Bioinformatics tools used for the identification of cis-regulatory elements
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",isbn:"978-1-83968-924-6",printIsbn:"978-1-83968-923-9",pdfIsbn:"978-1-83968-925-3",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"ea4ec0d6ee01b88e264178886e3210ed",bookSignature:"Dr. Hiran Wimal Amarasekera",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9500.jpg",keywords:"Bone Tumors, Oncology, Childhood Tumors, Cancer, Risk Factors, Modern Management, Benign Lesions, Tumor-Like Conditions, Immunology, Histochemistry, Cell Oncology, Tumor Markers",numberOfDownloads:308,numberOfWosCitations:0,numberOfCrossrefCitations:1,numberOfDimensionsCitations:1,numberOfTotalCitations:2,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 28th 2020",dateEndSecondStepPublish:"October 26th 2020",dateEndThirdStepPublish:"December 25th 2020",dateEndFourthStepPublish:"March 15th 2021",dateEndFifthStepPublish:"May 14th 2021",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Consultant Orthopaedic Surgeon from Sri Lanka currently working in University Hospitals of Coventry and Warwickshire, UK, trained at the National Hospital of Sri Lanka, at the Oldchurch Hospital in Essex UK and The Avenue Hospital Melbourne, Australia and University Hospitals of Coventry and Warwickshire, UK, obtained the FRCS from Royal College of Surgeons of Edinburgh, Scotland.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"67634",title:"Dr.",name:"Hiran",middleName:"Wimal",surname:"Amarasekera",slug:"hiran-amarasekera",fullName:"Hiran Amarasekera",profilePictureURL:"https://mts.intechopen.com/storage/users/67634/images/system/67634.jpg",biography:"Hiran Amarasekera is a Consultant Orthopaedic Surgeon from Sri Lanka currently working in University Hospitals of Coventry and Warwickshire, the UK as a hip preservation fellow. \r\nHis special interests include young adult hip and knee problems, sports injuries, Hip and knee arthroplasty, and complex arthroscopic procedures. \r\nHe completed the MBBS from Kasturba medical college Manipal, India and did his postgraduate in Trauma and Orthopaedics at the Post-graduate Institute of the Medicine University of Colombo obtained the MS. \r\nHe was initially trained at the National Hospital of Sri Lanka and then completed the further training at the Oldchurch Hospital in Essex UK and The Avenue Hospital Melbourne, Australia and University Hospitals of Coventry and Warwickshire, UK.\r\nHe obtained the FRCS from Royal College of Surgeons of Edinburgh in 2003 and was elected a fellow of Sri Lanka College of surgeons (FCSSL) 2012. \r\nHe has a keen interest in academia and research. Working as a clinical research fellow in Warwick Medical School he obtained the MPhil form University of Warwick and was elected for a research fellowship to University of California Los Angeles (UCLA). \r\nHis research interests include blood flow to the hip, failure of hip resurfacing, designing new hip prosthesis, and surgical approaches to the hip. \r\nHe has over 30 international publications and presentations and several book chapter. \r\nHe also works as a reviewer for international orthopedic journals and has reviewed over 35 papers and is a member of the editorial board of Sri Lanka Journal of Surgery.",institutionString:"University of Warwick Science Park",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"University of Warwick Science Park",institutionURL:null,country:{name:"United Kingdom"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:[{id:"73224",title:"Management of Early Osteoarthritis",slug:"management-of-early-osteoarthritis",totalDownloads:78,totalCrossrefCites:0,authors:[null]},{id:"71336",title:"Non-Surgical Regional Therapy for Osteoarthritis: An Update and Review of the Literature",slug:"non-surgical-regional-therapy-for-osteoarthritis-an-update-and-review-of-the-literature",totalDownloads:155,totalCrossrefCites:0,authors:[{id:"77195",title:"Dr.",name:"Charles",surname:"Mackworth-Young",slug:"charles-mackworth-young",fullName:"Charles Mackworth-Young"}]},{id:"72715",title:"Simultaneous Bilateral Joint Arthroplasties in Treatment of Osteoarthritis",slug:"simultaneous-bilateral-joint-arthroplasties-in-treatment-of-osteoarthritis",totalDownloads:75,totalCrossrefCites:1,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"247865",firstName:"Jasna",lastName:"Bozic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/247865/images/7225_n.jpg",email:"jasna.b@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"19008",title:"The Use of Functional Genomics in Synthetic Promoter Design",doi:"10.5772/20653",slug:"the-use-of-functional-genomics-in-synthetic-promoter-design",body:'The scope of this chapter is to examine how advances in the field of Bioinformatics can be applied in the development of improved therapeutic strategies. In particular, we focus on how algorithms designed to unravel complex gene regulatory networks can then be used in the design of synthetic gene promoters that can be subsequently incorporated in novel gene transfer vectors to promote safer and more efficient expression of therapeutic genes for the treatment of various pathological conditions.
A synthetic promoter is a sequence of DNA that does not exist in nature and which has been designed to control gene expression of a target gene. Cis-regulatory sequences derived from naturally-occurring promoter elements are used to construct these synthetic promoters using a building block approach; which can either be carried out by rational design or by random ligation (illustrated in figure 1A). The result is a sequence of DNA composed of several distinct cis-regulatory elements in a completely novel orientation that can act as a promoter enhancer; typically to initiate RNA polymerase II-mediated transcription.
Construction of synthetic promoters is possible because of the modular nature of naturally-occurring gene regulatory regions. This was cleverly demonstrated by a group that used synthetic promoters to evaluate the role of the TATA box in the regulation of transcription (Mogno et al., 2010). The authors looked at the role of the TATA box in dictating the strength of gene expression. They found that the TATA box is a modular component in that its strength of binding to the RNA polymerase II complex and the resultant strength of transcription that it mediates is independent of the cis-regulatory element enhancers upstream. Importantly, they also found that the TATA box does not add noise to transcription, i.e. it acts as a simple amplifier without altering specificity of gene expression dictated by the upstream enhancer elements. Thus implying that any combination of cis-regulatory enhancers could be coupled to a TATA box and it would be the enhancers that would mediate specificity without any interference from the TATA box. The implications from this study suggest that it should be possible to construct any type of synthetic promoter that is specifically engineered to display a highly restrictive pattern of gene regulation.
Synthetic promoters have been used in the study of gene regulation for more than two decades. In one of the first examples a synthetic promoter derived from the lipoprotein gene in E. Coli was used to efficiently drive the expression of a number of tRNA genes (Masson et al., 1986). In the years that followed a technique was developed that enabled the mutation of prokaryotic sequences flanking the essential -10 and -35 promoter elements (illustrated in figure 1B) and thus the efficient construction of synthetic promoters for use in bacteria (Jacquet et al., 1989). This approach was successfully used to produce promoters with much higher activity compared to naturally occurring sequences and it was immediately realised that such an approach would have important applications in the biotech industry, particularly in the enhanced production of biopharmaceuticals (Trumble et al., 1992).
Typical synthetic promoter layouts for prokaryotes and eukaryotes
Most of these studies were initially undertaken with a view to establish the important structural features of prokaryotic or eukaryotic promoters so that essential elements could be identified. In one example, the role of the Tat protein in the regulation of HIV gene expression was studied using synthetic promoters (Kamine et al., 1991). In this study a series of minimal promoters containing Sp1- binding sites and a TATA box were constructed and analysed to see if the Tat protein from HIV could activate them. The results demonstrated that Tat could only activate the synthetic promoters containing Sp1 sites and not promoters with the TATA box alone. The observations enabled the authors to propose that in vivo the Tat protein is brought to the promoter site by TAR RNA and then interacts with Sp1 to drive gene expression. In recent years more sophisticated studies using synthetic promoters have been undertaken to evaluate the important factors driving transcription factor binding to their corresponding cis-regulatory elements (Gertz et al., 2009a) and to thermodynamically model trans-factor and cis-element interactions (Gertz et al., 2009b).
As alluded to above, it was soon realised that synthetic promoter technology had direct implications in the improvement of the efficiency of gene expression. Indeed, one of the most widely used eukaryotic promoters employed for research purposes today is actually a synthetic promoter. The steroid-inducible Glucocorticoid Receptor Element (GRE) is a naturally occurring sequence that regulates the expression of a plethora of genes that are responsive to glucocorticoids. In a relatively early study several of these elements were linked together in order to construct a promoter with enhanced responsiveness to these steroids (Mader et al., 1993). This study detailed the construction of a 5 x GRE synthetic promoter linked to the Adenovirus type 2 major late promoter TATA region that displayed 50-fold more expression levels in response to steroid hormones when compared to the natural promoter sequence. This synthetic promoter is now a widely used constituent of a number of reporter constructs adopted in a variety of different research applications.
Finally, synthetic promoters have also been used in prokaryotic systems to reveal that regulation of gene expression follows boolean logic (Kinkhabwala et al., 2008). In this prototypical study the authors found that two transcription repressors generate a NOR logic; i.e. a OR b (on OR off), while one repressor plus one activator determines an ANDN logic; i.e. a AND NOT b (on AND NOT off). This idea was later expanded on to demonstrate that various combinations of synthetic promoters could combine to generate 12 out of 16 boolean logic terms (Hunziker et al., 2010). Most interestingly the results from these studies demonstrated that if a promoter does not follow a specific logic it is more likely to be leaky, in that it will drive gene expression under conditions where it is not expected to.
In this chapter we describe the evolution of synthetic promoter technology, its application in the development of improved tissue-specific promoters and its potential use for the development of effective disease-specific gene regulators; thus enabling the development of safer and more effective gene therapies.
In recent years some efforts have been made to construct synthetic promoters for tissue specific transcription based on the linking of short oligonucleotide promoter and enhancer elements in a random (Li et al., 1999; Edelman et al., 2000) or ordered (Chow et al., 1997; Ramon et al., 2010) fashion.
In what can be described as one of the first attempts to rationally design a tissue-specific synthetic promoter, Chow et al. describe the rearrangement of the cytokeratin K18 locus to construct a promoter mediating a highly restrictive pattern of gene expression in the lung epithelium (Chow et al., 1997). In this study the authors describe the generation of transgenic mice with this construct and demonstrate expression only in the lung. They also generated CMV (Cytomegalovirus) and SV40 (Sarcoma Virus 40) promoter based constructs and found lack of specificity and no expression in the lung epithelia. This study had important implications for researchers developing lung-based gene therapies, i.e. if CMV, one of the most widely used promoters, could not regulate gene expression in the lung epithelia then it is necessary to identify (or develop) new promoters that can efficiently regulate gene expression in this location. Indeed, it is now becoming increasingly apparent that traditional virus-derived promoters like CMV and RSV (Rous Sarcoma Virus) will have limited application in the development of modern gene therapeutics.
The random assembly of cis-regulatory elements has shown particular success as a means to develop synthetic promoters. In one such approach, which aimed to identify synthetic promoters for muscle-specific expression, duplex oligonucleotides from the binding sites of muscle-specific and non-specific transcription factors were randomly ligated and cloned upstream of a minimal muscle promoter driving luciferase (Li et al. 1999). Approximately 1000 plasmid clones were individually tested by transient transfection into muscle cells and luciferase activity was determined in 96-well format by luminometry. By this approach several highly active and muscle specific promoters were identified that displayed comparable strength to the most commonly used viral promoters such as CMV.
Typical procedure for generation of synthetic mammalian promoters (reproduced from PNAS, Vol. 97, No. 7, pp. 3038-3043 copyright (c) 2000 by the National Academy of Sciences, USA)
Retroviral vectors have also been used to screen for synthetic promoters in eukaryotic cells (Edelman et al., 2000). This study was the first description of a retroviral library approach using antibiotic resistance and FACS selection to isolate promoter sequences (illustrated in figure 2). The libraries generated using random oligonucleotides in an effort to identify new sequences as well as examining the effects of combinations of known elements and for uncovering new transcriptional regulatory elements. After preparing a Ran18 promoter library comprises random 18mer oligonucleotides, the authors analysed the sequences of the generated synthetic promoters by searching for known transcription factor binding motifs. They found that the highest promoter activities were associated with an increased number of known motifs. They examined eight of the best known motifs; AP2, CEBp, gre, ebox, ets, creb, ap1 AND sP1/maz. Interestingly, several of the promoter sequences contained none of these motifs and the author\'s looked for new transcription factors.
In a similar effort employed to examine one million clones, Sutton and co-workers adopted the FACS screening approach based on the establishment of a lentiviral vector-based library (Dai et al., 2004). In this study duplex oligonucleotides from binding sites of endothelial cell-specific and non-specific transcription factors were cloned in a random manner upstream of a minimal promoter driving expression of eGFP in a HIV self-inactivating expression vector. A pool of one million clones was then transfected into endothelial cells and the highest expressers were selected by FACS sorting. Synthetic promoters were then rescued from stable transfectants by PCR from the genomic DNA where the HIV vectors had integrated. The results from this study also demonstrated the possibility of isolating several highly active endothelial cell-specific synthetic promoter elements from a random screen.
Synthetic promoters active only in the liver have also been developed (Lemken et al., 2005). In this study transcriptional units from ApoB and OTC genes were used in a controlled, non-random construction procedure to generate a series of multimeric synthetic promoters. Specifically, 2x, 4x, 8x and 16x repeats of the ApoB and OTC promoter elements were ligated together and promoter activity analysed. The results indicated that the promoter based on 4xApoB elements gave the optimal levels of gene expression and that 8x and 16x elements gave reduced levels of expression, thus demonstrating the limitations of simply ligating known promoter elements together in a repeat fashion to achieve enhanced expression.
When adopting this type of methodology in the design of synthetic tissue-specific promoters it is important to use well-designed duplex oligonucleotides. For example, each element has to be spaced in such a way that the regulatory elements appear on the same side of the DNA helix when reassembled, relevant minimal promoter elements have to be employed so that the screen produces promoters capable of expressing efficiently only in the tissue of interest and there must be some sort of mechanism, such as the addition of Sp1 sites, for the protection against promoter silencing through methylation.
In addition to tissue-specific promoters, cell-type synthetic promoters have also been developed. In one study, researchers designed a synthetic promoter to be active in nonadrenergic (NA) neurones (Hwang et al., 2001). They authors randomly ligated cis-regulatory elements that were identified from the human dopamine beta-hydroxylase (hDBH) gene and constructed promoters with up to 50-fold higher activity than the original promoter. Specifically, two elements from the promoter were used to generate a multimeric synthetic promoter; PRS1 and PRS2 which are bound to by the Phox2a transcription factor. The results demonstrated that the PRS2 was responsible for higher levels of gene expression as it had higher affinity to Phox-2a. It was also found that eight copies of PRS2 in the same orientation yielded maximum activity.
In a similar type of study a synthetic promoter was constructed that was specifically active in myeloid cells (He et al., 2006). The promoter comprised myeloid-specific elements for PU.1, C/EBPalpha, AML-1 and myeloid-associated elements for Sp1 and AP-1, which were randomly inserted upstream of the p47-phox minimal promoter. Synthetic promoters constructed showed very high activity. Haematopoietic Stem Cells (HSC) were initially transduced then the expression in differentiated cells was examined; only myeloid cells were found to express the reporter construct. To test therapeutic applicability of these promoters apoE-/- mice were transplanted with HSC transduced with a lentiviral vector expressing apoE from CMV and synthetic promoters. Even though transduced cells containing CMV and synthetic promoters both corrected the artherosclerotic phenotype, the cells derived from lentiviral vectors harbouring the synthetic promoter did so with less variability. Thus highlighting the improved safety features when using synthetic promoters for gene therapy applications.
In addition to tissue- and cell type-specific constitutive promoters, inducible synthetic promoters can also be constructed. One group describe a synthetic promoter constructed by placing the EPO enhancer region upstream of the SV40 promoter. The result is a strong promoter that is active only under ischaemic conditions. The authors tested this promoter by developing Neural Stem Cells (NSC) responsive to hypoxia and proposed that this system could be used to deliver therapeutic stem cells to treat ischaemic events. The authors were able to demonstrate that transplantation of NSC modified with a hypoxia-sensitive synthetic promoter resulted in specific expression of the luciferase reporter gene in response to ischaemic events in vivo (Liu et al., 2010).
Synthetic promoters have direct applications in large-scale industrial processes where enzymatic pathways are used in the production of biological and chemical-based products (reviewed in Hammer et al., 2006). One of the most important limitations in industrial-scale processes that synthetic promoter technology addresses is the inherent genetic instability in synthetically engineered biological systems. For instance, in prokaryotic organisms designed to express two or more enzymes, mutations will invariably arise in very few generations resulting in the termination of gene expression. This is because there is the lack of evolutionary pressure keeping all the components intact. The result is that mutations accrue over generations resulting in the deactivation of the circuit. Homologous recombination in natural promoters driving high levels of gene expression is the main reason why this circuitry fails (Sleight et al., 2010). Therefore, the use of synthetic promoters in these systems should serve to lower gene expression to result in more genetic stability, allow the avoidance of repeat sequences to prevent recombination and allow the use inducible promoters (a feature that also reduces genetic instability). In summary, the use of synthetic promoter technology in complex genetically engineered synthetic organisms expressing a variety of components should serve to increase genetic stability and improve the efficiency of the processes that the components control.
One interesting therapeutic application for synthetic promoter technology that has been described is the generation of a class of replication-competent viruses that enable tumour cell-specific killing by specifically replicating in cancer cells. In this study a replication competent retrovirus was developed to selectively kill tumour cells (Logg et al., 2002). The authors added a level of transcriptional targeting by incorporating the prostate-specific probasin (PB) promoter into the retroviral LTR and designed more efficient synthetic promoters based on the PB promoter to increase the efficiency of retroviral replication in prostate cancer cells. The result was a retrovirus that could efficiency transduce and replicate only in cancer cells. This is an attractive therapeutic strategy for the treatment of cancer, as tumour virotherapy has actually been examined as a potential therapeutic strategy for several decades.
Synthetic promoters that are active only in cycling endothelial cells would be another attractive tool for the development of cancer gene therapies. The rationale being that by targeting new blood vessels growing into tumours we would be able to develop a cancer gene therapy that could cut off supply of nutrients to the growing cancer. In a study that adopted this approach the cdc6 gene promoter was identified as a candidate promoter active only in cycling cells and was coupled to the endothelin enhancer element to construct a promoter active in dividing endothelial cells (Szymanski et al., 2006). Four endothelin elements conjugated to the cdc6 promoter gave the optimal results in vitro. When introduced into tumour models in vivo, the synthetic promoter was more efficient at driving gene expression in cancerous tissues, when compared to a CMV promoter.
Perhaps one of the most impressive applications of synthetic promoter technology thus far was the development of a liver-specific promoter that could be used to essentially cure diabetes in a transgenic mouse model (Han et al., 2010). In this study a synthetic promoter active in liver cells in response to insulin was constructed. The authors designed 3-, 6- & 9-element promoters based on random combinations of HNF-1, E/EBP and GIRE cis-elements. In the 3-element promoters all 27 combinations of the three were tested and the highest activity promoters were used to generate the 6-element promoter and so on. Using this technique promoters with activity up to 25% of CMV were identified. Finally, the optimal promoter was chosen depending on its responsiveness to glucose. This promoter showed highest specificity to liver cells and in response to Glucose and yielded expression levels 21% that of CMV. Adenoviral vectors containing this promoter driving expression of insulin were injected into a mouse diabetic model. Injection with the highest dose of virus resulted in protection against hyperglycaemia for 50 days. Importantly, injection with adenovirus expressing insulin from a CMV promoter resulted in death of the animals due to hypoglycaemia, thus illustrating the importance of regulated expression in gene therapy. Importantly, the results from this study excellently illustrated why the clever design of synthetic promoters controlling restricted gene expression will be essential in the development of safe gene therapy.
Synthetic promoters are increasingly being used in gene therapy type of studies. In one recent study their potential application to the gene therapy of Chronic Granulomatous Disease (X-CGD; an X-linked disorder resulting from mutations in gp91-phox, whose activity in myeloid cells is important in mounting an effective immune response) was examined (Santilli et al., 2011). The authors cite a clinical trial using a retroviral vector, which was successful at correcting the phenotype, but expression was short-lived due to promoter inactivation. In order to address this issue a chimeric promoter was constructed that was a fusion of Cathepsin G and c-Fes minimal promoter sequences, which are specifically active in cells of the myeloid lineage. This promoter was used to drive the expression of gp91-phox in myeloid cells in mice using a SIN lentiviral vector and the results show effective restricted expression to monocytes and subsequent introduction of gp91 results in high levels of expression in target cells and restoration of wild type phenotype in vitro. X-CGD cells were then transduced with the lentiviral vector and grafted into a mouse model of CGD. The vector was able to sustain long-term expression of gp91-phox, resulting in levels of expression that could correct the phenotype. Expression was specifically seen in granulocytes and monocytes, and not B- and T-cells.
These studies serve to highlight the potential application of synthetic promoter technology in gene therapy. They particularly highlight the importance of achieving cell-type specific gene expression and address the common issue of promoter shutdown that is seen when using stronger viral promoters like those derived from the CMV and RSV. If gene therapy is to be a success in the clinic it will be imperative to develop promoters that are highly specific and which display a restrictive and predictable expression profile. Thus, synthetic promoter technology represents the ideal solution to achieve this goal and its use is likely to become an increasingly popular approach adopted by researchers developing gene therapeutics.
We first described how functional genomics experimentation and bioinformatics tools could be applied in the design of synthetic promoters for therapeutic and diagnostic applications several years ago (Roberts, 2007). Since then a number of scientists have also realised that this approach can be broadly applied across the biotech industry (Venter et al., 2007). In this section we discuss some of the tools that we use to analyse data obtained from large-scale gene expression analyses, which is subsequently used in the smart design of synthetic promoters conveying highly-specific regulation of gene expression.
To design a synthetic promoter it is essential to identify an appropriate number of cis-regulatory elements that can specifically bind to the trans factors that enhance gene transcription. This is where the importance of a number of bioinformatic algorithms becomes apparent. Over the past several years a number of databases and programs have been developed in order to identify transcription factor biding sites (TFBSs) on a variety of genomes. Below we introduce the most extensively used resources and discuss their application to the design of synthetic promoters, we pay particular attention to the identification of transcription networks active in cancer and how this information can be used to design cancer-specific promoters that can be used in the design of safer and more effective tumour-targeted gene therapies.
There is now a growing trend for researchers to analyse microarray data in terms of ‘gene modules’ instead of the presentation of differentially regulated gene lists. By grouping genes into functionally related modules it is possible to identify subtle changes in gene expression that may be biologically (if not statistically significantly) important, to more easily interpret molecular pathways that mediate a particular response and to compare many different microarray experiments from different disease states in an effort to uncover the commonalities and differences in multiple clinical conditions. Therefore, we are moving into a new era of functional genomics, where the large datasets generated by the evaluation of global gene expression studies can be more fully interpreted by improvements in computational methods. The advances in functional genomics made in recent years have resulted in the identification of many more cis-regulatory elements that can be directly related to the increased transcription of specific genes. Indeed, the ability to use bioinformatics to unravel complex transcriptional pathways active in diseased cells can actually serve to facilitate the process of choosing suitable cis-elements that can be used to design synthetic promoters specifically active in complex pathologies such as cancer.
In cancer the changes in the gene expression profile are often the result of alterations in the cell’s transcription machinery induced by aberrant activation of signalling pathways that control growth, proliferation and migration. Such changes result in the activation of transcription regulatory networks that are not found in normal cells and provide us with an opportunity to design synthetic promoters that should only be active in cancerous cells. If microarray technology is to truly result in the design of tailored therapies to individual cancers or even patients, as has been heralded, it is important that the functional genomics methodology that was designed for the identification of signalling and transcription networks be applied to the design of cancer-specific promoters so that effective gene therapeutic strategies can be formulated (Roberts & Kottaridis, 2007). The development of bioinformatics algorithms for the analysis of microarray datasets has largely been applied in order to unravel the transcription networks operative under different disease and environmental conditions. To this date there has been no effort to use this type of approach to design synthetic promoters that are operative only under these certain disease or environmental conditions.
The regulation of gene expression in eukaryotes is highly complex and often occurs through the coordinated action of multiple transcription factors. The use of trans-factor combinations in the control of gene expression allows a cell to employ a relatively small number of transcription factors in the regulation of disparate biological processes. As discussed herein, a number of tools have been developed that allow us to utilise microarray data to identify novel cis-regulatory elements. It is also possible to use this information to decipher the transcriptional networks that are active in cells under different environmental conditions. In yeast, the importance of the combinatorial nature of transcriptional regulation was established by specifically examining clusters of upregulated genes for the presence of combinations of cis-elements. By examining microarray data from yeast exposed to a variety of conditions the authors were able to construct a network of transcription revealing the functional associations between different regulatory elements. This approach resulted in the identification of key motifs with many interactions, suggesting that some factors serve as facilitator proteins assisting their gene-specific partners in their function. The idea that a core number of transcription factors mediate such a vast array of biological responses by adopting multiple configurations implies that it may be possible to hijack the transcriptional programs that have gone awry in multifactorial diseases in an effort to develop disease-specific regulatory elements. For instance, the meta-analyses of cancer datasets has permitted the identification of gene modules, allowing for the reduction of complex cancer signatures to small numbers of activated transcription programs and even to the identification of common programs that are active in most types of cancer. This type of analysis can also help to identify specific transcription factors whose deregulation plays a key role in tumour development. In one such study, the importance of aberrant E2F activity in cancer was reaffirmed during a search for the regulatory programs linking transcription factors to the target genes found upregulated in specific cancer types (Rhodes et al., 2005). It was shown that E2F target genes were disproportionately upregulated in more than half of the gene expression profiles examined, which were obtained from a multitude of different cancer types. It was thus proposed that integrative bioinformatics analyses have the potential to generate new hypotheses about cancer progression.
Different bioinformatics tools, examples of which are given in table 1, may be used to screen for cis-regulatory elements. In general, such tools function by comparing gene expression profiles between differentially regulated genes and examining upstream sequences, available through genome sequence resources. For the phylogenetic footprinting tools, the untranslated regions of specific genes are compared between species and the most highly conserved sequences are returned and proposed to be potential cis-elements. A combination of all available approaches may be employed in order to identify regulatory sequences that predominate in the profile of specific cell or tissue types. The most common sequences identified are then used as the building blocks employed in the design of synthetic promoters.
The ability to use gene expression data to identify gene modules, which mediate specific responses to environmental stimuli (or to a diseased state) and to correlate their regulation to the cis-regulatory elements present upstream of the genes in each module, has transformed the way in which we interpret microarray data. For instance, by using the modular approach it is possible to examine whether particular gene modules are active in a variety of different cancers, or whether individual cancers require the function of unique gene modules. This has allowed us to look for transcriptional commonalities between different cancers, which should aid in the design of widely applicable anti-cancer therapeutic strategies. In one early study, gene expression data from 1975 microarrays, spanning 22 different cancers was used to identify gene modules that were activated or deactivated in specific types of cancer (Segal et al., 2004). Using this approach the authors found that a bone osteoblastic module was active in a number of cancers whose primary metastatic site is known to be the bone. Thus, a common mechanism of bone metastasis between varieties of different cancers was identified, which could be targeted in the development of novel anticancer therapies.
It is also possible to identify the higher-level regulator that controls the expression of the genes in each module (Segal et al., 2003). Examination of the upstream regulatory sequences of each gene in a module may reveal the presence of common cis-regulatory elements that are known to be the target of the module’s regulator. Therefore, by identifying specific regulatory proteins that control the activation of gene modules in different cancers, it should be possible to extrapolate the important cis-elements that mediate transcription in the transformed cell. Thereby, allowing us to design and construct novel tumour-specific promoters based on the most active cis-regulatory elements in a number of tumour-specific gene modules. The ability to identify specific transcriptional elements in the human genome that control the expression of functionally related genes is transforming the application of functional genomics. Until recently the interpretation of data from microarray analysis has been limited to the identification of genes whose function may be important in a single pathway or response. How this related to global changes in the cellular phenotype had been largely ignored, as the necessary tools to examine this simply did not exist. With the advancement of bioinformatics we are now in a position to utilise all the data that is obtained from large-scale gene expression analysis and combine it with knowledge of the completed sequence of the human genome and with transcription factor, gene ontology and molecular function databases, thereby more fully utilising the large datasets that are generated by global gene expression studies.
For nearly two decades scientists have been compiling databases that catalogue the trans-factors and cis-elements that are responsible for gene regulation (Wingender et al., 1988). This has primarily been done in an effort to elucidate the various transcription programs that are activated in response to different biological stimuli in a range of organisms. The result is the emergence of useful tools that can be used to identify transcription factors and their corresponding cis-regulatory sequences that are useful in the design of synthetic promoters. In the remaining part of this chapter we briefly discuss each resource, indicating the unique aspect of its functionality.
TRANSFAC is perhaps the most comprehensive TFBS database available and indexes transcription factors and their target sequences based solely on experimental data (Matys et al., 2003). It is maintained as a relational database, from which public releases are made available via the web. The release consists of six flat files. At the core of the database is the interaction of transcription factors (FACTOR) with their DNA-binding sites (SITE) through which they regulate their target genes (GENE). Apart from genomic sites, ‘artificial’ sites which are synthesized in the laboratory without any known connection to a gene, e.g., random oligonucleotides, and IUPAC consensus sequences are also stored in the SITE table. Sites must be experimentally proven for their inclusion in the database. Experimental evidence for the interaction with a factor is given in the SITE entry in form of the method that was used (gel shift, footprinting analysis, etc.) and the cell from which the factor was derived (factor source). The latter contains a link to the respective entry in the CELL table. On the basis of those, method and cell, a quality value is given to describe the ‘confidence’ with which an observed DNA- binding activity could be assigned to a specific factor. From a collection of binding sites for a factor nucleotide weight matrices are derived (MATRIX). These matrices are used by the tool MatchTM to find potential binding sites in uncharacterized sequences, while the program PatchTM uses the single site sequences, which are stored in the SITE table. According to their DNA-binding domain transcription factors are assigned to a certain class (CLASS). In addition to the more ‘planar’ CLASS table a hierarchical factor classification system is also used.
TRANSCompel® originates from COMPEL, and functions to emphasize the key role of specific interactions between transcription factors binding to their target cis-regulatory elements; whilst providing specific features of gene regulation in a particular cellular content (Kel-Margoulis et al., 2002). Information about the structure of known trans factor and cis sequence interactions, and specific gene regulation achieved through these interactions, is extremely useful for promoter prediction. In the TRANSCompel database, each entry corresponds to an individual trans/cis interaction within the context of a particular gene and thus contains information about two binding sites, two corresponding transcription factors and experiments confirming cooperative action between transcription factors.
ABS is a public database of known cis-regulatory binding sites identified in promoters of orthologous vertebrate genes that have been manually collated from the scientific literature (Blanco et al., 2006). In this database some 650 experimental binding sites from 68 transcription factors and 100 orthologous target genes in human, mouse, rat or chicken genome sequences have been documented. This tool allows computational predictions and promoter alignment information for each entry and is accessed through a simple and easy-to-use web interface; facilitating data retrieval and allowing different views of the information. One of the key features of this software is the inclusion of a customizable generator of artificial datasets based on the known sites contained in the whole collection and an evaluation tool to aid during the training and the assessment of various motif-finding programs.
JASPAR is an open-access database of annotated, high-quality, matrix-based TFBS profiles for multi-cellular eukaryotic organisms (Sandelin et al., 2004). The profiles were derived exclusively from sets of nucleotide sequences that were experimentally demonstrated to bind transcription factors. The database is accessible via a web-interface for browsing, searching and subset selection. The interface also includes an online sequence analysis utility and a suite of tools for genome-wide and comparative genome analysis of regulatory regions.
HTPSELEX is a public database providing access to primary and derived data from high-throughput SELEX experiments that were specifically designed in order to characterize the binding specificity of transcription factors (Jagannathan et al., 2006). The resource is primarily intended to serve computational biologists interested in building models of TFBSs from large sets of cis-regulatory sequences. For each experiment detailed in the database accurate information is provided about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, assembled clone sequences and complete sets of in vitro selected protein-binding tags.
TRED is a database that stores both cis- and trans-regulatory elements and was designed to facilitate easy data access and to allow for the analysis of single-gene-based and genome- scale studies (Zhao et al., 2005). Distinguishing features of TRED include: relatively complete genome-wide promoter annotation for human, mouse and rat; availability of gene transcriptional regulation information including TFBSs and experimental evidence; data accuracy is ensured by hand curation; efficient user interface for easy and flexible data retrieval; and implementation of on-the-fly sequence analysis tools. TRED can provide good training datasets for further genome-wide cis-regulatory element prediction and annotation; assist detailed functional studies and facilitate the deciphering of gene regulatory networks.
Databases of known TFBSs can be used to detect the presence of protein-recognition elements in a given promoter, but only when the binding site of the relevant DNA-binding protein and its tolerance to mismatches in vivo is already known. Because this knowledge is currently limited to a small subset of transcription factors, much effort has been devoted to the discovery of regulatory motifs by comparative analysis of the DNA sequences of promoters. By finding conserved regions between multiple promoters, motifs can be identified with no prior knowledge of TFBSs. A number of models have emerged that achieve this by statistical overrepresentation. These algorithms function by aligning multiple untranslated regions from the entire genome and identifying sequences that are statistically significantly overrepresented in comparison to what it expected by random.
YMF is a program developed to identify novel TFBSs (not necessarily associated with a specific factor) in yeast by searching for statistically overrepresented motifs (Sinha et al., 2003; Sinha & Tompa, 2002). More specifically, YMF enumerates all motifs in the search space and is guaranteed to produce those motifs with the greatest z-scores.
SCORE is a computational method for identifying transcriptional cis-regulatory modules based on the observation that they often contain, in statistically improbable concentrations, multiple binding sites for the same transcription factor (Rebeiz et al., 2002). Using this method the authors conducted a genome-wide inventory of predicted binding sites for the Notch-regulated transcription factor Suppressor of Hairless, Su(H), in drosophila and found that the fly genome contains highly non-random clusters of Su(H) sites over a broad range of sequence intervals. They found that the most statistically significant clusters were very heavily enriched in both known and logical targets of Su(H) binding and regulation. The utility of the SCORE approach was validated by in vivo experiments showing that proper expression of the novel gene Him in adult muscle precursor cells depends both on Su(H) gene activity and sequences that include a previously unstudied cluster of four Su(H) sites, indicating that Him is a likely direct target of Su(H).
At present these tools are mainly applied in the study of lower eukaryotes where the genome is less complex and regulatory elements are easier to identify, extending these algorithms to the human genome has proven somewhat more difficult. In order to redress this issue a number of groups have shown that it is possible to mine the genome of higher eukaryotes by searching for conserved regulatory elements adjacent to transcription start site motifs such as TATA and CAAT boxes, e.g. as catalogued in the DBTSS resource (Suzuki et al. 2004; Suzuki et al., 2002), or one can search for putative cis-elements in CpG rich regions that are present in higher proportions in promoter sequences (Davuluri et al., 2001). Alternatively, with the co-emergence of microarray technology and the complete sequence of the human genome, it is now possible to search for potential TFBSs by comparing the upstream non-coding regions of multiple genes that show similar expression profiles under certain conditions. Gene sets for comparative analysis can be chosen based on clustering, e.g. hierarchical and k-means (Roth et al., 1998), from simple expression ratio (Bussemaker et al., 2001) or functional analysis of gene products (Jensen et al., 2000). This provides scientists with the opportunity to identify promoter elements that are responsive to certain environmental conditions, or those that play a key role in mediating the differentiation of certain tissues or those that may be particularly active in mediating pathologic phenotypes.
Phylogenetic footprinting, or comparative genomics, is now being applied to identify novel promoter elements by comparing the evolutionary conserved untranslated elements proximal to known genes from a variety of organisms. The availability of genome sequences between species has notably advanced comparative genomics and the understanding of evolutionary biology in general. The neutral theory of molecular evolution provides a framework for the identification of DNA sequences in genomes of different species. Its central hypothesis is that the vast majority of mutations in the genome are neutral with respect to the fitness of an organism. Whilst deleterious mutations are rapidly removed by selection, neutral mutations persist and follow a stochastic process of genetic drift through a population. Therefore, non-neutral DNA sequences (functional DNA sequences) must be conserved during evolution, whereas neutral mutations accumulate. Initial studies sufficiently demonstrated that the human genome could be adequately compared to the genomes of other organisms allowing for the efficient identification of homologous regions in functional DNA sequences.
Subsequently, a number of bioinformatics tools have emerged that operate by comparing non-coding regulatory sequences between the genomes of various organisms to enable the identification of conserved TFBSs that are significantly enriched in promoters of candidate genes or from clusters identified by microarray analysis; examples of these software suites are discussed below. Typically these tools work by aligning the upstream sequences of target genes between species thus identifying conserved regions that could potentially function as cis-regulatory elements and have consequently been applied in the elucidation of transcription regulatory networks in a variety of models.
TRAFAC is a Web-based application for analysis and display of a pair of DNA sequences with an emphasis on the detection of conserved TFBSs (Jegga et al., 2002). A number of programs are used to analyze the sequences and identify various genomic features (for example, exons, repeats, conserved regions, TFBSs). Repeat elements are masked out using RepeatMasker and the sequences are aligned using the PipMaker-BLASTZ algorithm. MatInspector Professional or Match (BioBase) is run to scan the sequences for TFBSs. TRAFAC then integrates analysis results from these applications and generates graphical outputs; termed the Regulogram and Trafacgram.
CORG comprises a catalogue of conserved non-coding sequence blocks that were initially computed based on statistically significant local suboptimal alignments of 15kb regions upstream of the translation start sites of some 10793 pairs of orthologous genes (Dieterich et al., 2003). The resulting conserved non-coding blocks were annotated with EST matches for easier detection of non-coding mRNA and with hits to known TFBSs. CORG data are accessible from the ENSEMBL web site via a DAS service as well as a specially developed web service for query and interactive visualization of the conserved blocks and their annotation.
CONSITE is a flexible suite of methods for the identification and visualization of conserved TFBSs (Lenhard et al., 2003). The system reports those putative TFBSs that are both situated in conserved regions and located as pairs of sites in equivalent positions in alignments between two orthologous sequences. An underlying collection of metazoan transcription-factor-binding profiles was assembled to facilitate the study. This approach results in a significant improvement in the detection of TFBSs because of an increased signal-to-noise ratio, as as the authors demonstrated with two sets of promoter sequences.
CONFAC enables the high-throughput identification of conserved TFBSs in the regulatory regions of hundreds of genes at a time (Karanam et al., (2004). The CONFAC software compares non-coding regulatory sequences between human and mouse genomes to enable identification of conserved TFBSs that are significantly enriched in promoters of gene clusters from microarray analyses compared to sets of unchanging control genes using a Mann–Whitney statistical test. The authors analysed random gene sets and demonstrated that using this approach, over 98% of TFBSs had false positive rates below 5%. As a proof-of-principle, the CONFAC software was validated using gene sets from four separate microarray studies and TFBSs were identified that are known to be functionally important for regulation of each of the four gene sets.
VAMP is a graphical user interface for both visualization and primary level analysis of molecular profiles obtained from functional genomics experimentation (La Rosa et al., 2006). It can be applied to datasets generated from Comparative Genomic Hybridisation (CGH) arrays, transcriptome arrays, Single Nucleotide Polymorphism arrays, loss of heterozygosity analysis (LOH), and Chromatin Immunoprecipitation experiments (ChIP-on-chip). The interface allows users to collate the results from these different types of studies and to view it in a convenient way. Several views are available, such as the classical CGH karyotype view or genome-wide multi-tumour comparisons. Many of the functionalities required for the analysis of CGH data are provided by the interface; including searches for recurrent regions of alterations, comparison to transcriptome data, correlation to clinical information, and the ability to view gene clusters in the context of genome structure.
CisMols Analyser allows for the filtering of candidate cis-element clusters based on phylogenetic conservation across multiple gene sets (Jegga et al., 2005). It was previously possible to achieve this for individual orthologue gene pairs, but combining data from cis-conservation and coordinate expression across multiple genes proved a more difficult task. To address this issue, the authors extended an orthologue gene pair database with additional analytical architecture to allow for the analysis and identification of maximal numbers of compositionally similar and phylogenetically conserved cis-regulatory element clusters from a list of user-selected genes. The system has been successfully tested with a series of functionally related and microarray profile-based co-expressed orthologue pairs of promoters and genes using known regulatory regions as training sets and co-expressed genes in the olfactory and immunohematologic systems as test sets. A significant amount of effort has been dedicated to the cataloguing of transcription factors and their corresponding cis-elements. More recently, these databases have been compiled with the aim to utilise them to unravel regulatory networks active in response to diverse stimuli.
PreMod was developed in an effort to identify cis-regulatory modules (CRM) active under specific environmental conditions (Blanchette et al., 2006; Ferretti et al., 2007). Starting from a set of predicted binding sites for more than 200 transcription factor families documented in the Transfac database (described above), the authors describe an algorithm relying on the principle that cis-regulatory modules (CRMs) generally contain several phylogenetically conserved binding sites for a small variety of transcription factors. The method allowed the prediction of more than 118,000 CRMs within the human genome. During this analysis, it was revealed that CRM density varies widely across the genome, with CRM-rich regions often being located near genes encoding transcription factors involved in development. Interestingly, in addition to showing enrichment near the 3’ end of genes, predicted CRMs were present in other regions more distant from genes. In this database, the tendency for certain transcription factors to bind modules located in specific regions was documented with respect to their target genes, and a number of transcription factors likely to be involved in tissue-specific regulation were identified.
CisView was developed to facilitate the analysis of gene regulatory regions of the mouse genome (Sharov et al., 2006). Its user interface is a browser and database of genome-wide potential TFBSs that were identified using 134 position-weight matrices and 219 sequence patterns from various sources. The output is presented with information about sequence conservation, neighbouring genes and their structures, GO annotations, protein domains, DNA repeats and CpG islands. The authors used this tool to analyse the distribution of TFBSs and revealed that many TFBSs were over-represented near transcription start sites. In the initial paper presenting the tool they also identified potential cis-regulatory modules defined as clusters of conserved TFBSs in the entire mouse genome. Out of 739,074 CRMs identified, 157,442 had a significantly higher regulatory potential score than semi-random sequences. The CisView browser provides a user-friendly computer environment for studying transcription regulation on a whole-genome scale and can also be used for interpreting microarray experiments and identifying putative targets of transcription factors.
BEARR is web browser software designed to assist biologists in efficiently carrying out the analysis of microarray data from studies of specific transcription factors (Vega et al., 2004). Batch Extraction and Analysis of cis-Regulatory Regions, or BEARR, accepts gene identifier lists from microarray data analysis tools and facilitates identification, extraction and analysis of regulatory regions from the large amount of data that is typically generated in these types of studies.
VISTA is a family of computational tools that was built to assist in the comparative analysis of DNA sequences (Dubchak & Ryaboy, 2006). These tools allow for the alignment of DNA sequences to facilitate the visualization of conservation levels and thus allow for the identification of highly conserved regions between species. Specifically, sequences can be analysed by browsing through pre-computed whole-genome alignments of vertebrates and other groups of organisms. Submission of sequences to Genome VISTA enables the user to align them to other whole genomes; whereas submission of two or more sequences to mVISTA allows for direct alignment. Submission of sequences to Regulatory VISTA is also possible and enables the predication of potential TFBSs (based on conservation within sequence alignments). All VISTA tools use standard algorithms for visualization and conservation analysis to make comparison of results from different programs more straightforward.
PromAn is a modular web-based tool dedicated to promoter analysis that integrates a number of different complementary databases, methods and programs (Lardenois et al., 2006). PromAn provides automatic analysis of a genomic region with minimal prior knowledge of the genomic sequence. Prediction programs and experimental databases are combined to locate the transcription start site (TSS) and the promoter region within a large genomic input sequence. TFBSs can be predicted using several public databases and user-defined motifs. Also, a phylogenetic footprinting strategy, combining multiple alignments of large genomic sequences and assignment of various scores reflecting the evolutionary selection pressure, allows for evaluation and ranking of TFBS predictions. PromAn results can be displayed in an interactive graphical user interface. It integrates all of this information to highlight active promoter regions, to identify among the huge number of TFBS predictions those which are the most likely to be potentially functional and to facilitate user refined analysis. Such an integrative approach is essential in the face of a growing number of tools dedicated to promoter analysis in order to propose hypotheses to direct further experimental validations.
CRSD is a comprehensive web server that can be applied in investigating complex regulatory behaviours involving gene expression signatures, microRNA regulatory signatures and transcription factor regulatory signatures (Liu et al., 2006). Six well-known and large-scale databases, including the human UniGene, mature microRNAs, putative promoter, TRANSFAC, pathway and Gene Ontology (GO) databases, were integrated to provide the comprehensive analysis in CRSD. Two new genome-wide databases, of microRNA and transcription factor regulatory signatures were also constructed and further integrated into CRSD. To accomplish the microarray data analysis at one go, several methods, including microarray data pre-treatment, statistical and clustering analysis, iterative enrichment analysis and motif discovery, were closely integrated in the web server.
MPromDb is a database that integrates gene promoters with experimentally supported annotation of transcription start sites, cis-regulatory elements, CpG islands and chromatin immunoprecipitation microarray (ChIP-chip) experimental results within an intuitively designed interface (Sun et al., 2006). Its initial release contained information on 36,407 promoters and first exons, 3,739 TFBSs and 224 transcription factors; with links to PubMed and GenBank references. Target promoters of transcription factors that have been identified by ChIP-chip assay are also integrated into the database and thus serving as a portal for genome-wide promoter analysis of data generated by ChIP-chip experimental studies.
A comprehensive list of the all the databases described above with a summary of their features and a reference to the original citation are shown in table 1.
Each of the aforementioned databases can be used when searching for potential regulatory sequences for inclusion in the design of synthetic promoters. Indeed, these resources can be used in order to identify cis-regulatory elements that may play a role in the formation of a particular cellular phenotype, or those that may be important in driving differentiation in developing organs. Synpromics, an emerging synthetic biology company recently incorporated in the United Kingdom, has cleverly utilised these tools in developing a proprietary method of synthetic promoter production where identified elements are incorporated into the design of promoters that are able to specifically regulate gene expression in a particular cellular phenotype. This method harnesses a cell’s gene expression profile in order to facilitate the design of highly specific and efficient promoters. The result is a range of promoters that are inducible, tissue (or cell)-specific, active in response to a particular pathogen, chemical or biological agent and even able to mediate gene expression only under certain pathological conditions, such as cancer. Indeed, Synpromics has successfully generated a range of synthetic promoters that specifically drive high levels of gene expression in colorectal cancer and are looking to apply these promoters in the development of safer gene therapies (manuscript in preparation).
Resource | Description | Citation |
DBTSS | Database of transcriptional start sites | Suzuki et al., (2002) |
TRAFAC | Conserved cis-element search tool | Jegga et al., (2002) |
TRANSCompel | Database of composite regulatory elements | Kel-Margoulis et al., (2002) |
TRANSFAC | Eukaryotic transcription factor database | Matys et al., (2003) |
Phylofoot | Tools for phylogenetic footprinting purposes | Lenhard et al., (2003) |
CORG | Multi-species DNA comparison and annotation | Dieterich et al., (2003) |
CONSITE | Explores trans-factor binding sites from two species | Lenhard et al., (2003) |
CONFAC | Conserved TFBS finder | Karanam et al., (2004) |
CisMols | Identifies cis-regulatory modules from inputed data | Jegga et al., (2005) |
TRED | Catalogue of transcription regulatory elements | Zhao et al., (2005) |
Oncomine | Repository and analysis of cancer microarray data | Rhodes et al., (2005) |
ABS | Database of regulatory elements | Blanco et al., (2006) |
JASPAR | Database of regulatory elements | Sandelin et al., (2004) |
HTPSELEX | Database of composite regulatory elements | Jagannathan et al., (2006) |
PReMod | Database of transcriptional regulatory modules in the human genome | Blanchette et al., (2006) |
CisView | Browser of regulatory motifs and regions in the genome | Sharov et al., (2006) |
BEARR | Batch extraction algorithm for microarray data analysis | Vega et al., 2004) |
VISTA | Align and compare sequences from multiple species | Dubchak et al., (2006) |
PromAn | Promoter analysis by integrating a variety of databases | Lardenois et al., (2006) |
Bioinformatics tools used for the identification of cis-regulatory elements
Importantly, synthetic promoters often mediate a level of gene expression with much greater efficiency than that seen with viral promoters, such as CMV, or compared to naturally occurring promoters within the genome. Given that the entire Biotech industry is centred on the regulation of gene expression, it is likely that synthetic promoters will eventually replace all naturally-occurring sequences in use today and help drive the growth of the synthetic biology sector in the coming decades.
In summary, synthetic promoters have emerged over the past two decades as excellent tools facilitating the identification of important structural features in naturally occurring promoter sequences and allowing enhanced and more restrictive regulation of gene expression. A number of early studies revealed that it was possible to combine the cis-regulatory elements from promoters of a few tissue-specific genes and use these as building blocks to generate shorter, more efficient tissue-specific promoters. Several simple methodologies to achieve this emerged and have been applied in a multitude of organisms; including plant, bacteria, yeast, viral and mammalian systems.
Recent advances in bioinformatics and the emergence of a plethora of tools specifically designed at unravelling transcription programs has also facilitated the design of highly-specific synthetic promoters that can drive efficient gene expression in a tightly regulated manner. Changes in a cell’s gene expression profile can be monitored and the transcription programs underpinning that change delineated and the corresponding cis-regulatory modules can be used to construct synthetic promoters whose activity is restricted to individual cell types, or to single cells subject to particular environmental conditions. This has allowed researchers to design promoters that are active in diseased cells or in tissues treated with a particular biological or chemical agent; or active in cells infected with distinct pathogens.
A number of institutions, such as Synpromics, have taken advantage of these advances and are now working to apply synthetic promoter technology to the enhanced production of biologics for use in biopharmaceutical, greentech and agricultural applications; the development of new gene therapies; and in the design of a novel class of molecular diagnostics. As the synthetic biology field continues to develop into a multi-billion dollar industry, synthetic promoter technology is likely to remain at the heart of this ever-expanding and exciting arena.
Coronaviruses are large, enveloped, single-stranded, positive-sense RNA viruses with a genome of approximately 30 kilobases in length. The genus Coronavirus belongs to the family Coronaviridae in the order Nidovirales. They are classified into three groups. Group 1 contains various mammalian viruses including porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and human coronaviruses 229E and NL63. Group 2 includes canine respiratory coronavirus among other mammalian viruses and human coronavirus OC43. Human severe acute respiratory syndrome coronavirus (SARS-CoV-1) is considered a distant relative of this group. Group 3 contains solely avian coronaviruses. Human coronaviruses (HCoVs) cause respiratory infections, mainly, but gastroenteritis and neurological disorders may also occur. So far, at least seven human coronaviruses have been described including SARS-CoV-2, which was just sequenced in 2020, and two of these coronaviruses (OC43 and 229E) are responsible for 10–30% of all common colds. HCoV-HKU1 is mostly associated with bronchiolitis and pneumonia [1, 2, 3].
The gross life cycle of the SARS-CoV-1 has been observed in Vero E6 cells (African green monkey kidney cells) following inoculation with the virus under an electron microscope. The SARS-CoV-1 enters the cells through membrane fusion. Then, the nucleocapsids are assembled in the rough endoplasmic reticulum (RER) and mature by budding into the smoothe vesicles derived from the Golgi apparatus. Finally, the smoothe vesicles fuse with the cell membrane and the mature virus particles are released [4]. SARS-CoV-2 displays a similar life cycle.
Recent molecular studies have revealed that in order to facilitate entry of the virus into a human cell, the “S” spike surface glycoprotein of SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE-2) cellular receptor. Binding of the virus occurs via the S1 subunit of the S protein to a receptor and entry requires S protein priming by the cellular serine protease in order to allow fusing together of viral and cell membranes, a process which is initiated by the S2 subunit [5]. Following the fusion of viral and plasma membranes, the virus RNA undergoes transcription and replication inside the cell cytoplasm. Viral proteins are synthesized and the new RNA genomes are assembled and packaged in the endoplasmic reticulum, in the Golgi apparatus, and in the endoplasmic reticulum-Golgi intermediate compartment prior to virion release in vesicles. In fact, the S protein of SARS-CoV-2 binds to ACE-2 receptors with an approximately 10–20 fold higher affinity than that of SARS-CoV-1 and this added feature may aid in the efficient spread of SARS-CoV-2 among human populations. However, SARS-CoV-2 does not employ the other usual CoV receptors such as aminopeptidase N and dipeptidyl peptidase 4 to enter human cells [6].
ACE-2 is a membrane-associated aminopeptidase that converts angiotensin II to angiotensin 1–7 and plays a role in the cleavage of peptides [3]. Expression of ACE-2 in human tissues correlates with known sites of SARS-CoV-1 infection including lungs (particularly airway epithelia), heart, kidneys, small intestine, testes, and vascular endothelia [7]. These same tissues also overlap with the sites of SARS-CoV-2 infection in humans due to ACE-2 receptor availability.
On a personal note, as a biochemist, I have been following every bit of new research on any chemical compound that might successfully combat the virus. Around January 6th, 2020, I developed a very bad flu while in India after meeting with a friend who had just travelled to Wuhan in China. Overnight, I got a sore-throat that lasted a few days followed by a severe head cold with sinus congestion and mucous and, finally, it developed into a dry cough. During this debilitating flu, I also had some loose bowel movements with mucous. In the aftermath of the flu that lasted around 14 days, I was plagued with dizziness and weakness for two more weeks.
Although we had heard of the novel coronavirus in China, there was no reason to believe that was what I had just experienced since there had been no unusual respiratory distress. So, it did not seem to overlap with the pneumonia-like symptoms of the new coronavirus from China. Moreover, the friend had returned at the beginning of January and, as far as we knew at that time, the virus had only appeared in December. Therefore, it seemed unlikely that the friend had been exposed to any infected individuals while in China. Furthermore, the traveller from China never became sick (although one other person who attended the same meeting as myself developed a very bad flu within two weeks of coming in contact with this person). At the same time, there are also many seasonal flus like swine flu (H1N1) that are endemic in India, so there was no reason to consider that it was a coronavirus infection. Finally, the only medicines I took initially were some herbal Ayurvedic cold remedies mainly with a licorice-root base (a potent anti-inflammatory), aspirin at night, and an electrolyte solution to prevent dehydration from diarrhea. When I had a relapse of the gastrointestinal symptoms in March including stomach pain after I returned to Canada, a course of azithromycin helped to resolve the symptoms.
However, it was only when the weakness and malaise persisted for 3–4 months after the initial illness and new data started to emerge about the differing patterns of COVID-19 infection, that I started to consider another possible cause. Firstly, all my symptoms were consistent with the disparate effects of the novel coronavirus including the lingering apathy. Secondly, it became apparent that the new coronavirus had appeared in Wuhan some time before December. Thirdly, unlike other flu viruses, the phenomenon of asymptomatic spreaders became widely known. So, now, even though I had not been tested for the new virus or COVID-19 antibodies, I started to suspect that I could have experienced a form of coronavirus infection.
Finally, I had my COVID-19 test in August 2020 and, although it was negative, it did not preclude the possibility that I had the disease in January 2020 and that my body had formed and shed antibodies to the novel coronavirus (antibody testing was also negative). Since it is not known exactly how long antibodies persist following infection, even these may not be detected after a certain recovery period (there are recent reports antibodies decline after three months). Studies in rhesus monkeys show that re-infection does not occur in the recovered macaques up to 28 days after initial infection [8]. Nevertheless, prolonged inflammation and reports of re-infection in recovered humans are a surprising aspect of this virus. In my case, one additional negative C-reactive protein (inflammatory marker) test decisively clinched the matter.
Some scientists have opined that COVID-19 is highly contagious and highly lethal to a small subset of the population, while it produces milder symptoms in most people. Although the SARS-CoV-2 virus infects people of all ages, the World Health Organization (WHO) has determined that the evidence to date suggests that older adults and adults with underlying medical conditions are at a higher risk of developing severe COVID-19 disease [9].
One large study out of New York State seems to indicate that obesity, high blood pressure, and diabetes are strong risk factors for COVID-19 [10]. It has also been observed that cardiovascular disease and respiratory diseases could greatly affect the prognosis [11]. In fact, in an interesting German study involving autopsies on 12 COVID-19 patients the results revealed that coronary heart disease and asthma were common comorbid conditions in 50% of the deceased [12].
Other research suggests that cancer patients are more vulnerable to COVID-19 infection. A multicenter study showed that patients with cancer had higher risks in all severe outcomes of the disease tested. Hematologic cancer, lung cancer, or metastatic cancer (stage IV) cases experienced the highest frequency of severe events, while nonmetastatic cancer cases experienced similar frequencies to patients without cancer. Moreover, cancer patients who received surgery had higher risks of severe events than patients without cancer or those who underwent radiotherapy [13].
In addition, a surprising gender disparity appears to be present in relation to SARS-CoV-2 infection. Statistics from Australia, Belgium, Germany, Italy, the Netherlands, South Korea, Spain, the U.K and the US reveal that mortality rates from the virus are significantly higher in infected males than in infected females. In New York, approximately 60% of COVID-19-related deaths occurred in men. This may partly reflect biological characteristics since women produce stronger immune responses than men and are physically better at warding off viral and other types of infections. Nevertheless, biochemical differences in sex hormones are also likely to play a role in determining this dichotomy [14] and certain researchers have suggested it may be due to the presence of ACE-2 receptors in the testicles [15].
In the largest Chinese study to date assessing severity of coronavirus infection in smokers, it was found that higher percentages of current and former smokers needed ICU support or mechanical ventilation. Higher percentages of smokers among the severe cases also died [16]. Therefore, ultimately, the risk of any one individual is determined by the number of risk factors they display. For example, a ninety year old male smoker with diabetes and hypertension displaying five risk factors (age, gender, smoke inhalation, high blood pressure, and diabetes) would have an extremely high risk of contracting a terminal case of COVID-19.
However, genetic risk factors as a result of ethnic origin can only be considered once all these other significant risk factors have been taken into consideration. So far, despite attempts by various institutions to prove an ethnic link to COVID-19 infection, there is no compelling evidence to suggest that any one human group is genetically more susceptible to the novel coronavirus than any other beyond mitigating factors such as socioeconomic status or environmental conditions [17]. In order to establish a true genetic component, rigorous genetic testing must be undertaken to identify predisposing genes in susceptible ethnic groups. Prior to gene isolation and identification of a specific genetic polymorphism, a biochemical reaction resulting in a higher percentage of the disease is often demonstrated in a particular human population. As an example, the human sunburn cycle in response to UVA/B radiation only occurs in a minority of people with fair skin; however, most people simply tan when they are exposed to sunlight. In fact, these represent two separate physiological processes (burning and tanning). The former condition, scientific sunburn as a result of the human sunburn cycle, is mostly due to a genetic polymorphism involving the expression of very low levels of melanin in human skin since it can be corrected by wearing a sunscreen containing black sesame melanin [50 mg/ml] in a zinc oxide cream base [7.5%] [18, 19, 20]. It is also correlated with a high risk for skin cancer. Nonetheless, there may be other genetic factors like differences in DNA repair enzyme activity which can contribute to this unusual trait in certain individuals, as well [21].
Simultaneously, a surprising recent genetic association study has revealed that a major genetic risk factor for severe COVID-19 in humans may actually be inherited from Neanderthals. Outside the continent of Africa (0.3%), modern humans have inherited significantly more genetic material from other hominid species including Neanderthals (approximately 2%) and Denisovans [22]. Europeans and South Asians appear to have the greatest complement of Vindija Neanderthal genes from Croatia and a gene cluster on chromosome 3 inherited from this species has been identified as a risk locus for respiratory failure after infection with SARS-CoV-2. Among certain South Asian populations, up to 50% can carry at least one copy of this risk haplotype and the highest carrier frequency occurs in Bangladesh where 63% of the population carries it. In the UK it has been reported that individuals of Bangladeshi origin have roughly a two times higher risk of dying from COVID-19 than people of other nationalities [23].
Interestingly, there are high levels of air pollution in the two regions of China and Northern Italy that were hardest hit by the virus suggesting that environmental conditions can have an impact on the infectiousness of the disease [24]. Italian researchers have recently proposed an association between higher mortality rates in Northern Italy and peaks of particulate matter concentrations in this region. The most polluted northern provinces of Italy were found to have more infection cases than the less polluted southern provinces and this correlated well with ambient particulate matter concentrations that often exceeded the legal limit in these areas. All data for this study was collected prior to the lockdown [25]. Surprisingly, further research by the same group demonstrated that SARS-CoV-2 RNA was present on outdoor airborne particulate matter that was collected from an industrial site in Bergamo, Italy. This evidence suggests that, under the right atmospheric conditions, SARS-CoV-2 could create clusters with particulate matter and enhance persistence of the virus in the atmosphere by facilitating its capacity for diffusion. However, the vitality and virulence of the coronavirus diffused via this method remain to be confirmed [26].
This could have been a significant factor in the spread of the coronavirus in highly polluted and populated cities like Mumbai, India. Social conditions such as crowding in slums have also been considered contributory to dispersal of the virus in developing countries like Brazil and India. Proximity to infected individuals increases the risk of person-to-person transmission since the SARS-CoV-2 virus is spread mainly by respiratory droplets, but can be aerosolized, too [3].
No matter how healthy an individual may be, the more exposure they have to a particular virus, the greater risk they have of contracting the disease. The greater the number of particles of the virus one is exposed to, the greater the chance that they will overwhelm the body and immune responses. This is the reason that young doctors and other frontline healthcare workers are getting serious cases of COVID-19 and dying at a higher frequency than the general population.
View of Downtown Mumbai – December 2019
View of Mumbai Harbour – December 2019
In general, COVID-19 infection is associated with the increased production of pro-inflammatory cytokines, C-reactive protein, increased risk of pneumonia, sepsis, acute respiratory distress syndrome, and heart failure [24]. In fact, a cluster of unexplained pneumonia cases were first reported in Wuhan, China in late December 2019. A few days later, the cause of this pneumonia was identified as a new member of the coronavirus family. Since then, the virus has spread throughout China and precipitated a global pandemic [6].
Early reports from China suggested the most common symptoms of COVID-19 infection were fever (88%) and dry cough (67.7%). Rhinorrhea (4.9%) and gastrointestinal symptoms (diarrhea 4–14%) were less common. At the same time, a majority of patients (81%) had only mild symptoms (no pneumonia or mild pneumonia). Among patients with more pronounced symptoms, 14% experienced severe symptoms while 5% were critically ill with respiratory failure, septic shock, or multiorgan dysfunction or failure [3].
Although the novel coronavirus preferentially infects cells in the respiratory tract, autopsy results from Germany showed that it can be detected in multiple organs. The highest levels of the virus were detected in the lungs and the respiratory tract, while lower levels were usually present in the heart, liver, brain, kidneys, and spleen. This data suggests that SARS-CoV-2 may spread via the bloodstream and infect other organs. It also appears that COVID-19 may predispose patients to venous thromboembolism in several different ways including via endothelial dysfunction and promotion of a procoagulatory state by tissue factor pathway activation. High plasma levels of proinflammatory cytokines were observed in a small subset of patients with severe COVID-19 and, therefore, direct activation of the coagulation cascade by a cytokine storm is also plausible [12].
In one study, it was found that 22% of critically ill patients experienced myocardial injury from the infection [3]. In another study, the incidence of thrombotic complications in ICU patients with COVID-19 infections was reported to be 31%. It was concluded that COVID-19 may predispose to both venous and arterial thromboembolism due to excessive inflammation, hypoxia, immobilization, and diffuse intravascular coagulation [27].
In addition, the COVID-19 pandemic is surprisingly associated with neurological symptoms and complications including anosmia, hypogeusia, seizures, and stroke. Although statistics are not widely available at this point, the clinical course of COVID-19 is most severe in elderly male patients with comorbidities such as hypertension, diabetes, heart disease, and obesity which are all risk factors for stroke. There appears to be hypercoagulability associated with COVID-19 as a result of a “sepsis-induced coagulopathy” that may be a predisposing factor due to the formation of blood clots in the body [28]. COVID-19 complications in the brain can include delirium, inflammation, and encephalitis. A new study from UCL suggests that serious problems can occur even in individuals with mild cases of the virus [29].
A temporary loss of smell (anosmia) can be a consistent indicator of COVID-19 infection. An interesting finding is that the virus seems to change the sense of smell in patients by infecting and affecting the function of non-neural cells that support olfactory neurons. However, the neurons themselves do not appear to be infected as they do not express ACE-2 receptors [30].
Diabetes is already known to be a risk factor for COVID-19 and diabetics are more likely to die from the disease. Now, mounting evidence suggests that not only does diabetes make patients more vulnerable to the novel coronavirus, but the virus may actually trigger diabetes in some. Preliminary tissue studies indicate that the virus may act by damaging insulin-producing cells in the pancreas of affected individuals [31].
Even though, initially, children were thought to be unaffected by the novel coronavirus, a cluster of children with hyperinflammatory shock and features similar to Kawasaki disease and toxic shock syndrome was first reported in England. This hyperinflammatory condition could lead to severe illness, multiorgan failure, and even death in extreme cases. New reports out of the UK and US suggest that symptoms in young children (mainly toddler to elementary school age) can include inflammation of the blood vessels and coronary arteries. Almost all these pediatric cases had positive SARS-CoV-2 test results. As a result, this illness has been termed COVID-19-associated multisystem inflammatory syndrome [32].
Historically, there is no doubt that vaccines have provided a tremendous tool against infection for a variety of microbes including those causing small pox, tetanus, typhoid, cholera, and polio. Vaccines are an effective way for a population to achieve herd immunity (the concept that a pandemic will end once 60–70% of people become immune to any particular virus or microorganism). However, more recently, there are instances in which the production of viral vaccines has not been so successful as in the case of human immunodeficiency virus (HIV) and human coronaviruses (HCoVs) possibly due to their complex genomes. Virologists and immunologists maintain that it takes up to ten years to prepare a really good vaccine that has been properly tested. In fact, some of these specialists are skeptical about the race to find the first vaccine for the novel coronavirus within one year and often strike a cautionary note.
There are a number of things to consider in connection with a SARS-CoV-2 vaccine. Firstly, even if a safe and effective vaccine is made against the novel coronavirus, it may not be widely available in time to make a significant difference to the pandemic. Secondly, no successful vaccine against any coronavirus has been produced so far despite seventeen years of research. Moreover in March, the British Society for Immunology published an open letter stating that it is unknown whether this virus will induce long-term immunity in affected individuals as other related viruses do not [33]. Thirdly, certain vaccines can protect against a disease, but not against infection, so vaccinated individuals could potentially become asymptomatic carriers of SARS-CoV-2. Fourthly, some vaccines developed against SARS-CoV-1 (a close viral relative of SARS-CoV-2) actually exacerbated the disease in mice. Fifthly, although the easiest way to make a vaccine is to inactivate the pathogen, there are new vaccines in current trials based on RNA from coronaviruses or other RNA viruses that have never before been approved or tested in humans. Therefore, there could conceivably be unintended or irreversible consequences. Finally, at least one of the novel coronavirus vaccines approved for clinical trials so far has caused severe adverse events in three of eight healthy, young individuals that were tested [34] and other trials have been suspended. Unfortunately, the contamination of vaccines which are mass produced for a burgeoning human population also seems to be a potential problem and an ideal tool for rival countries to conduct biological warfare upon each other. Oral or nasal vaccines may be safer in this respect [35]. In addition, there is a physical limit to the number of vaccines a person can safely receive as new and deadlier viruses arise in the environment.
The action of UVB radiation striking and reacting thermally with 7-dehydrocholesterol in human skin results in the production of Vitamin D3 in the human body. This form of Vitamin D is converted to the hormonal metabolite, calcitriol, in a set of biochemical reactions in the liver, kidneys, and other organs as required. Then, calcitriol binds with the nuclear vitamin D receptor, which is a DNA binding protein, that interacts directly with regulatory sequences near target genes and affects their transcriptional output.
Vitamin D also enhances cellular innate immunity partly through the induction of antimicrobial peptides, including human cathelicidin, and, defensins. Cathelicidins exhibit direct antimicrobial activities against a spectrum of microbes including many types of bacteria, enveloped and nonenveloped viruses, and fungi. The main action of these host-derived peptides is to kill the invading pathogens by perturbing their cell membranes. Moreover, vitamin D is effective in reducing concentrations of pro-inflammatory cytokines that produce the inflammation that injures the lining of the lungs leading to pneumonia during viral infections like COVID-19 and increasing concentrations of anti-inflammatory cytokines [24].
According to a recent clinical study with a large sample size taken from different countries around the world, vitamin D supplements were found to protect against respiratory tract infections including colds and influenza. The most benefit was observed in patients who were very vitamin D deficient. This protective effect is likely provided by the capacity of vitamin D to boost levels of antimicrobial peptides in the lungs [36].
Vitamin D deficiency is a world-wide problem, but is particularly pronounced in the elderly, who are at greatest risk of contracting severe COVID-19 infection. The release of pro-inflammatory cytokines is one of the major causative factors in serious COVID-19 infections. However, vitamin D modulates their presence in the body by preventing macrophages from releasing too many inflammatory cytokines and chemokines. Calcitriol has also been found to exert an influence on ACE-2 receptors. Thus, it is not surprising that vitamin D deficiency has been correlated with COVID-19 cases and an increased risk of mortality in a European study [37].
RNA synthesis occurs in the life cycle of the SARS-CoV-1 virus in order to reproduce its genetic material and is catalyzed by an RNA-dependent RNA polymerase, which is the core enzyme of a multiprotein replication/transcription complex. In the case of SARS-CoV-1, an excess of intracellular zinc ions has been found to efficiently inhibit the RNA-synthesizing activity of this replication and transcription multiprotein. Enzymatic studies in vitro have revealed that zinc directly blocks the activity of the RNA polymerase by inhibiting elongation and reducing template binding. This RNA polymerase core, which is a central component of the coronaviral replication/transcription machinery, is well conserved among the members of the coronavirus family including SARS-CoV-2 [38, 39]. Therefore, it is quite possible that zinc treatment would have a similar biochemical effect on SARS-CoV-2 and interfere with its ability to replicate.
Since current research indicates that the mineral, zinc, can inhibit the replication of coronavirus and a variety of other RNA viruses in cell culture, it has become a potentially important and interesting supplement to study at this time. In the human body, zinc performs a variety of vital antioxidant functions and is required for maintaining good health. Inside the cell, the harmful effects of free radicals are balanced by the action of antioxidant enzymes (such as copper-zinc superoxide dismutase) and non-enzymatic antioxidants (such as metallothioneins). As zinc cannot pass easily through membranes, zinc-transporting proteins, ZIPs (Zrt-Irt-like protein or Zinc Iron permease) and ZnTs (Zinc transporters) help to facilitate this process. Metallothionein also aids in the regulation of zinc levels and the distribution of this metal in the extracellular space. The presence of zinc within the cell causes an increase in metallothionein, which is the major zinc-binding protein, and together they form a thermodynamically stable complex [40, 41]. Thus, low risk ways of increasing zinc bioavailability in the body can be safely considered.
In rats, rice fortified with zinc oxide or zinc carbonate is a feasible vehicle for zinc absorption, although zinc oxide displays lower bioavailability than zinc carbonate [42]. In young adults, zinc absorption from supplemental zinc citrate is comparable with that from zinc gluconate, but higher than from zinc oxide [43]. It is already known that zinc can be absorbed from topical (non-nano) zinc oxide by human skin in small quantities (nano forms of zinc oxide are not associated with significant zinc absorption) [44]. One of our recent studies suggests that zinc is absorbed by the human body from our suncare products (all with the same basic formula containing a medicinal form of zinc oxide) in sufficiently large quantities with regular use [45].
So, recently, when our company received an inquiry from Health Canada regarding any innovations that may benefit Canadian health workers at this critical time during the novel coronavirus pandemic, the answer was that we do have a product that may be useful to medical professionals and health workers in the field. It is a natural, award-winning suncare product specially formulated to block apoptotic sunburn (Skin Protector Plus). Its active ingredient is a non-nano, medicinal form of zinc oxide. The novel thing about this product is that it appears to be an efficient delivery system for boosting zinc levels in the whole body in a relatively short period of time. There is no toxicity associated with this product due to the use of high grade zinc oxide and natural ingredients. Since it is so safe and contains no harsh chemicals (already tested on human volunteers), no pre-clinical trials would be required to test its efficacy in protecting subjects from COVID-19 in a clinical study. The objective of such a comprehensive study would be to test and confirm the hypothesis outlined above, in vivo; namely, if maximum zinc levels are maintained in the human body via percutaneous zinc absorption from a topically applied zinc oxide cream, then it may provide one suitable defense against SARS-CoV-2 infection. Although oral supplementation is also an option, this type of topical application on the surface of the skin may be a faster method of ensuring even zinc distribution throughout the body and delivery to the various potential points of viral entry. Moreover, it may actually provide a physical barrier or blockade against entrance of the virus into the body by allowing suffusion and accumulation of zinc pools directly beneath the skin.
Quinine, an alkaloid derived from the bark of the cinchona tree, is most commonly found in South America, Central America, the islands of the Caribbean, and parts of the western coast of Africa. It is an important antimalarial drug and a synthetic form with a similar mode of action is known as chloroquine [46]. Chloroquine has been reported to inhibit the SARS-CoV-1 virus in infected cell cultures in vitro at doses equivalent to those used in the treatment of acute malaria in humans. Its antiviral effect appears to depend on the fact that chloroquine is a weak base that increases the pH of acidic vesicles when added extracellularly. The nonprotonated portion of chloroquine enters the cell where it becomes protonated and concentrated in acidic, low-pH organelles such as endosomes, Golgi vesicles, and lysosomes. The subsequent antiviral activity of the chloroquine depends partly on the extent to which a particular virus utilizes endosomes for entry into the cell [47]. In addition, this drug appears to interfere with terminal glycosylation of the angiotensin-converting enzyme 2 (ACE-2) cellular receptor, which is engaged by the virus for extracellular binding. This step may have a negative effect on the ability of the virus to gain entry into the host cell and, therefore, to initiate its replication cycle. Thus, infection may be deterred at clinically admissible concentrations [48]. Chloroquine also displays an immunomodulatory activity by suppressing the production and release of tumour necrosis factor alpha and interleukin 6 [49].
Furthermore, chloroquine was demonstrated to have strong antiviral activity against HCoV-OC43 in vitro. The anticoronaviral properties of chloroquine were also tested against HCoV-OC43 infection in newborn mice in vivo. Treatment with daily doses of chloroquine were found to have a long-lasting protective effect against lethal coronavirus OC43 infection in the newborn mice [1].
These favourable results suggest that chloroquine may be considered for use at antimalarial doses in the prevention of infections caused by coronaviruses, particularly SARS-CoV-2, which utilizes ACE-2 receptors in order to gain entry into host cells like its close relative, SARS-CoV-1.
Licorice root has been a commonly used ingredient in both Ayurvedic and traditional Chinese medicine for centuries, particularly in cough and cold remedies. Twenty triterpenoids and nearly three hundred flavonoids have been isolated from this herb. Scientific studies have shown that these metabolites possess many pharmacological activities including antiviral, antimicrobial, anti-inflammatory, and anti-tumour properties. However, glycyrrhizic acid or glycyrrhizin (GL), 18β-glycyrrhetinic acid (GA), liquiritigenin (LTG), licochalcone A (LCA), licochalcone E (LCE) and glabridin (GLD) are the main active components which possess antiviral and antimicrobial activities [50].
It has been known for some time that glycyrrhizic acid extracted from licorice (Glycyrrhiza glabra) root is active against viruses. This chemical is able to disrupt the growth and cytopathology of several unrelated DNA and RNA viruses without harming the host cell or its ability to replicate. Glycyrrhizic acid has also been demonstrated to inactivate herpes simplex virus particles irreversibly [51].
In a more recent study, the anti-SARSCoV activity of 15 glycyrrhizic acid derivatives was tested. Glycyrrhizin was shown to inhibit SARS-CoV-1 replication in vitro [52]. GL has also been reported to act by inhibiting viral gene expression and replication, reducing adhesion force and stress, and reducing High mobility group box 1 protein (HMGB1) binding to DNA. In addition, GL can enhance host cell activity by blocking the degradation of IκB, activating T lymphocyte proliferation and/or suppressing host cell apoptosis [50]. Thus, the potential for this licorice root component (GL) against SARS-CoV-2 infection is plausible.
Isoflavones and their related flavonoid compounds, particularly genistein, exert antiviral properties against a wide range of DNA and RNA viruses in vitro and in vivo [53]. The biological properties of the flavonoids are well studied, but the mechanisms of action underlying their antiviral properties are not fully understood. Isoflavones appear to have a combination of negative effects on viruses including affecting virus binding, entry, replication, viral protein translation and formation of certain viral envelope glycoprotein complexes. A variety of host cell signalling processes can also be affected by isoflavones including induction of gene transcription factors and secretion of cytokines. All these effects are dependent on dose, frequency of administration, and different combinations of isoflavones employed in bioassays in vitro. Genistein may be able to mimic the action of 17-beta-estradiol [E2] due to its similar structure or to act as an E2 antagonist and its activity as a broad-spectrum tyrosine kinase inhibitor may contribute to its ability to influence estrogen receptor-independent mechanisms [54]. Despite their unique effect on immune function and anti-inflammatory activity, there is still a lack of data confirming the antiviral efficacy of such soy isoflavones in vivo against coronaviruses and other viruses thereby forming a worthwhile subject for biochemical study.
At least seven human coronaviruses have been described to date including SARS-CoV-2, which is closely related to and resembles SARS-CoV-1 in many respects. Both viruses bind to ACE-2 receptors on human cells. ACE2 is a membrane-associated aminopeptidase that converts angiotensin II to angiotensin 1–7 and plays a general role in the cleavage of peptides. Expression of ACE2 in human tissues correlates with known sites of SARS-CoV-1 infection including lungs (particularly airway epithelia), heart, kidneys, small intestine, testes, and vascular endothelia. These same tissues overlap with known sites of SARS-CoV-2 infection in humans.
A cluster of unexplained pneumonia cases were first reported in Wuhan, China and, a few days later, the cause of this pneumonia was identified as a new member of the coronavirus family. SARS-CoV-2 infection appears to be associated with a puzzling array of symptoms and complications. The major symptoms noted in China were fever (88%) and dry cough (67.7%), while rhinorrhea (4.9%) and gastrointestinal symptoms (diarrhea 4–14%) were less common. A majority of patients (81%) had only mild symptoms (no pneumonia or mild pneumonia). Among patients with more pronounced symptoms, 14% experienced severe symptoms while 5% were critically ill with respiratory failure, septic shock, or multiorgan dysfunction or failure.
New data suggests that SARS-CoV-2 may spread via the bloodstream to infect other organs. In addition to the lungs, other target organs can include the heart, liver, brain, kidneys, and spleen. It also appears that COVID-19 may predispose patients to venous thromboembolism in several different ways including via endothelial dysfunction and promotion of a procoagulatory state. In fact, it was found that a significant percent of critically ill patients experienced myocardial injury from the infection and it has been concluded that COVID-19 may predispose to both venous and arterial thromboembolism due to excessive inflammation, hypoxia, immobilization, and diffuse intravascular coagulation. The COVID-19 pandemic is associated with various neurological symptoms and complications including anosmia, hypogeusia, seizures, and stroke, as well. COVID-19 complications in the brain can include delirium, inflammation, and encephalitis. Despite initial reports that children were unaffected by the novel coronavirus, it has emerged that pediatric patients are susceptible to a COVID-19-associated multisystem inflammatory syndrome that can cause serious inflammation of the blood vessels.
Several internal risk factors have been identified for SARS-CoV-2 infection. The main ones include age (older adults are more vulnerable to serious infection by the virus), gender (the virus is significantly more deadly in men than in women), obesity, heart disease, diabetes, cancer status, and smoking. However, there is no convincing evidence to date that any particular ethnic group displays a stronger genetic susceptibility to the virus (although, there may be a possible link to an inherited Neanderthal gene locus). Nevertheless, specific genetic variants such as those for the gene that encodes a protein that interacts with the ACE-2 receptor may be involved in determining individual patient responses to the disease. Simultaneously, external risk factors like environmental pollution, social conditions such as crowding, and frequency of exposure to infected persons also seem to play an important role.
Reports of re-infection in recovered humans is a surprising aspect of this virus. Recently, a team from the University of Hong Kong reported the first case of re-infection of COVID-19 within a period of approximately four and a half months. Genomic analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Moreover, there was a difference of 24 nucleotides between both viruses that infected the patient suggesting two different viral strains were involved [55].
Even though the virus is associated with positive COVID-19/COVID-19 antibody and high C-reactive protein test results, antibody levels may decline soon after infection. Consequently, it is quite possible that a lasting resistance to the virus will not be achievable. In the event that long-term immunity cannot be induced to the novel coronavirus by a vaccine, an annual, bi-annual, or even tri-annual inoculation may be required (current data suggests that antibodies begin to decrease or disappear three months after infection). This means that other modes of protection and prevention like supplementation may be more relevant in this case. Some candidates include Vitamin D, zinc, chloroquine/quinine, glycyrrhizic acid, and genistein due to anti-viral properties such as the ability to inhibit replication and reproduction of coronaviruses.
Scientists have concluded that drastic social distancing, quick detection and isolation of infected individuals and travel restrictions were the most effective steps for containment of COVID-19 in China. Genome sequencing has also helped to track and control COVID-19 infections quickly. However, if people do not continue to be careful, certain places may become vulnerable to further rounds of this disease. WHO recently reported that coronavirus infections among younger populations were skyrocketing. The proportion of cases in teens and young adults increased six-fold, while the proportion in young children and babies increased seven-fold by August. This may be attributable in part to the resurgence of large parties and social gatherings attended by young people following the relaxation of restrictions during the summer. Therefore, it seems very likely that the denouement of the COVID-19 story will be largely dictated by our social habits and ability to adapt to a new set of societal norms and conditions. This will include wearing face masks in public places, possibly, with a thin zinc coating along with a special zinc oxide crème formulation applied to the skin underneath [56].
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