it for urgent work (such as those responding to pandemics and other disasters); apply an Intel-made programming framework that allows loads to be shared across heterogeneous computers (those involving more than one type of processor, which should make upgrades easier because any new processors do not have to be exactly matched to the existing ones); install faster storage systems (based on solid-state memory chips); improve fast communication between the parts of the computer; and work on new types of graphics to visualise the data produced by the supercomputer.

Cambridge Open Exascale Lab works with a broad range of industry, government, University and other partners. Its industry partners include Dell and Intel Corporation. With its aim to recruit 20 more staff in 2021, hopefully the lab will have sufficient support from talented people to achieve its goal in developing an exascale computing system very shortly.

Cambridge Open Exascale Lab is situated in the West Cambridge Data Centre (WCDC) built by the University of Cambridge at a cost of £20m. This centre is designed to accommodate the rapid growth in demand for high performance computing, and is one of UK’s most energy-efficient high performance computer data centres. It has a high level of security and provides research computing services at a national level.



Most information written in this blog post is mainly from the website of Cambridge Open Exascale Lab.

References:
1. Adam Mann. Core Concept: Nascent exascale supercomputers offer promise, present challenges. PNAS, September 15, 2020 117 (37) 22623-22625).

Coronavirus (46) NHS website on vitamins and minerals

Coronavirus (46) NHS website on vitamins and minerals
After reading the last two blog posts, you might be eager to start a balanced diet in order to strengthen your immune system. The NHS website on vitamins and minerals provides examples of food that can be easily found in UK supermarkets.¹ Here in this blog post, I would like to tabulate the information from the website to make it easier for you to have a look. The harmful effects if we have too much of the vitamins/minerals and the suggested maximum daily intake amount for an adult in the website are also included in the table.

As the daily intake requirement for the vitamins/minerals between adults and children, male and female are different, you might have an interest to have a look at the report from the Public Health of England on dietary recommendations for both children and adults before you plan for your diet.²

Table 1. Sources of vitamins and minerals

	Sources of Vitamin	Can it be stored in the body?	Effects if having too much
Vitamin A	cheese, eggs, oily fish such as trout, salmon, sardines, pilchards, fortified low-fat spreads milk and yoghurt, liver and liver products such as liver pâté	Yes	Having more than an average of 1.5 mg a day of vitamin A over many years may affect your bones, making them more likely to fracture when you're older..Having large amounts of vitamin A can harm your unborn baby.
Beta-carotene (Precursor of vitamin A)	Yellow, red and green (leafy) vegetables, such as spinach, carrots, sweet potatoes and red peppers, yellow fruit, such as mango, papaya and apricots	Yes	If you eat more beta-carotene, less is converted, and the rest is stored in fat reserves in the body. So too much beta-carotene can make you turn yellow, but will not kill you with hypervitaminosis.
Vitamin B1 (Thiamine)	Peas, some fresh fruits (such as bananas and oranges), nuts, wholegrain breads, some fortified breakfast cereals, liver	No	There's not enough evidence to know what the effects might be of taking high doses of thiamin supplements each day. Taking 100mg or less a day of thiamin supplements is unlikely to cause any harm.
Vitamin B2 (Riboflavin)	Milk, eggs, fortified breakfast cereals, mushrooms, plain yoghurt	No	There's not enough evidence to know what the effects might be of taking high doses of riboflavin supplements each day. Taking 40mg or less a day of riboflavin supplements is unlikely to cause any harm.
Vitamin B3 (Niacin: nicotinic acid and nicotinamide. )	Meat, fish, wheat flour, eggs	No	Taking high doses of nicotinic acid supplements can cause skin flushes. Taking high doses for a long time could lead to liver damage. Taking 17mg or less of nicotinic acid supplements a day, or 500mg or less of nicotinamide supplements a day, is unlikely to cause any harm.
Vitamin B5 (Pantothenic acid)	Chicken, beef, liver and kidney, eggs, mushrooms, avocado	No	If you take supplements, do not take too much as this might be harmful. Taking 200mg or less a day of pantothenic acid in supplements is unlikely to cause any harm.
Vitamin B6 (Pyridoxine)	Pork, poultry, such as chicken or turkey, some fish, peanuts, soya beans, wheatgerm, oats, bananas, milk, some fortified breakfast cereals. The bacteria that live naturally in your bowel are also able to make vitamin B6.	No	Taking 200mg or more a day of vitamin B6 can lead to a loss of feeling in the arms and legs known as peripheral neuropathy. This will usually improve once you stop taking the supplements. But in a few cases when people have taken large amounts of vitamin B6, particularly for more than a few months, the effect can be permanent.
Vitamin B7 (Biotin)	Biotin is also found in a wide range of foods, but only at very low levels. The bacteria that live naturally in your bowel are able to make biotin, so it's not clear if you need any additional biotin from the diet.	No	If you take biotin supplements, do not take too much as this might be harmful. Taking 0.9mg or less a day of biotin in supplements is unlikely to cause any harm.
Vitamin B9 (Folate or folic acid)	Broccoli, brussels sprouts, leafy green vegetables such as cabbage, kale, spring greens and spinach, peas, chickpeas and kidney beans, liver (but avoid this during pregnancy), breakfast cereals fortified with folic acid	No	Taking doses of folic acid higher than 1mg can mask the symptoms of vitamin B12 deficiency, which can eventually damage the nervous system Taking 1mg or less a day of folic acid supplements is unlikely to cause any harm.
Vitamin B12	Meat, fish, milk, cheese, eggs, some fortified breakfast cereals	No	There's not enough evidence to show what the effects may be of taking high doses of vitamin B12 supplements each day. Taking 2mg or less a day of vitamin B12 in supplements is unlikely to cause any harm.
Vitamin C (Ascorbic acid)	Citrus fruit, such as oranges and orange juice, peppers, strawberries, blackcurrants, broccoli, brussels sprouts, potatoes	No	Taking large amounts (more than 1,000mg per day) of vitamin C can cause stomach pain, diarrhoea, flatulence. These symptoms should disappear once you stop taking vitamin C supplements.
Vitamin D	The body creates vitamin D from direct sunlight on the skin when outdoors. Vitamin D is also found in a small number of foods. Sources include oily fish – such as salmon, sardines, herring and mackerel, red meat, liver, egg yolks, fortified foods – such as some fat spreads and breakfast cereals	Yes	Taking too many vitamin D supplements over a long period of time can cause too much calcium to build up in the body (hypercalcaemia). This can weaken the bones and damage the kidneys and the heart. If you choose to take vitamin D supplements, 10 micrograms a day will be enough for most adults.
Vitamin E	Plant oils – such as rapeseed (vegetable oil), sunflower, soya, corn and olive oil; nuts and seeds; wheatgerm-found in cereals and cereal product	Yes	There is not enough evidence to know what the effects might be of taking high doses of vitamin E supplements each day. Taking 540mg (800 IU) or less a day of vitamin E supplements is unlikely to cause any harm.
Vitamin K	Green leafy vegetables such as broccoli and spinach, vegetable oils, cereal grains. Small amounts can also be found in meat and dairy foods.	n.a	There's not enough evidence to know what the effects might be of taking high doses of vitamin K supplements each day. Adults need approximately 1 microgram a day of vitamin K for each kilogram of their body weight.
Calcium	Milk, cheese and other dairy foods, green leafy vegetables such as curly kale, okra but not spinach (spinach does contain high levels of calcium but the body cannot digest it all), soya drinks with added calcium, bread and anything made with fortified flour, fish where you eat the bones such as sardines and pilchards	n.a.	Taking high doses of calcium (more than 1,500mg a day) could lead to stomach pain and diarrhoea. Adults aged 19 to 64 need 700mg of calcium a day.
Chromium	Meat, nuts, cereal grains	n.a.	There's not enough evidence to know what the effects might be of taking high doses of chromium each day. Having 10mg or less a day of chromium from food and supplements is unlikely to cause any harm.
Copper	Nuts, shellfish, offal	n.a.	Taking high doses of copper could cause stomach pain, sickness, diarrhoea, damage to the liver and kidneys (if taken for a long time). Having 10mg or less a day of copper supplements is unlikely to cause any harm.
Iodine	Sea fish, shellfish, plant foods such as cereals and grains (the levels vary depending on the amount of iodine in the soil where the plants are grown)	n.a.	Taking high doses of iodine for long periods of time could change the way your thyroid gland works. This can lead to a wide range of different symptoms, such as weight gain. However, taking 0.5mg or less a day of iodine supplements is unlikely to cause any harm.
Iron	Liver (but avoid this during pregnancy); red meat; beans such as red kidney beans, edamame beans and chickpeas; nuts; dried fruit such as dried apricots; fortified breakfast cereals; soy bean flour	n.a.	Side effects of taking high doses (over 20mg) of iron include: constipation, feeling sick, being sick, stomach pain. Very high doses of iron can be fatal, particularly if taken by children. Taking 17mg or less a day of iron supplements is unlikely to cause any harm. But continue taking a higher dose if advised to by a GP.
Manganese	Bread, nuts, breakfast cereals (especially wholegrain), green vegetables such as peas	n.a.	Taking high doses of manganese for long periods of time might cause muscle pain, nerve damage and other symptoms, such as fatigue and depression. For most people, taking 4mg or less of manganese supplements a day is unlikely to cause any harm. For older people, who may be more sensitive to manganese, taking 0.5mg or less of manganese supplements a day is unlikely to cause any harm.
Molybdenum	Molybdenum is found in a wide variety of foods. Foods that grow above ground tend to be higher in molybdenum than foods that grow below the ground, such as potatoes or carrots.	n.a.	There's some evidence to suggest taking molybdenum supplements might cause joint pain.
Phosphorus	Red meat, dairy foods, fish, poultry, bread, brown rice, oats	n.a.	Taking high doses of phosphorus supplements for a short time can cause diarrhoea or stomach pain. Taking high doses for a long time can reduce the amount of calcium in the body, which means bones are more likely to fracture. Taking 250mg or less a day of phosphorus supplements on top of the phosphorous you get from your diet is unlikely to cause any harm.
Potassium	Bananas, some vegetables such as broccoli, parsnips and brussels sprouts, beans and pulses, nuts and seeds, fish, beef, chicken, turkey	n.a.	Taking too much potassium can cause stomach pain, feeling sick and diarrhoea. Taking 3,700mg or less of potassium supplements a day is unlikely to have obvious harmful effects. But older people may be more at risk of harm from potassium because their kidneys may be less able to remove potassium from the blood.
Selenium	Brazil nuts, fish, meat, eggs	n.a.	Too much selenium causes selenosis, a condition that, in its mildest form, can lead to loss of hair and nails. Taking 350μg or less a day of selenium supplements is unlikely to cause any harm.
Sodium chloride (salt)	Ready meals, meat products such as bacon, some breakfast cereals, cheese, tinned vegetables with added salt, some bread, savoury snacks	n.a.	Having too much salt is linked to high blood pressure, which raises your risk of serious problems like strokes and heart attacks. You should have no more than 6g of salt (around 1 teaspoon) a day.
Zinc	Meat, shellfish, dairy foods such as cheese, bread, cereal products such as wheatgerm	n.a.	Taking high doses of zinc reduces the amount of copper the body can absorb. This can lead to anaemia and weakening of the bones. Do not take more than 25mg of zinc supplements a day unless advised to by a doctor.

By now, you might have an idea what you would like to have in your meals. However, before you start the next meal, you can think about preparing it by yourself: buy fresh and unprocessed foods, add less salt and sugar, and use moderate amounts of oil for cooking. This way, you can get the most value of vitamins/minerals from the meal. Last but not least, don’t forget to drink enough water and exercise regularly.³



References
1. Vitamins and minerals. NHS. https://www.nhs.uk/conditions/vitamins-and-minerals/
2. Government dietary recommendations. Government recommendations for energy and nutrients for males and females aged 1 – 18 years and 19+ years. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/618167/government_dietary_recommendations.pdf
3. Nutrition advice for adults during the COVID-19 outbreak. WHO. http://www.emro.who.int/nutrition/news/nutrition-advice-for-adults-during-the-covid-19-outbreak.html

Coronavirus (45) Nutrients help to combat COVID-19 (cont'd)

Coronavirus (45) Nutrients help to combat COVID-19 (cont’d)
In addition to the vitamins mentioned in my last blog post, minerals such as iron, zinc, selenium and copper are also essential for good immunity. They are required in smaller quantities and are therefore called trace minerals. Let us have a look at the same two review articles^1,2 used in the last blog post, on how different trace minerals can protect ourselves from infectious diseases.

Copper
Copper itself is an antimicrobe. Copper supports neutrophil, monocyte and macrophage function and natural killer cell activity.

People on a low copper diet have decreased lymphocyte proliferation and decreased production of IL-2, which is important for immune response. Children with Menke’s syndrome, a rare congenital disease with no circulating copper-carrying protein caeruloplasmin, show immune impairments and have increased bacterial infections and pneumonia. Analysis of studies on Chinese children showed that those with recurrent respiratory tract infection were more likely to have low levels of copper in their hair.¹

Iron
Iron is a trace mineral that we should be careful about the amount we take in. Iron is required for both host and pathogen. Iron deficiency can impair host immunity, while iron overload can cause oxidative stress to propagate harmful viral mutations.²

Iron deficiency has harmful effects on immune function, including impairment of: 1. the ability to generate reactive oxygen species for the killing of harmful microorganisms; 2. bacterial killing; 3. natural killer cell activity; 4. T lymphocyte proliferation, and 5. production of T helper 1 cytokines. These in turn increase susceptibility to infection.¹

On the other hand, infections caused by organisms that spend part of their life-cycle intracellularly may actually be enhanced by iron. In the children living in tropical regions, iron at doses above a particular threshold has been associated with increased risk of malaria and other infections, including pneumonia. Thus, iron intervention in malaria-endemic areas is not advised.¹ Moreover, a study giving iron (50 mg on each of 4 days a week) to iron-deficient schoolchildren in South Africa increased the risk of respiratory infections.¹

In general, the harmful consequences of iron overdoses on infections include: 1. Impairment of immune function; 2. Excess iron favours damaging inflammation; 3. Helping the growth of pathogens that require iron.

Selenium
Selenium deficiency adversely affects several components of both innate* and acquired immunity,** and increases susceptibility to infections.¹

It is of concern to find that dietary selenium deficiency induces rapid mutation of benign variants of RNA viruses to virulence. Deficiency in selenium can cause oxidative stress in the host, and can alter a viral genome so that a normally benign or mildly pathogenic virus can become highly virulent.² Selenium could assist a group of enzymes that, in concert with vitamin E, work to prevent the formation of free radicals and prevent oxidative damage to cells and tissues.

It was reported that combination of selenium with ginseng stem-leaf saponins could induce immune response to a live bivalent infectious bronchitis coronavirus vaccine in chickens.² Therefore, the review article written by Zhang et al suggests that selenium supplementation could be an effective choice for the treatment of novel variants of COVID-19.²

You may wonder how much selenium we need to maintain the normal function of our immunity. It was found that selenium supplementation with 100 to 300 µg/day could improve various aspects of immune function in humans including in the elderly. Selenium supplementation of 50 or 100 µg/day in adults in the UK with low selenium status improved some aspects of their immune response to a poliovirus vaccine.¹

Zinc
Zinc has an important role in maintaining and developing immune cells of both the innate* and adaptive immune system.***

Especially you may find it interesting that zinc seems to play an important role in antiviral defence. It was found to inhibit the RNA polymerase required by RNA viruses to replicate. Moreover, zinc supports proliferation of CD8+ cytotoxic T lymphocytes, key cells in antiviral defence. These findings suggest that zinc might play a key role in host defence against the RNA virus SARS-CoV-2 that cause COVID-19.¹ In fact, the combination of zinc and pyrithione at low concentrations inhibits the replication of SARS coronavirus.²

Zinc deficiency has a marked impact on bone marrow by decreasing the number of immune precursor cells. Therefore, zinc is important in maintaining T and B lymphocyte numbers. Moreover, antibody production is decreased in zinc deficiency. Zinc deficiency also impairs many aspects of innate immunity, including phagocytosis and natural killer cell activity. Patients with the zinc malabsorption syndrome, acrodermatitis enteropathica, display severe immune impairments and increased susceptibility to bacterial, viral and fungal infections.¹

Correcting zinc deficiency lowers the likelihood of respiratory and skin infections. Recent reviews and analysis of trials with zinc reported shorter durations of common cold in adults, reduced incidence and prevalence of pneumonia in children, and reduced mortality when given to adults with severe pneumonia.¹

Conclusion
After reading the two blog posts on the different nutrients and their importance in fighting against infection, we understand we should have a balanced diet in order to maintain our immune system to prevent respiratory diseases such as COVID-19. No single nutrient should be left out in order to attain the optimum condition of our immune system for health.

As new pathogens responsible for influenza continually emerge, and outbreaks of new variants of the SARS-CoV-2 virus are highly possible, it is especially necessary to have a dietary regimen that includes all the nutrients in order to reduce the adverse effects from new or mutating pathogens.



*The innate immune system is the body’s first line of defense against germs. The innate immune system consists of 1. skin and mucous membranes that forms a physical barrier against germs; 2. immune system cells (defense cells) and proteins that are activated upon inflammation; 3. white blood cells (leukocytes) that kill bacteria or viruses, by phagocytosis, that enter the body; 4. natural killer cells specialized in identifying cells that are infected by a virus, and then destroy the cell surface using cell toxins.³
Since the innate immune system responds in the same way to all germs and foreign substances, it is also referred to as the "nonspecific" immune system. It acts very quickly: it makes sure that bacteria that have entered the skin through a small wound are detected and destroyed on the spot within a few hours. However, the innate immune system has only limited power to stop germs from spreading.³
**“Acquired immunity is a type of immunity that develops when a person’s immune system responds to a foreign substance or microorganism, or that occurs after a person receives antibodies from another source. The two types of acquired immunity are adaptive and passive. Adaptive immunity occurs in response to being infected with or vaccinated against a microorganism. The body makes an immune response, which can prevent future infection with the microorganism. Passive immunity occurs when a person receives antibodies to a disease or toxin rather than making them through his or her own immune system.” (from online NCI (National Cancer Institute) dictionary. https://www.cancer.gov/publications/dictionaries/cancer-terms/def/acquired-immunity)
***The adaptive immune system takes over if the innate immune system is not able to destroy the germs. The adaptive immune system is made up of 1. T lymphocytes in the tissue between the body's cells; 2. B lymphocytes which are also found in the tissue between the body's cells; 3. antibodies in the blood and other bodily fluids.
The adaptive immune system specifically targets the type of germ that is causing the infection. It first identifies the germ, which makes it slower to respond than the innate immune system, and then it destroys it. It can "remember" germs, so the next time a known germ is encountered, the adaptive immune system can respond faster.



References
1. P.C. Calder. Nutrition, immunity and COVID-19. Review. BMJ Nutrition, Prevention & Health. 2020 May 20;3(1):74-92.
2. L. Zhang, and Y. Liu. Potential interventions for novel coronavirus in China: A systematic review. Journal of Medical Virology, 2020 May 92(5):479-490.
3. The innate and adaptive immune systems. InformedHealth.org. Cologne, Germany: Institute for Quality and Efficiency in Health Care (IQWiG); 2006-. https://www.ncbi.nlm.nih.gov/books/NBK279396/

Coronavirus (44) Nutrients help to combat COVID-19

Coronavirus (44) Nutrients help to combat COVID-19
Since the outbreak of COVID-19 in the UK, which led to the first national lockdown in March 2020, the pandemic has already lasted for more than 15 months. During this period of time, many countries in the world experienced several waves of COVID-19 outbreaks, and quite a few major variants of the SARS-CoV-2 virus emerged. For the UK, the country has been attacked by the wild type, the variant from South Africa (Beta variant, B1.351), the variant from Kent (Alpha variant, B.1.1.7), and recently the variant that originated from India (Delta variant, B1.617.2).

The existing therapies and vaccines against COVID-19 were designed based on the SARS-CoV-2 virus first identified. The longer the virus spread among people, the higher the chances that the virus would mutate. The mutated variants that later become dominant are usually more virulent, and they are more resistant, to various degrees, to the existing therapies and vaccines. Unless we could produce a specific therapy or vaccine in time for an emerged variant, a good immune system, which is able to respond promptly and appropriately to different challenges, is very important to protect us against any SARS-CoV-2 variants.

Optimal nutritional status and lifestyle habits are essential to keeping our immune systems working properly. Here in this blog post, I would like to share with you findings from two review articles on how different nutrients can help us protect ourselves from infectious diseases.^1,2 This might give us an idea of what we could prepare for our meals in order to strengthen our health to combat coronavirus-related diseases.

Vitamin A
Vitamin A has been called an “anti-infective vitamin”. It is essential for body's defences against infection as it is important for normal differentiation of epithelial tissue.*^1,2 Lack of vitamin A is associated with increased susceptibility to respiratory infections, diarrhoea and severe measles.

Moreover, vitamin A is important for immune cell maturation and function: Vitamin A controls maturation of neutrophils, macrophages, natural killer cells, dendritic cells, and CD4+ T lymphocyte, which are involved in the killing of pathogens.¹

As we are undergoing a national vaccination program in the UK, it is also relevant to note that vitamin A deficiency can impair the body’s response to vaccination. Vitamin A’s metabolite, retinoic acid, is required for normal functioning of B lymphocytes, including antibody generation. An example from Indonesian children with vitamin A deficiency showed a higher antibody response to tetanus vaccination after providing them with vitamin A, suggesting that lack of vitamin A can impair the response to vaccination.¹

B group vitamins
B vitamins are water-soluble vitamins and work as part of coenzymes.² B vitamins are generally involved in intestinal immune regulation, thus contributing to gut barrier function.¹ Vitamins B6 and B12 and folate (Vitamin B9) all support the activity of natural killer cells and CD8+ cytotoxic T lymphocytes, effects which would be important in antiviral defence.¹ Lack of vitamin B6 deficiency causes thymus and spleen atrophy, low blood T lymphocyte numbers, and impaired lymphocyte proliferation and T lymphocyte-mediated immune responses,¹ while vitamin B12 deficiency decreases phagocytic** and bacterial killing capacity of neutrophils. In general, shortage of B vitamins weakens the host immune response.

Other B vitamins also has their special functions.1 Vitamin B2 (riboflavin) and UV light effectively reduced the amount of MERS-CoV in human plasma products.^2,3 Vitamin B3 (nicotinamide) could enhance the killing of Staphylococcus aureus (bacteria which often cause skin infections, pneumonia, heart valve infections, and bone infections). Moreover, lung injury during mechanical ventilation is usually seen in the severe cases of COVID-19 who need ventilators to get oxygen into body. Vitamin B3 treatment to these patients has a strong anti-inflammatory effect as it significantly inhibits neutrophil infiltration into the lungs.² Neutrophil infiltration in inflamed lung causes damage to the lung, and is a hallmark of Acute Respiratory Distress Syndrome in severe COVID-19 cases.

Vitamin C
Vitamin C is involved in collagen biosynthesis in connective tissues and is important for maintaining epithelial integrity (tissue in glands and linings).***^1,2

Its roles in immunity include leucocyte migration to sites of infection, phagocytosis** and bacterial killing, natural killer cell activity, T lymphocyte function (especially of CD8+ cytotoxic T lymphocytes) and antibody production1 (similar to the function of vitamin A).

Vitamin C supplementation has been shown to decrease the duration and severity of upper respiratory tract infections such as the common cold.¹ People deficient in vitamin C are susceptible to severe respiratory infections such as pneumonia.¹ This suggests that vitamin C might prevent the susceptibility to lower respiratory tract infections. Furthermore, vitamin C may also protect against infection caused by a coronavirus, as vitamin C increased the resistance of cultures of chick embryo tracheal organ to avian coronavirus infection.² As COVID-19 is related to lower respiratory tract infection, the Chinese researchers of a review article even suggest vitamin C could be one of the choices for COVID-19 treatment.²

Vitamin D
Vitamin D receptors are found in most immune cells. Vitamin D stimulates the maturation of many immune cells, and enhances epithelial integrity. Vitamin D also induces antimicrobial peptide synthesis in epithelial cells and macrophages, directly enhancing host defence.¹ Moreover, vitamin D increases phagocytosis, superoxide^# production and bacterial killing by innate immune cells.¹

A study from Taiwan found that people with vitamin D deficiency has lower antibody response, after vaccination with influenza A virus subtype H3N2 and B strain, than the group of people with normal vitamin D levels.¹ Studies using data from British and American populations suggested that vitamin D levels is inversely correlated with respiratory infection. This means the lower the vitamin D levels, the higher the risk of viral respiratory tract infection.¹

Vitamin D can be synthesized in our body with the help of sunlight. Summer is the time with sufficient sunlight, but over a year it is only a short time, so a high proportion of healthy adults in the UK are reported to have low levels of vitamin D. Moreover, the reduced outdoor journies due to the COVID-19 pandemic, further decreases the chance of people to absorb sunlight. Therefore, in addition to absorbing vitamin D from food, vitamin D deficient patients in the UK are usually prescribed vitamin D supplements by a GP. Meanwhile, a study in Japan found that supplementation of Japanese schoolchildren with vitamin D for 4 months during winter reduces the risk of influenza A by about 40%.¹

Vitamin E
Vitamin E is a lipid-soluble antioxidant that plays an important role in reducing oxidative stress through binding to free radicals.²

Vitamin E also plays a role in immune response and enhances antibody production.¹ The effect of vitamin E is especially obvious in healthy adults over 60 years of age. Research found a positive association between plasma vitamin E and cell-mediated immune response, and a negative association between plasma vitamin E and the risk of infections in this age group.¹ Studies by the Nutrition Research Center on Ageing at Tufts University in Boston demonstrated that vitamin E supplementation at high doses (800 mg/day) enhanced T helper 1 cell-mediated immunity (lymphocyte proliferation and IL-2 production), and improved vaccination response to the hepatitis B virus.¹ The same research group also reported that a daily intake of vitamin E supplement (135mg/day) for a year decreased upper respiratory tract infections, particularly the common cold, in elderly residents of a nursing home.¹ A study from Spain provided further evidence that supplementation of older adults with vitamin E improved their immunity defences.¹

Information on other nutrients to protect ourselves from infectious diseases will be presented in my next blog post.



*Epithelial tissue covers most of the external and internal surfaces of the body and its organs. These tissues serve as the first line of defence against inorganic, organic, and microbial intruders. Epithelial cells are the main cell type of these tissues.⁴
**Phagocytosis is a process of ingesting harmful foreign particles, bacteria, and dead or dying cells.
***Epithelial cells are the main cell type of epithelial tissues, which cover most of the external and internal surfaces of the body and its organs.⁴ Epithelial integrity is very important as a first line of defence against inorganic, organic, and microbial intruders.
^#Superoxide is a reactive oxygen species. It is generated by the immune system to kill invading pathogens in oxygen-dependent killing mechanisms.




References
1. P.C. Calder. Nutrition, immunity and COVID-19. Review. BMJ Nutrition, Prevention & Health. 2020, May 20;3(1):74-92.
2. L. Zhang, and Y. Liu. Potential interventions for novel coronavirus in China: A systematic review. Journal of Medical Virology, 2020, May 92(5):479-490.
3. S.D. Keil, R. Bowen, and S. Marschner. Inactivation of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment. Transfusion. 2016;56:2948-2952.
4. J. Gunther, & H-M. Seyfert. The first line of defence: insights into mechanisms and relevance of phagocytosis in epithelial cells. Semin Immunopathol. 2018; 40(6): 555–565.

Coronavirus (43) Mass asymptomatic testing of SARS-CoV-2 using lateral flow devices (cont’d)

Coronavirus (43) Mass asymptomatic testing of SARS-CoV-2 using lateral flow devices (cont’d)
Continued from my last blog post.
Limitation of lateral flow tests
The lateral flow test kit from Innova for detection of SARS-CoV-2 infections has been tested by Public Health England and validated by the UK government.^1,2 It was initially tested among 132 candidates when the UK government were considering the use of lateral flow devices (LFDs) in mass-testing for COVID-19 in an asymptomatic population.¹

The test report showed that all the tested lateral flow devices have a viral antigen detection rate of >90% at 100,000 RNA copies/ml (for comparison, only 3,600 to 10,000 copies/ml of virus in the sample is already enough to be detected by RT-PCR, which is more than ten times as sensitive). The study found a kit failure rate of 5.6% from 8951 Innova SARS-CoV-2 Antigen Rapid Qualitative Tests. The most common reason for kit failure was poor transfer of the liquid within the device from the reservoir onto the test strip. ¹

The false positive rate was 0.32% from 6954 Innova tests. This means that for every 1,000 people tested, only 3 people would get a false positive result. The study also found that the sensitivity (the detection rate of positive cases) across the sampling cohort is significantly dependent on the test operator. Sensitivity of the tests performed by laboratory scientists was 78.8%, trained healthcare-workers was 70%, while the self-trained members of the public was 57.7%.¹

The above study showed that the kit failure rate for the tests is not low, and they tend to give a more accurate result only if a sample with higher viral concentration is being tested and the user performing the test is well-trained. This means the overall accuracy of the lateral flow tests is generally lower among asymptomatic people who are not well-trained and who have a generally lower viral concentration.

When it comes to the real world evaluation of Innova SARS-CoV-2 Antigen Rapid Qualitative Test, the sensitivity of the test is found to be much lower than the above report. The Innova lateral flow test was used in a mass test of the population in Liverpool last November.³ By evaluation of the performance of the Innova lateral flow test against RT-PCR testing using data from 5,869 people, it was found that 60% of infected people could not be detected by the lateral flow tests. On the other hand, the test performed better for detecting cases in people with higher viral loads, with test sensitivity in this group at 66.7%.³ Similar to the result from the other study, the specificity was 99.9% in this study.^1,3 This means the positive results from the lateral flow tests are highly accurate.

Based on these research studies, the general sensitivity of the lateral flow tests are 40–76%, which means that about half of infected people may be missed.^1,3,4 Those carrying COVID-19 who were wrongly told they were free of the virus could transmit to more people than those who do not have the tests, due to a false sense of security. Therefore, many scientists called on the government at least to pause the rollout of rapid asymptomatic testing using the Innova tests, as they are sceptical that the lateral flow tests are able to control effectively the transmission of infection.^5,6

Can the lateral flow test detect COVID-19 variants?
There has no research paper or data available yet on the efficiency of the Innova SARS-CoV-2 Antigen Rapid Qualitative Test to detect the Kent and Indian variants which are now prevalent in the UK. The planned rollouts of lateral flow tests in schools were paused because of concerns about the risk of missing cases caused by the new and more transmissible SARS-CoV-2 variants.⁷

Where can we get the free lateral flow test?
After reading all the information about the free of charge lateral flow test scheme, you might want to try to have one yourself. The free rapid lateral flow tests are being made available by the government in England. As long as you are in England, aged 11 or above, and have no COVID-19 symptoms, you can order one pack of lateral flow tests per day online and it will be delivered to your home. Each pack contains 7 tests.⁶

​ The tests can also be collected at local PCR test sites and most of the local pharmacies in England. It is very important to remember that the tests are just for asymptomatic people: if you have COVID-19 symptoms, you should not go outside to collect a test; instead, you should order a PCR test and self-isolate.⁶

Who could benefit from the free lateral flow tests?
The sensitivity of lateral flow tests are in doubt, and the usefulness of the lateral flow tests being used as a tool to control the transmission of infection is being questioned by some scientists. However, as long as the tests are repeated twice a week as suggested by the government, there is still a chance of the asymptomatic infections being detected. Therefore, the free lateral flow test scheme available in England provides certain degree of protection for the households who have members working in patient-facing or customer-facing sectors.

However, you have to remember that the test on average misses about half of the COVID-19 infectious cases, i.e. a negative result does not rule out a COVID-19 infection. If your result is negative from the lateral flow test, it is very important to still follow all the current restrictions imposed by the government.



References
1. Peto T & UK COVID-19 Lateral Flow Oversight Team. COVID-19: rapid antigen detection for SARS-CoV-2 by lateral flow assay: a national systematic evaluation for mass-testing. medRxiv. 2021; (published online Jan 26.) (preprint). https://doi.org/10.1101/2021.01.13.21249563
(Later published online in Lancet, May 29, 2021. DOI:https://doi.org/10.1016/j.eclinm.2021.100924)
2. Order coronavirus (COVID-19) rapid lateral flow tests. GOV.UK website. https://www.gov.uk/order-coronavirus-rapid-lateral-flow-tests
3. Liverpool COVID-19 community testing pilot. Interim evaluation report by the University of Liverpool. 23 December, 2020. https://www.liverpool.ac.uk/media/livacuk/coronavirus/Liverpool,Community,Testing,Pilot,Interim,Evaluation.pdf
4. RT-LAMP assay: Fail to detect positive cases more than 50% in November in Manchester. “Covid-19: Rapid test missed over 50% of positive cases in Manchester pilot. BMJ 2020; 371 doi: https://doi.org/10.1136/bmj.m4323 (Published 06 November 2020) Cite this as: BMJ 2020;371:m4323”
5.UK government must urgently rethink lateral flow test roll out, warn experts. The BMJ news, 11/01/21. https://www.bmj.com/company/newsroom/uk-government-must-urgently-rethink-lateral-flow-test-roll-out-warn-experts/
6. Covid-19: UK regulator approves lateral flow test for home use despite accuracy concerns. BMJ news, 2020; 371 doi: https://doi.org/10.1136/bmj.m4950 (Published 23 December 2020)
7. Mass testing of COVID-19: January update on lateral flow tests. UK Parliament Post, 29 January 2021. https://post.parliament.uk/mass-testing-for-covid-19-january-update-on-lateral-flow-tests/

Coronavirus (42) Mass asymptomatic testing of SARS-CoV-2 using lateral flow devices

Coronavirus (42) Mass asymptomatic testing of SARS-CoV-2 using lateral flow devices
In order to contain the spread of COVID-19, in addition to the national lockdown and nationwide COVID-19 vaccination, the UK government also use reverse-transcription polymerase chain reaction (RT-PCR) testing of nose/throat swabs, contact tracing procedures, and mobile applications to identify close contacts of infected symptomatic individuals.

In addition to the above measures, the government also rolled out mass asymptomatic testing on 9th April, aiming to identify people with COVID-19 who are not displaying any symptoms. This testing programme allows everyone in England to access two COVID-19 tests a week, free of charge, even if they do not have any symptoms.¹ Ideally, by quickly identifying the asymptomatic patients and having these people self-isolate, and through the rapid finding and testing of their close contacts, the spread through the community could be interrupted.¹

The programme uses the lateral flow test kit, SARS-CoV-2 Rapid Antigen Lateral Flow Qualitative Test, from Innova. Since many may have heard of the programme but not used the test kit, this and the coming blog posts provide more information about the test so that you can have a better idea of it.

What are lateral flow tests?
Lateral flow tests are basically assays designed to test for different protein targets. A sample is placed on a conjugation pad where the analyte (or antigen) of interest is bound by conjugated antibodies. The analyte-antibody mix then migrates along a membrane by capillary flow, across both ‘test’ and ‘control’ strips. These strips are coated with antibodies detecting the analyte of interest, and a positive test is confirmed by the appearance of coloured control and test lines.²

As no laboratory processing is needed, lateral flow tests can be performed in the area convenient to the person being tested. Moreover, a short turnaround time, relatively higher test accuracy and the economic affordability make the tests suitable for mass testing. In fact, the tests have been used for rapid testing in communities and workplaces.³

Innova SARS-CoV-2 Antigen Rapid Qualitative Test is a disposable test kit, like a home pregnancy test kit. It detects nucleocapsid protein antigen from SARS-CoV-2 in direct nose and/or throat swabs. A positive result is seen as a dark band or a fluorescent glow on the “test” strip after about 30 minutes.⁴ The appearance of two lines on both the “test” and “control” strips indicates that the test is positive. The appearance of one line on the “control” strip only indicates a negative result. However, if the “control” strip line does not appear after 30 minutes of waiting, this means the test has failed to work and should be retaken.

The whole procedure to perform the test by yourself is written clearly in the instruction booklet included in each pack of the test kit. It is interesting to find that, according to the instruction booklet distributed to NHS staff, NHS recommends only a nasal swab is used and the sample is taken in a different way to that described in the packaged instructions for use, with more rotation of the swab at a lower level of penetration, to enable easier self-administration of the test.⁵

Storage precautions
Test kits should be stored at room temperature. It is very important to keep the test kits in an area with no direct sunlight, neither should the test kit be kept in a fridge or freezer. High or low temperatures can denature or inactivate the antibodies in the kit and affect the result.

Instructions after knowing the results
If you are taking the lateral flow test at home, you should register the results, whether positive or negative, online or by calling 119. If you get a positive test result, everyone in your household must self-isolate according to the government guidelines.⁶ Moreover, you need to take an RT-PCR test to further confirm the result.^1,6

If the test result is invalid, you need to retake the test with a new test kit.



References
1. Mass asymptomatic COVID-19 testing: Strategy and accuracy. Research briefing, House of Commons Library, 12 May, 2021. https://commonslibrary.parliament.uk/research-briefings/cbp-9223/
2. O'Farrell B. Evolution in lateral flow-based immunoassay systems. Lateral Flow Immunoass. 2009; p 1-33. https://doi.org/10.1007/978-1-59745-240-3_1.
3. Peto T & UK COVID-19 Lateral Flow Oversight Team. COVID-19: rapid antigen detection for SARS-CoV-2 by lateral flow assay: a national systematic evaluation for mass-testing. medRxiv. 2021; (published online Jan 26.) (preprint). https://doi.org/10.1101/2021.01.13.21249563
(Later published online in Lancet, May 29, 2021. DOI:https://doi.org/10.1016/j.eclinm.2021.100924)
4. Primary Care Supply webpage for Innova SARS-CoV-2 Antigen Rapid Qualitative Test. https://www.primarycaresupplies.co.uk/innova-sars-cov-2-antigen-lateral-flow-rapid-test-kit-box-of-20/
5. A guide for healthcare staff self-testing for coronavirus using a Lateral Flow Device (LFD). By NHS. https://www.england.nhs.uk/coronavirus/wp-content/uploads/sites/52/2020/11/LFD_NHSStaff_A4_161120_.pdf
6. Order coronavirus (COVID-19) rapid lateral flow tests. GOV.UK website. https://www.gov.uk/order-coronavirus-rapid-lateral-flow-tests