Targeted Gene Therapy: A Fantasy or a Reality?

Introduction

Current advancements in the medical field have made reality the prospect of gene therapy, which was considered impossible not too long ago. While in the past, this was considered some sort of science fiction, the current leaps and bounds in technological advancements are making it possible to treat diseases from the very core structure, DNA. Initially, this therapy was confined only to the hematopoeitic stem cell population. However, now virtually any part of the human body can be treated with DNA therapy. The current scope of gene therapy is limitless, with a focus primarily on genetic and congenital disorders, cancer therapy and management of chronic pain and other degenerative conditions.

Many fields now come under the single heading of gene therapy. By definition, gene therapy is defined as a method whereby a normal gene can be introduced into the DNA sequence to replace any faulty genes leading to genetic disorders or diseases. This is, however, the primary and the initial type of genetic experiment and experience that was carried out when gene therapy initiated. The therapy can be carried out in two ways, either through germ cell gene therapy or somatic cell gene therapy.

The former is still in the experimental stages of development due to the many legal and ethical issues involved; however, by far, this method is the most effective one in treating and curing genetic conditions and diseases. (Rovephoenix, 2005, np) Now there are multiple models of genetic transfer and manipulation available, all showing some promise in the progression of the field.

The most common mode of introduction of a new gene into the DNA sequence is through the use of viruses. Many viruses are currently being used to carry out this function, and among them are included retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.d this method has been used successfully in the past and continues to be used in many of the research works. However, the focus is also shifting towards non-viral methods of genetic transfer.

Non-viral methods may include the direct introduction of the gene into the target cells, liposomes, or binding the DNA to a specific target attaching molecule. (HGPI, 2007, np) the newer techniques are continuously developing, and with time only will the efficacy of each one understood.

The non-viral therapy, in many ways, increased the efficiency and results of the gene therapy that was facing hurdles due to the only mode of viral transfer. Viral modes of genetic transfer posed many problems such as virus recombination, oncogenic effects and unexpected immune response. The non-viral methods helped by increasing the simplicity of the introduction of the DNA into the body, the relatively less costly making of the drugs, and the absence of any immune response common to the viral model of gene delivery. (Niidome and Huang, 2002, pp 1648)

The non-viral techniques can be categorized into two forms, naked DNA delivery by any physical method and chemical mediated delivery. The physical mechanism is usually carried out on the tissues of skeletal muscle, liver, thyroid, heart muscle, urological organs, skin and tumours. Features such as electroporation, gene gun delivery method, ultrasound, hydrodynamic injection etc., are some of the techniques that are used to assist the physical introduction of DNA into the body. (Niidome and Huang, 2002, pp 1649)

The chemical methods of gene delivery include lipid-mediated gene delivery, peptide-mediated gene delivery, and polymer mediated gene delivery, respectively. Alongside, many new methods are being studied to improve the gene therapy procedures without the reliance on viral vectors. (Niidome and Huang, 2002, pp. 1650) These methods, however, are not completely perfect as yet and therefore, more research is required before any right methods can be categorized.

Currently, there are four therapeutic strategies that are used in genetics to treat diseases. Gene therapy proper is the method whereby a healthy gene is introduced into the target organ to replace the defective gene. Most of the development in this area refers to stem cell transplantation and is widely used in the brain. The second method works by reducing the expression of the mutated genes. For this purpose, some of the genes used include antisense oligonucleotides and ribozymes.

The third technique includes positional cloning strategies, where a mutation in the genes is used to create drugs for the treatment of the condition. Finally, the introduction of the embryonic tissue into the cell population is the fourth option, whereby the lost tissue is replenished. Except for the last one, all three techniques are being used successfully in clinical practise with promising results and better outcomes than conventional therapeutic methods. (Carter and Schuchman, 2001, pp 392)

The advantages of gene therapy can be appreciated from every aspect and in the various organs that it is applied. Among the various organs, the skin has achieved increased interest among genetic researchers as the skin has many capabilities of healing itself. Skin can also produce many substances that are necessary for the various healing processes of the body. Therefore, this is the first area in dermatology where gene therapy is currently working.

With this technology, the skin can be made to produce a variety of therapeutically effective substances that can be used in other therapeutic interventions. (Hengge, 1999, pp 419) Skin is a very good detoxifier and can act as a metabolic sink. Skin especially has the capability of producing immunity-related products that are being widely cultivated through genetic therapy. (Hengge, 1999, pp 420)

Through gene therapy, the researchers are now able to achieve improved transduction rates by pseudotyping, which can help in target cell recognition, in bicistronic expression, can help in reducing the immunogenicity of virus vectors and can help in phenotypic correction of various diseases. (Hengge, 1999, pp 420)

Another area where gene therapy is showing its efficacy is in the relief of various treatments such as chronic pain conditions or arthritis etc. This particular therapy is working by stimulating the painkilling effects of opiates which are commonly prescribed in such patients. This therapy is in many ways superior to other medications, which can cause sleepiness or other side effects with limited efficacy in reducing pain. This therapy has been able to provide pain relief for more than 3 months in the patients studied with higher satisfaction levels. This therapy, therefore, may also be useful for chronic pain conditions such as cancer (Science Daily, 2008, np)

Role of Targetted Gene Therapy in Various Immunodeficiency Disorders

Immunodeficiency states and diseases have long been treated symptomatically. As such, there has been no particular therapy that could cure such patients. Most of these patients had a very short life span characterized by multiple and recurrent diseases and infections. (Mackiewicz, 2001, pp 200) The introduction of stem cell research in gene therapy has been very effective in the severe combined immunodeficiencies. Immunodeficiency states have been successfully treated with various genetic therapies. It was very difficult in the initial stages of the therapy because of a lack of knowledge about various gene functions. With gaining knowledge in this area, it is now possible to treat the correct gene and rectifying it.

Hematopoeitic stem cell research is one of the most contributing areas in this research, and new techniques such as gene transfer may help in improving the outcomes. (Mackiewicz, 2001, pp 204) These cells are mostly used in gene therapy due to their ability to constantly renew themselves, thereby improving themselves. These cells also retain the ability to differentiate themselves into various populations. However, there are certain technical difficulties currently being encountered in the HSC. For this purpose, the first challenge concerned is to improve the engraftment of the cells. Mostly the aim is to purify the transduced cells from the competitors.(Hossle, Seger and Steinhoff, 2002, pp 89)

Cell surface marker genes can be introduced into the cell cultures, which would then express themselves on the successfully transduced cells. These cells can then be selected and introduced in the patients, making the probability of successful treatment higher. However, the human experiments seriously flaw at this point since the selection of the transduced cells may, in fact, result in grafts that are unable to support normal hematopoiesis. There are now clearer concepts about the type of grafting to be used as well. For example, it is now known that “engraftment is dependant on the ratio of the host to donor HSC but not on any actions in the clearing space”.(Hossle, Seger and Steinhoff, 2002, pp 89)

Earlier on, it was thought that HSC niches must be cleared for successful chances of engraftment. These and many other concepts are now being changed as more information is gathered, signifying that genetic therapy still lies in its infancy stages. (Hossle, Seger and Steinhoff, 2002, pp 90)

Potential of Gene Therapy in the Treatment of Cancer

Cancer therapy stands to benefit the most from gene therapy. It stands to have more potential than inherited disorders have from gene therapy. However, there are certain challenges that cancer therapy has. These include the inability to equally distribute genetic material into the whole of the tumour mass. Understanding the various aspects of cellular multiplication and biological activities is of help.

However, the simultaneous requirement is for the development of good vectors for the proper delivery of the genes. Among the most common therapies used in cancer treatments are the suicide genes. Suicide genes include coding for particular enzymes which modify “a non-toxic pro-drug into a toxic molecule, thus leading to the death of the cells expressing the suicide gene” (Kouraklis, 1999, pp 674).

The conditions for which this treatment is being given include malignant mesothelioma, brain tumours, ovarian cancer, metastatic colon cancer, malignant melanoma, and breast cancer. Another treatment in wide use is tumour suppressor gene therapy and oncogene inactivation. This therapy is being used for head and neck squamous cell carcinoma, hepatocellular carcinoma, breast cancer, acute leukaemia and lung cancer, and primary and metastatic liver cancer. Immunogene therapy has shown promise against metastatic ovarian cancer, brain tumours, malignant melanoma, renal and squamous cell carcinomas, and breast cancer. (Kouraklis, 1999, pp 676)

However, for any progress in the field of cancer and its treatment, there is needed constant understanding about the various processes of cancer and how these are genetically influenced and controlled. There are still questions about the direct introduction of genes into the nuclear DNA or whether there should be an extrachromosomal gene transfer system. Tumour suppressor genes are currently being used for the treatment of a variety of cancer, and more research in this area is needed. Since viral vectors are an important mode of transfer of the genes, there is a need for further research and development in the area. The aim is to reduce the toxicity of these viral vectors and increase the transduction efficiency of the non-viral vectors. (Kouraklis, 1999, pp 680)

Genetic Therapy and Osteology

Current orthopaedic surgeons are also looking into the possibility of regenerating the disc tissue in the bone structure of old patients and those suffering from such disorders. Gene therapy, therefore, is being researched in the areas of disc regeneration by knowing the basic fundamentals of disc formation. It is known that disc tissue is made from chondrocytes which synthesize proteoglycans and collagen type II. By improving their production, there is hope that disc degeneration can be achieved successfully. For this purpose, viral vector gene therapy is used, which incorporates TGD beta 1 into the DNA. (Vaccaro, 2006, pp 449)

This gene has the capability to stimulate the formation of proteoglycan and collagen synthesis. A dose dependant response has been learned, and multiple uses of growth factors and affecting genes have shown enhanced results. The introduction of the Sox-9 gene as a promoter of chondrogenesis is also helping in the advancement of the technology. However, there are certain challenges in this area as well. For example, while the growth factors may be successful in slowing the degeneration process, they may not reverse it, bringing to question the possibility of regenerating disc tissue. However, even so, the development is good news for patients, who may not need frequent surgeries for disc replacements etc. (Vaccaro, 2006, pp 449)

So Is Targetted Gene Therapy a Fact or a Fiction?

In many ways, the fictitious nature of gene therapy is present, as it opens up to us the options of cures for all ills. Now there is hope that even the most deadly of medical conditions such as cancer and HIV etc. can be cured. With increased knowledge in the various disciplines of the human cellular mechanisms, there is an improved understanding of how gene therapy may provide answers to questions that were never known.

However, many facts still place the researchers in doubt about the practical implications of genetic therapy. For example, genetic therapy is still in its infancy stages, and there is a lot to be known about the advantages and disadvantages of various procedures before any attempt should be made on human subjects. These attempts are, however, being made, and critics still caution against using these new therapies. Therefore, genetic therapy is currently both fact and fiction since for fiction to become a reality, time is required.

References

Janet E Carter and Edward H Schuchman, 2001. Gene Therapy for Neurodegenrative Diseases: Fact or Fiction? The British Journal Of Psychiatry 178:392-394.

Hengge UR, Taichman LB, Kaur P, Rogers G, Jensen TG, Goldsmith LA, Rees JL, Christiano AM. How realistic is cutaneous gene therapy? Exp Dermatol 1999: 8: 419–431. C Munksgaard.

Johann P Hossle, Reinhard A Seger, and Dirk Steinhoff, 2002. Gene Therapy of Hematopoeitic Stem Cells: Strategies for Improvement. News Physiological Sciences, Vol. 17, No. 3, 87-92.

Human Genome Project Information, 2007. Gene Therapy. Web.

Gregory Kouraklis, 1999. Progress in Cancer Gene Therapy. Acta Oncologica, Vol 38, No. 6, pp 675-683.

Andzej Mackiewicz, Maciej Kurpisz, and Jan Zeromski, 2001. Progress in Basic and Clinical Immunology.

Mount Sinai Hospital / Mount Sinai School of Medicine. “Targeted Gene Therapy Provides Relief For Chronic Pain, Study Shows.” ScienceDaily 2008. Web.

T Niidome and L Huang, 2002. Gene Therapy Progress and Prospects: Non Viral Vectors. Gene Therapy, Vol 9, No 24, pp 1647-1652.

Rovephoenix, 2005. An Overview of Gene Therapy. Web.

A R Vaccaro, 2006. Disc Regeneration: Is It Fact or Fiction? Journal of Bone and Joint Surgery, British Volume Vol 88B Issue SUPP_III, 449.

The Gay Gene: Understanding Human Sexuality

A gene is a molecular unit of heredity of a living organism. In simpler terms, sections of DNA that contain complete instructions are known as genes. Since they are units of heredity, they can be passed on from one generation to the next. Genes determine which species a living organism belongs to (Arnqvist 1061). Genes usually encode for proteins. Proteins go out into the body and carry out the instructions given to them by the genes. Molecular engineering has been used to tinker with genes and to change what they code for. Sometimes these genes are removed entirely and replaced with different ones. In the long debate on homosexuality, many wonder if it is a lifestyle choice or if it is determined by one’s genes. Religious sects condemn it as evil and some even believe that prayers can ‘cure’ homosexuality. Scientists have carried out studies to try and find out if a gay gene exist and lay the debate to rest.

In his article, Burr compares human sexual orientation to human handedness. Both traits have a majority orientation and a minority orientation. People do not choose to be left-handed and there is no gene that has been found for left-handedness (Burr para. 4). If there is a gene for left-handedness, it shows a lot of variation. Some left handed people use the left hand for writing while using the right for other activities while others use the left for particular activities only (Lalumière, Blanchard and Zucker 576). If this gene existed and it was similar to a gay gene, it would explain the difference in gay people. Some gay individuals’ exhibit highly feminine characters while others seem to be just on the edge between heterosexual and homosexual. Where then would one draw the line between being gay and being straight? Discovery of a gay gene would be a very important factor in eliminating doubt.

If a gay gene is real, what would be the probability of inheriting it? In pedigree analysis, traits that are genetically influenced aggregate in families and in the case of dominant or sex-linked inheritance, are transmitted from one generation to the next (Dean 322). Since males receive the X chromosome from their mothers, if the X chromosomes contain a gene that increases the probability of being homosexual, then there would be a higher incidence of homosexuality in brothers. Human sexuality is complex and there is no single genetic locus that has been found to account for homosexuality (Dean et al. 323).

Some scientists argue that it is not the presence of a gay gene but the absence of certain genes that causes homosexuality. There are certain proteins that control sex orientation in mice. A good example is an enzyme known as fucose mutarotase whose absence in mice was proven to cause an abnormal sexual receptivity. This is according to research carried out by Dongkyu et al (2). They were able to observe that mice lacking in the above enzyme could not utilize and incorporate fucose into protein. This led to masculine behavior and a preference for female urine. In a different study by Yan et al (para. 4), the neurotransmitter 5-hydroxytryptamine was discovered as being a requirement for male sexual preference. Previous studies have implicated this neurotransmitter in male sexual behavior but not in sexual preference (Yan et al para. 5). Certain mutations are a biological explanation for sex orientation. In a study by Kimchi et al (4), it is reported that Trpc2 female mice show a reduction in female-specific behavior. These mutant females are said to have displayed unique characteristics of male sexual and courtship behaviors like mounting, pelvic thrust, solicitation and anogenital olfactory investigation. In mice, pheromone detection is mediated by the vomeronasal organ (VNO) and the main olfactory epithelium. Male mice that are deficient for TRPC2, an ion channel specifically expressed in VNO neurons and essential for VNO sensory transduction, are impaired in sex discrimination and male-male aggression (Kimchi et al.). Other studies show that there are rare alleles encoding either male ornaments or female preferences for them that are better protected against loss in species (Hudson and Pfenning 1090). In this study, they argue that in evolution, mutation may lead to males evolving elaborate secondary sexual traits as seen in some animals.

Hormones may also play a role in sexual orientation. Right after conception, it is hard to tell male and female zygotes apart. The female brain is the default. The ‘masculinisation’ of the male brain by sex hormones initiates sex changes (Swidey para. 4). The theory of how hormones affect male sexual orientation is based on this fact. If the brain develops as female in the absence of testosterone, what leads to the loss of female characteristics leading to lesbianism? Research in mammals has demonstrated that pheromone sensing in the periphery is important for sexual preference. Pinker (3) differs with this finding stating that when people check out a prospective partner, they seek out words or pictures, not dirty laundry.

Scientists have studied mice behavior and anatomy to try and discover the gay gene. There are various ways that homosexual mice differ from heterosexual mice in anatomy. Mice lacking the fucose mutarotase enzyme exhibit male-like sexual behavior. This has been attributed to neurodevelopmental changes in the preoptic area of the mutant brain resembling a wild type male (Dongkyu et al 2). Males lacking central serotonergic neurons lost sexual preference (Yan et al. para. 8).

Reproductive behaviors (for example, receptivity or mounting) are one of the characteristics of sexual differences. Mice are usually the animal of choice in experiments because they are mammals and reproduce fast. They are more ethically acceptable than primates and are genetically similar to humans. On the other hand, there is a difference between specific hormones in mice and those found in human beings (Liu para. 5). As such, studies carried out on mice may not entirely be compatible with those carried out on human beings.

What would happen if a gay gene in humans was discovered in humans? If this happened, there would be a great change in the way gay people are perceived and treated. There would be a reduction in homophobic behavior since being gay would not be regarded as an option anymore (Swidley para. 7). Most believe that a gay gene would go against the theory of evolution. Natural selection is based on the fact that the best traits are passed on and persist in order to keep the species alive. The gay gene, if there was one, would not lead to reproduction and would lead to the dwindling of the human species. In this case, nature may have found a way of reducing human population in a less gruesome way like floods, famine and disease in the past.

There would be disadvantages too in that some mothers would probably opt to change their child’s sexuality or to abort a child with this gene. The presence of a gay gene in people who believe they are heterosexual would also likely cause confusion. It may also lead to discrimination because it would be possible for an organization to determine one’s sexual preference scientifically. Religious sects would have to come up with a way to accept gay people, not as sinners, but as a creation of God which would go against most popular religious teachings.

Works Cited

Arnqvist, Goran, Edvardson, Martin, Friberg, Urban and Nilsson, Tina. “Sexual Conflict Promotes Speciation in Insects.” Proc Natl Acad Sci, 97(2000): 10460-10464

Burr, Chandler. “The “Gay Gene” Hits the Big Time”. The Harvard Gay & Lesbian Review. 26.1996

Dean H. Hamer, Stella Hu, Victoria L. Magnuson, Nan Hu, Angela M. L. Pattatucci. “A Linkage Between DNA Markers on the X Chromosome and Male Sexual Orientation.” Science, Science 261. 5119 (1993): 321–7

Dongkyu, Park, Dongwook, Choi, Junghoon, Lee, Dae-sik. Lim and Chankyu, Park. “Male—like sexual behavior of female mouse lacking fucose mutarotase.” BMC Genetics, 11.62 (2010):1-2

Hudson Kern Reeve and David W Pfenning. “Genetic Biases for showy males: Are some genetic systems especially conducive to sexual selection?” PNAS, 100 (2003):1089-1094

Kimchi, Tali, Jennings, Xu and Dulac, Catherine. “A functional circuit underlying male sexual behavior in the female mouse brain.” Nature 95.472 (2011)

Lalumière, Martin, Blanchard, Ray and Zucker, Kenneth. “Sexual orientation and handedness in men and women: a meta-analysis”. Psychol Bull, 126. 4(2000): 575–92.

Pinker, Steven. “Sniffing Out the Gay Gene.” Boston Globe, 2005. Web.

Swidey, Neil. “What Makes People Gay?” Boston Globe, 2011 Web.

Yan, Liu, Jiang Yun’ai, Si Yunxia, Kim Ji-Young,Chen Zhou-Feng and Rao Yi. “Molecular regulation of sexual preference revealed by genetic studies of 5H-T in the brains of male mice.” Nature, 10.1038 (2011).

Going Public: IPO Capital and Execution Strategy

After careful analysis of what has been achieved within the current infrastructure of Gene One, the founding members of Gene One and the current board members are in agreement with the idea that Gene One is prepared and capable of becoming a public entity. Gene one has revolutionized the manner in which farmers preserve tomatoes and potatoes. Gene One is currently conducting research that will rapidly expand the crops that can benefit from Gene One’s gene technology that prevents crops from becoming diseased and is hopeful that their research will speed up the growth rate of multiple crops. The success that Gene One has had in eight short years has presented a quandary concerning the strategic path of Gene One’s future. Should Gene One execute a strategy of constancy and continuity? Will this type of strategy keep Gene One’s competitors from gaining ground on Gene One’s share of the market place over the next several years? After careful consideration of these perplexing questions, the leadership of Gene One has decided on an alternate strategy, as Gene One has not achieved all that they have by continuity alone. Gene One has taken risks that were founded on sound strategy and faith that intelligent men and women with innovative ideas and unsurpassed drive and passion cannot be stopped. These types of people are winners and they accomplish their goals. These factors set the stage for constantly evolving ideas that have provided multiple benefits that have been accomplished in record time. Seldom has this type of success been achieved in the biotech industry which is considered a risky business in some cases. That said, the Gene One leadership has decided that Gene One must be given an opportunity to experience its maximum growth potential before some piggy back organization with lucrative investors siphon off strategic market areas previously held by Gene One.

Gene One 3

As with any strategy, there are risks involved. However, due to Gene One’s financial stability, strong leadership and growth potential, these risks are just obstacles that can be turned into positive opportunities. Please note the following:

Economic Packages (founders of Gene One, board members and essential personnel)

While IPO capital is of extreme importance concerning Gene One’s preparations to go public, this topic can not be considered if it does not include job security and economic packages that reward the founders, board members and essential personnel at Gene One for their contributions that have led to the meteoric rise of Gene One over an eight-year timeframe. This is not a difficult problem to solve due to the strategy that will restructure and diversify Gene One while maintaining its technological and competitive edge over the competition.

IPO Capital

The following is a brief bio of Charles Jones, Gene One’s marketing officer:

“Two years after Gene One’s start-up, Don Ruiz, Chief Executive Officer for Gene One, recruited 35-year-old Charles because of his reputation for “smart” risk taking and his biotechnology connections. Don saw him as the perfect person to develop and implement Gene One marketing strategy. Self-confident and moral, Charles easily garners trust for himself and the company.”

Ruiz, Gene One Company Overview Report

The Gene One leadership feels that while Charles is limited in his abilities to personally design and implement a marketing infrastructure, his overall talents and track record suggests that with proper training he could be successful in this type of endeavor, as it would be critical to Gene

One’s intentions to implement innovative, cost efficient initiatives. The leadership of Gene One has believes that Charles would benefit from assistance and training from an outsourced consulting firm that would train him to design and implement a marketing infrastructure that meets the future needs of Gene One. This type of initiative would be cost efficient while providing Charles with the skills to provide holistic diversifications that are strategic in nature when needed. This would streamline budgetary spending as well.

The leadership at Gene One has an annual growth target of 40 percent. The Gene One leadership believes the following areas will allow Gene One to realize that growth target:

Target Headquarters

The Gene One leadership has decided that Lynchburg, Virginia would be the perfect headquarters for Gene One’s public entity endeavors. This provides Gene One with a headquarters that is much cheaper than the current headquarters through a reduced leasing cost while providing Gene One with a strategic conduit to the southern states. This will expedite a cost efficient expansion throughout the south which will streamline the operational budget at Gene One. This will provide Gene One with IPO capital for new development, advertising, and the previously mentioned market restructuring initiatives.

Gene One 5

Southern Based Modular Strategy

By implementing a strategy that maximizes the cheap leasing benefits of modular expansion throughout the south, Gene One will streamline its operational budget of the past three years in addition to hiring employees at a fraction of the cost of the operational budget that would be allotted in several states that are not within the southern region of America. This strategy will keep the leadership team at Gene One in touch with their core clients and supporters and will positively impact the economy in the southern states they expand to by employing thousands of new applicants.

Training and Doctrine Development

This area will need someone who is intelligent, experienced and capable of plotting the course for Gene One’s future employees. Greg Thoman, Chief Human Resources Officer has fourteen years of human resources experience. Greg has gone through thousands of applications and has decided who gets employed at Gene One for the past six years. Greg knows what type of person is capable of flourishing in a Gene One system that provides critical and innovative solutions to previously unsolved dilemmas in the biotech industry.

Note

Correlative Analysis

The cumulative effect of these innovative moves will streamline cost, expand the client base and expand Gene One’s market share. The savings to the operational budget alone will facilitate an environment where Gene One can safely go public within their target date of 36 months from now.

IPO Execution Strategy

The outsourced consultants that will be working with Charles in designing and implementing a marketing infrastructure should see that initiative come to fruition within 18 months. That would give Gene One 12 months of operational experience with the new marketing infrastructure before going public.

Gene One has had unparalleled success in the biotech industry. Innovative strategies and ever evolving research initiatives have propelled Gene One into a lucrative business that was not an easy sell when Gene One was founded eight years ago. The leadership of Gene One has opted to explore the positive suggestions of their profit margins concerning growth potential in order to protect themselves from being victimized by their success, as success always breeds duplication that can threaten a company’s market share variables. The future looks bright for Gene One as they explore the cost cutting strategy of a modular expansion through the south. Gene One also has an innovative strategy that is geared towards streamlining their budget while simultaneously diversifying their operations. The Gene One leadership believes that the cumulative effect of these moves will translate into an annual growth rate of 40 percent if they are implemented within three years time.

References

  1. Ghemawat, Pankaj, (2004), “”, Harvard Business Online. Web.
  2. Minford, John., (2002),”The Art of War”, New York: Penguin Group
  3. Renaissance Capital., (2006), “IPO”, (Online)
  4. Share Builder Corporation., (2006), “Share Builder”, (Online)
  5. Yahoo Finance., (2006), “”, (Online) Web.

A Development and Characteristics of Vivo Gene Modification Techniques

Artificial genes engineering is one of the most recent innovation in biomedical field. To isolate the pure genes, the scientists used bacterial viruses called bacteriophages, agents that are capable of extracting various genes from the bacteria. Once the genes were sucked into the bacterial virus, the researchers went about the difficult task of separating the ones they wanted from the rest of the genes in the “soup mix.” They did this with an enzyme that digested only the unwanted portion of the DNA, leaving the strand of genetic material they were after.

The article describes the development and characteristics of vivo gene modification techniques developed by NIEHS scientists. The researchers use so callused yeast artificial chromosomes (used in engineering large DNC fragments).Yeast artificial chromosomes contains such sequences as telomeric, centromeric and replication origin. The researchers succeeded in isolating a unit of heredity, the genetic system of which more closely resembles a human’s. The accomplishments have, of course, paved the way toward a better understanding of genes’ behavior and the role they play in defects and disease. There are practical possibilities of another kind. What if scientists could separate the gene that directs the manufacture of silk in a silkworm? Inserted into a bacterium, or into a host of bacteria, such a gene might be stimulated into producing large quantities of the substance for which it is coded. The bacteria would be transformed into busy minifactories, producing insulin to treat diabetes. The steps are not existent now, but when one stops to think about it, with work in higher organisms, it is not inconceivable that in not too long a time this sort of technique could be used. The more you think about it, the more it becomes frightening. “Delitto Perfetto provides a new dimension to YAC cloning of human DNA,” Resnick says, “because it gives one the opportunity to modify genes directly on the YAC without any subcloning process” (Medlin 2002, p. 88). The synthesis is just the beginning step in the investigation of that particular gene. Scientists interested in synthesizing a human gene have some formidable obstacles to overcome. Furthermore, it has been estimated that the chances of getting a virus to transport the right piece of DNA into a cell, using the easiest method known, are about one in one hundred thousand. And even if the DNA does get in, no one really knows how many of an ailing individual’s cells have to be so treated to cure a disease or defect. These problems will undoubtedly be resolved. There is something else about this restriction enzyme. When it is used to slice up DNA, the pieces it produces have sticky ends. This means that when a specific bit of DNA is cut out of a plasmid from a bacterium and then mixed with DNA cut, let us say, from a virus, the sticky ends of each DNA piece glue together. It makes little difference that the DNA sources are different, bacterium or virus. The point is that something new has been constructed. After the two bits of DNA are fused into one, this tiny new genetic package, now known as recombinant DNA, may be easily introduced into bacteria. As the bacteria multiply by division, each new cell that is produced contains recombinant DNA that is exactly the same as that made when the original was glued together. So simple is the technique that any one of you could do it, as long as you or your school chemistry laboratory had some of the enzyme.

The article depicts new ways of biomedical engineering applied to DNA and genes. These developments propose great opportunities for medicine to treat diseases and improve human health. Science can take genetic material from two different sources, join it, and then grow as much of it as desired. This will enable cell biologists to scrutinize genes as never before, increasing their understanding of basic biological processes.

Bibliography

Medlin, J. Delitto Perfetto: Foreign DNA Disappears without a Trace. Environmental Health Perspectives, 110 (1), 2002, 88.-90.

Gene Therapies: The Market Access

Role of Clinical Development in Reimbursement of Gene Therapies

Although, to date, there are hundreds of developing gene therapy medicinal products (GTMPs), only a tiny part of them will be able to receive marketing authorization (MA) and gain reimbursement. Clinical development can be effective and innovative, but it does not guarantee that it will be funded. In addition, it is essential but difficult to “strengthen clinical evidence with additional data from systematic reviews, meta-analyses of studies and registries” (Van Overbeeke et al. 407). Thus, the level of clinical development affects the possibility of reimbursement: the better characteristics it has, there are more chances that GTMPs can be funded.

Regulatory Requirements

The set of regulatory requirements complicates the access of GTMPs to market and, correspondingly, their reimbursement. The FDA, guided by these standards that are sometimes “unclear and differ between jurisdictions,” has approved only several products, making them available and giving them the right to gain reimbursement (Van Overbeeke et al. 404). The role of regulation is quite ambiguous: on the one hand, it strives to ensure that gene products are high-quality, safe, and effective; on the other hand, it creates additional challenges of their receiving MA.

Providing Gene Therapy Reimbursement

The gene therapy should be provided with reimbursement: the invention of a practical and helpful product capable of helping individuals involves the hard work of many people and a large number of clinical trials. Perhaps the FDA should reconsider its requirements: it is challenging to believe that only a dozen developed GTMPs are effective and safe. The reimbursement can be provided by different public-private collaboration platforms and by state bodies financing medical institutions.

The Process of Determining the Quality

Determining the quality of GTMP should be based on reconsidered FDA’s primary requirements and three main criteria: safety, effectiveness, and long-term functioning. In addition, it would be helpful to define the quality and consider the issue of reimbursement in half of the year after GTMP’s start of work to ensure that product meets all criteria. Thus, financing should be provided for all achievements of gene therapy that demonstrate their beneficial features.

Work Cited

Van Overbeeke, Eline, et al. “Market Access of Gene Therapies across Europe, USA, and Canada: Challenges, Trends, and Solutions.” Drug Discovery Today, vol. 26, no. 2, 2021, pp. 399-415.

Embryonic Gene Testing and Manipulation

Back in 1997, Hollywood envisioned a world where “designer babies” would proliferate the world in the movie Gattaca. The so-called perfect babies are the product of what was then an imaginary genetic testing method. during that time, genetic testing was not even a real word yet. Who would have known that a decade later, Hollywood’s imagination would be turned into reality by fertility specialists? Originally envisioned to aid parents in discovering what genetic illnesses they may have passed on to the unborn child, these days, the original goal of the project, which is to help parents to make informed decisions about pregnancy has taken a negative trend. Due to the technical advancements in the area, the possibility to choose the sex of a child, choosing the most healthy embryos, using donated sperms and eggs, has given man an almost godlike quality to create a life or prevent life from coming to be.

This quest to have the most perfect child is something that I totally disagree with. Diseases or imperfections in a people’s genetic makeup are what make us human beings. If God had meant us to be perfect, he would have created us that way. But the reality is that mankind will always have imperfections and there is nothing science can do to prevent it or lessen its occurrences. Every embryo has a right to become a fetus and then eventually a human being. Nobody has the right to dictate which fetuses pass or fail via genetic testing. A disease most often stems from a genetic or development malfunction on the part of the parent’s DNA. It is this imperfection that makes one a biological child of a parent. Without the hereditary imperfection, a parent cannot call a child his for they no longer share common traits.

Due to the newness of the field of genetic testing, there is no uniform governing policies to be followed by the agencies that implement them. Consider it a wild frontier where anything a potential parent wants to do can happen and nobody can stop them from doing it. However, I believe that it is not the right of anybody to dictate what traits they wish to see in their potential child. Even if it were true that genetic manipulation became a reality and we can kill off the disease gene or the criminal gene, the reality is that we will only open the door to another genetic evolution that future human beings will be prone to and have to deal with.

Should it become important and relevant to choose the characteristics of a potential child though, The characteristics that are chosen should be based upon moral and social concepts, as well as parental choices. Perhaps the most important characteristic would pertain to the genetic variations and mutations that cause severe physical and mental disabilities in children. To be specific, I would concentrate on the genes that cause DNA mutations that are harmful and could prevent the normal functions of a specific DNA base sequence. Perhaps even eliminate the single-gene diseases that produce autosomal diseases, autosomal recessive diseases, and X-linked diseases. Aside from these aforementioned DNA base sequence mutations, everything else, from the color of the eyes of the child to his height, should all be left in the hands of nature and heredity.

Again, I must reiterate that I do not favor the genetic manipulation of embryos in order to suit the wants and whims of a parent. Being a parent is not something that can be designed to suit our wants and needs, it is a responsibility for another human being who came from our very own DNA sequence. Regardless of the characteristics and shortcomings of the child, he or she is still the flesh and blood of the parent.

Work Cited

Baruch, S., Javitt G., Scott, J., Hudson, K. (2008). Reproductive Genetic Testing: Issues And Options For Policy Makers. Web.

Clayton, Ellen Wright, M.D., J.D. (2003). Ethical, legal, and social implications of genomic medicine. The New England Journal of Medicine. 349;6, 562-568.

Mallia, Pierre. (2003). Biomedical ethics: genetics. StudentBMJ. 11, 320-321.

New York State, Department of Health. (2001). Genetic Testing And Screening In the Age Of Genomic Medicine. 2008. Web.

Activation and Repression of Gene Expression

Introduction

Activation and repression of gene expression in both eukaryotes and prokaryotes is a complex process that many factors mediate. Scientists have formulated theories and hypotheses that elucidate a number of mechanisms through which activation and repression occur in both in vivo and in vitro environments. In the activation and repression of gene expression, histones play a central role, as they are proteins that associate with and bind to the DNA material in cells. The research questions sought to establish if acetylation of histones and chromatin remodeling complexes influence gene expression. According to the Agalioti, Chen, and Thanos, “chromatin modifying complexes recruited by transcription factors covalently modify the N-terminal tails of histones by adding or removing phosphate, methyl, or acetyl groups,” (381). On this basis, the research article hypothesizes that existence of chromatin modeling complexes and transcription factors that catalyze acetylation of histones, which have an overall impact of activating expression of genes. Hence, the histone code hypothesis predicts that a functional interaction exists between histone acetylase (GCN5) and chromatin remodeling complexes (SWI/SNF) in the cells. In proving the histone code hypothesis, the study hypothesized that histone acetylation is specific to certain residues that are responsible for gene activation through in vivo mechanism. The basis of the study is that modification of histones at the promoter and enhancer sites is critical in the activation of gene expression. Hence, to prove the hypothesis, the research sought to establish if acetylation of histones has a regular pattern that has a significant impact in enhancing activation of genes. Examination of the technologies employed in the study shows that they are appropriate in proving the histone code hypothesis.

Hypothesis

The major hypothesis of the study is that of histone code hypothesis. Histone code hypothesis states that functional interaction between histone acetylase (GCN5) and chromatin remodeling complexes (SWI/SNF) activates expression of genes. Modification of histones at the promoter and enhancer sites enhances gene expression. Agalioti, Chen, and Thanos state that the aim of the research paper is to “provide direct evidence for both the existence of a histone acetylation code and its interpretation by the transcriptional apparatus during human IFN-β gene activation” (381). Acetylation of lysine moieties in histones causes modification of histones, hence, bringing about activation of gene expression. Thus, the study assumes that acetylation of histones generates cascades of reactions that lead to enhanced expression of genes.

Punch Line

Acetylation of histones plays a significant role in regulation of gene expression. Specifically, “acetylation of histone H4K8 mediates recruitment of the SWI/SNF complex whereas acetylation of K9 and K14 in histone H3 is critical for the recruitment of TFIID” (Agalioti, Chen, and Thanos 381). Recruitment of transcriptional factors in a cascade manner explains the mechanism of gene expression via acetylation process. Acetylation of histones induces ATP-dependent process that causes SWI/SNF complexes to slide and initiate the transcription process at the downstream. Additionally, acetylation of histones causes recruitment of extra complexes such as TFIID, which are bromodomains that regulate interaction of transcription factors with the promoter and enhancer sites. According to Agalioti, Chen, and Thanos, acetylation of histones enhances activation of IFN-β gene expression by facilitating recruitment of SWI/SNF and TFIID complexes. Therefore, to prove acetylation of histones, the study sought to ascertain how Sendai virus induces acetylation of lysine residues present in histones H3 and H4 in the transcription of IFN-β gene.

Methodology

To determine the role of histone acetylation, the study employed the methodology of conducting an in vitro infection of cells with viruses and then assessing the extent of histone acetylation. In the in vitro experiment, the researchers infected the HeLa cells with Sendai virus. The virus infection then induced acetylation of lysine residues that are present in histones H4 and H3. After incubation for different periods, formaldehyde treatment was added to cause protein-DNA complexes and protein-protein cross-linkages to form. Immunoprecipitation of the linkages and complexes formed was then performed using antibodies that are specific to acetylated lysine residues in H4 and H3. While acetylation of H4 peaks at between 4-6 hours of incubation, H3 peaks at 6-10 hours after incubation. To determine the acetylated residues, electrophoresis by SDS-PAGE was performed to separate histone residues according to their sizes, thus separating acetylated ones. Eventually, Western blot was employed to identify acetylated lysine residues in histones using specific immunoprobes. The methodology used in ascertaining acetylation of lysine residues in histones due to virus infection is appropriate as it targets proteins, which are products of gene expression.

Technologies Employed

The experiment employed in vitro culture of HeLa cells as a technology for assessing the impact of virus infection on gene expression via acetylation of histones. The acetylation mechanism that occurs in an in vivo environment is similar to the one that occurs in an in vitro environment. Therefore, the use of HeLa cells reflects the actual mechanism that occurs in the cells. Activation of gene expression due to virus infection was then detected using RT-PCR, which measures the message level of IFN-β gene (IFN-β mRNA). Immunoprecipitation technology was also applicable in cross-linking chromatin material that is rich in acetylated lysine residues. To separate different types of histones depending on their size, SDS-PAGE was a critical technology. After separation of histones based on their sizes, Western blotting was essential as a technology that uses immunoprobes, which are very specific to acetylated lysine residues in histones. Thus, Western blotting is an important technology that aided detection of acetylated lysine residues in histones, following activation of IFN-β gene in human.

Data Analysis

The data show that acetylation of histones activates expression of IFN-β gene. The virus infection induces expression of the IFN-β gene, thus causing enhanced transcription process in cells. After infection of HeLa cells with Sendai virus, there was increased expression of IFN-β gene in both H3 and H4 histones. Agalioti, Chen, and Thanos report that acetylation of H3 and H4 histones occur in different patterns, as acetylation of H3 peaks at 6-10 hours after incubation while acetylation of H4 peaks at 4-6 hours after incubation (382). However, the acetylation process begins at 3 hours after incubation in both H3 and H4 histones. Since the virus induces acetylation of histone at the IFN-ß promoter, it enhances gene expression leading to the increased message level of the IFN-β in the HeLa cells. RT-PCR done indicated the IFN-β mRNA increased with time after infection, thus peaking at 4-6 hours in H4 and 6-10 hours for H3. SDS-PAGE electrophoresis and Western blotting do support the histone code hypothesis that acetylation of histones activates expression of IFN-β gene in human.

Derived Conclusions

Histone code is critical in regulation of IFN-β gene, which is in human. The acetylation of histones enhances expression of IFN-β gene as shown by RT-PCR analysis. According to Agalioti, Chen, and Thanos, GCN5 acetyltranferase causes acetylation of H3 and H4 histones leading to activation of the IFN-β gene in human. Acetylation of histones causes recruitment of SWI/SNF complexes and TFIID complexes, which are responsible for triggering a series of mechanisms that lead to activation of IFN-β gene. Thus, the study proved that the histone code hypothesis is applicable in elucidating the activation of IFN-β gene in human.

Interpretation of Figures

In the article, figure 1 depicts how virus infection induces activation of IFN-β in human by acetylation of histones. RT-PCR shows that there is an increased message level following infection by the Sendai virus. Immunoprecipitation techniques also indicate that proteins associated with H3 and H4 increase due to the infection. Ultimately, SDS-PAGE and Western blot confirmed that lysine residues in H3 and H4 histones were acetylated, thus promoting expression of IFN-β gene in human. The figure 4 elucidates the mechanism of acetylation from a functional perspective. Since recombinant nucleosome cannot undergo acetylation process, the IFN-β gene activation cannot occur, as in the case with native nucleosome. Hence, these findings proved that acetylated histones initiate recruitment of SWI/SNF and TFIID complexes necessary for translation of histone code as shown in the model, figure 5.

Summary

Histone code helps in regulation IFN-β gene expression in human. Specifically, acetylation of H3 and H4 histones activates expression of the IFN-β gene. Acetylated histones initiate the recruitment of SWI/SNF and TFIID complexes, which are essential in the downstream activation of gene expression cooperatively. The use of recombinant nucleaosomes proved that lysine residues in histones are critical regions of acetylation, because, without them, acetylation will not take place. Hence, acetylation of histone code is central in activation of IFN-β gene in human.

Recommendation

  • Histone code hypothesis predicts that acetylation of histones in IFN-β gene provides means through which activation of gene expression occurs. As the study hypothesizes that acetylation of histone activates expression of IFN-β gene in human, more studies are necessary to establish if other factors that mediate remodeling of histone structures exist.
  • Viruses have the ability to induce acetylation of histones. Since Sendai virus induces acetylation of histones in HeLa cells, other viruses and stem cells should be used to ascertain the role of histone code in gene expression.
  • Transcription factors and other complexes determine the expression of genes. Given that acetylation of histones initiates downstream processes that are critical in recruitment of SWI/SNF and TFIID complexes, a study is necessary to determine if additional complexes that mediate IFN-β gene expression exist.

Works Cited

Agalioti, Theodora, Guoying Chen, and Dimitris Thanos. “Deciphering the Transcriptional Histone Code for a Human Gene.” Cell 111.3 (2002): 381-392. Web.

Discussion on Lab Report an E. Coli Bacteria Lacz Gene

The objective of this experiment was to reassert that mutation of PUC19 really does occur. The PUC19 sample was obtained from an E. coli bacteria whose LacZ gene had been mutated and hence it was non functional. Production of β-galactosidase had, therefore, stopped. The main idea of this experiment was to reverse this mutation so that the gene can regain its ability to produce β-galactosidase. The production of β galactosidase is indicated by presence of blue colonies on the plate which results from the cleavage of X-galactosidase which is colorless into galactosidase which is blue in color.

The Puc19M was heated to denature the double stranded DNA and placed on a tube containing ice. An annealing buffer, together with a mutagenesis primer was added during which, the annealing buffer helped in bringing the primer and the single strand DNA into consistency. The mutagenesis primer was added to perform reverse functions to the mutated gene in order to make it functional. A selection primer was also added to alter the restriction site of the plasmid thus allowing the synthesis of a new circular DNA strand.

A synthesizing buffer was added to provide the suitable environment required for the synthesis of the new DNA strand. The addition of T4 DNA polymerase was to facilitate the hybridization of the old and the new DNA strands. T4 DNA ligase was as well used to join the two ends of the new DNA strand. The DNA obtained was introduced into E.coli mutS since they lack the gene responsible for repair of mismatched sequences. Hence, the new DNA strand could not be repaired.

After electroporating the mixture overnight, a pellet was formed onto which, a cell suspension was added to prevent enzymatic reactions. The cell was lysed to obtain the plasmid DNA. Addition of an alkaline solution was to get rid of all the membrane proteins that could have been present. The solution was then neutralized.

The column that had been prepared during centrifugation was inserted into a tube as the supernatant was discarded so that the DNA could be left stuck in the resin. Ethanol was used to dissolve away any impurities but after the solution had been centrifuged two more times. Centrifugation was performed one more time to further remove any unwanted materials. This was followed by the transfer of the spin into a smaller centrifuge tube where water that had been freed of nuclease, was added to wash away resin from the DNA. This process was carried out in the microcentrifuge tube for one minute.

A NdeI digest procedure, using the newly formed DNA, was then carried out. The new DNA was used because, unlike the old DNA strand, it does not have restriction sites for NdeI. The procedure involved addition of the plasmid DNA sample into a buffer containing sterilized water and incubating the solution for 2 hours.

Due to the lack of restriction site for NdeI in the new DNA strand, the new Puc19 will remain in its circular form and as a result, the LacZ genes present in the Puc19 will still be active due to the lack of NdeI restriction site which would have, otherwise, hindered the functioning of the LacZ genes. As a result, the ability of the genes to produce β galactose would be restored which, as discussed earlier, was evidenced by the appearance of blue colonies on the plate.

Gene-Environment Interaction: Personality Development

Genes and the environment mutually influence one another. One way to investigate these bidirectional interactions between genes and environment is to examine monozygotic twins. One of the studies dedicated to the issue of the gene-environment interaction of fragile x syndrome in twins was performed by Willemsen et al. (2000). The authors analyzed monozygotic twin sisters’ case, one of whom is affected by a full mutation in the FMR1 gene and is mentally handicapped. The production of an FMRP protein, which is presented in the brain and develops connections between synapses, depends on this FMR1 gene. Another sister is not affected by the mutation and has normal mental development. Willemsen et al. (2000) analyzed the blood cells of the twins. This analysis showed that the normal sister’s X chromosome is active in all blood cells, whereas her sister’s X chromosome “inactive in approximately 50% of her blood cells” (Willemsen et al., 2000, p. 603). The work concludes that inactivation of the X chromosome when the embryos of the twins became separated.

It is essential to study gene-environment interactions because it could improve population health. The investigation of these interactions is possible through the examination of monozygotic twins reared apart. This method was used by Bergeman et al. (1988) to test how different environments affect identical genotypes. Bergeman et al. (1988) analyze 99 pairs of such twins to see the differences in personalities between tweens who were parented separately.

Another method to explore interactions between genes and environment is multivariate modeling. This method could be illustrated by the research conducted by Gillespie et al. (2004). The scholars analyzed the Junior Eysenck Personality Questionnaire scores taken by more than 540 twin pairs via genetic simplex modeling. Gillespie et al. (2004) discovered that genetic variations in twins at the age of 14 – 16 years could be explained by genetic variations present in them at the age of 12.

References

Bergeman, C. S., Plomin, R., McClearn, G. E., Pedersen, N. L., & Friberg, L. T. (1988). Genotype Environment interaction in personality development: Indentical twins reared apart. Psychology and aging, 3(4), 399–406.

Gillespie, N. A., Evans, D. E., Wright, M. M., & Martin, N. G. (2004). Genetic simplex modeling of Eysenck’s dimensions of personality in a sample of young Australian twins. Twin Research and Human Genetics, 7(6), 637-648.

Willemsen, R., Olmer, R., Otero, Y. D. D., & Oostra, B. A. (2000). Twin sisters, monozygotic with the fragile X mutation, but with a different phenotype. Journal of medical genetics, 37(8), 603-604.

Whole-Genome Sequencing for Identification and Gene Function Prediction of Bacterial Genomes

Introduction

When it comes to identifying a pathogen, whole-genome sequencing (WGS) can provide much useful information. This tool sequences the whole genome and compares it to the stored data of other sequenced genomes to find the specific pathogen among the identified matches. One of the most useful applications of the given approach is to determine the original source of an outbreak, and that is why WGS is actively used in epidemiology studies. Joensen et al. (2018) use WGS to analyse outbreaks of Campylobacter jejuni in the modern world by studying pathogen isolates. The scientists “support clonal linkage between isolates and highlight that outbreaks potentially occur more frequently than previously assumed” (Joensen et al., 2018, para. 14). It means that researchers should draw more attention to preventing and managing epidemics.

Simultaneously, Yang et al. (2017) use WGS to analyse the outbreak of Mycobacterium tuberculosis in Shanghai, China. The researchers stipulate that WGS “can be used to identify putative source cases, super-spreaders and transmission directions in the absence of, or complementary to, extensive epidemiological data” (Yang et al., 2017, p. 275). Even though these two examples demonstrate that WGS addresses human beings, the tool can also be used to investigate poultry pathogens.

Concerning chickens, spotty liver disease (SLD) is a severe problem. According to Van et al. (2017, p. 226), this condition leads to egg production losses and mortality in birds. That is why numerous studies have been conducted to identify the bacteria that is responsible for the given disease. Van et al. (2017, p. 226) mention that Campylobacter hepaticus is the cause of SLD in chicken, which is an initial hypothesis of the practical. However, additional research is needed to obtain more data on the SLD bacteria and their pathogenesis. Consequently, the aim of the paper is to isolate the unknown pathogen’s DNA from chicken with an SLD and use WGS to identify this specific pathogen. Thus, the Rapid Annotation using Subsystem Technology (RAST) will annotate bacterial genomes, and the Basic Local Alignment Search Tool (BLAST) will identify similarities between sequences.

Material and Methods

This work deals with an unknown bacterial sample that was obtained from chickens with SLD. As for methods, the given practical relies on Practical manual: whole-genome sequencing for identification and gene function prediction of bacterial genomes (2020). This document highlights all the steps required to decode a pathogen, including three separate practicals. Thus, Practical 1 explains tagmentation of genomic DNA, Practical 2 comments on library qualification and Practical 3 discusses bioinformatics issues. Consumables and equipment are mentioned at pages 6, 8 and 13 of the Practical manual (2020). Thus, it is possible to conclude that the given practical follows specific manual regulations.

Results

As has been mentioned above, the sequenced genome is submitted to the RAST for annotation and to the BLAST for identification. The given section is going to present the results of these procedures. Figure 1, a BLAST screenshot, demonstrates that the search has concluded that Campylobacter hapaticus strain HV10 is an SLD pathogen, while Campylobacter jejuni is the closest related species. The average nucleotide identity (ANI) value of the result is more than 99%.

Figure 2 represents the Seed Viewer data for the annotated genome. This information is useful to define the number of coding sequences and RNAs of the given isolate. Thus, Figure 2 shows that the isolate has 1,580 coding sequences and 45 RNAs. It means that the sample under analysis implies many genes and RNAs that can be matched against the given database. This fact should increase the reliability of the obtained results.

At the same time, Figure 2 indicates that the isolate has 191 subsystems, and Figure 3 below presents their analysis. The latter shows that the genome under investigation does not have any phages, prophages, transposable elements or plasmids. However, Figure 3 indicates 16 virulence, disease and defence genes. This subsystem is essential because it can include antibiotic resistance elements that, in turn, determine disease treatment options.

Discussion

WGS has demonstrated that Campylobacter hepaticus is an unknown pathogen that is isolated from chickens with an SLD. It is the same species as the bacteria that usually cause the disease. Thus, the practical results have supported the initial hypothesis and proved that Campylobacter hepaticus is a typical cause of an SLD among chickens. This finding is useful because it can help prevent SLD outbreaks and treat ill birds more successfully.

Since the identified isolate is one of Campylobacter species, it is necessary to comment on the genomics and proteomics features of the group. Thus, Van et al. (2019, p. 6) indicate that a typical structure of a Campylobacter genome includes “many genes encoding chemotaxis (11 genes), motility (47 genes) and adherence/surface protein (59 genes).” In addition to that, this genome acquires carbohydrates and metals with the help of appropriate metabolism loci (Van et al., 2019, p. 6). These are the features that are typical for any of the Campylobacter species.

However, Campylobacter hepaticus is a separate genome that implies unique coding sequences. Firstly, the given isolate has “genes with predicted roles in chemotaxis, capsule and lipooligosaccharide synthesis and metabolism” (Van et al., 2019, p. 6). It results in the fact that the pathogen tends to move from the gastrointestinal tract to the liver. Secondly, the lipooligosaccharide locus (LOS), a region that experience rearrangements, is unique to Campylobacter hepaticus because of “2 kb of the inserted sequence” (Van et al., 2019, p. 7). It means that the genome has a significant part that is unique compared to other Campylobacter species. Thirdly, it is reasonable to comment on antibiotic resistance of the isolate because this phenomenon determines treatment options. In general, the risk of antibiotic-resistant plasmids is present in bacteria isolated from poultry sources. However, the subsystem statistics above has indicated that the isolate does not have any plasmids but contains 16 virulence, disease and defence genes. These elements can be a source of antibiotic resistance, meaning that they deserve additional attention.

Conclusion

Whole-genome sequencing is a useful tool to analyse genomes and identify their specific pathogens. This approach is used in epidemiology because it helps to determine a virus or bacterium that has caused an outbreak. That is why this method is utilised to investigate chickens with spotty liver disease. The initial hypothesis stipulated that Campylobacter hepaticus causes this disease, but additional research was necessary to prove it. Thus, whole-genome sequencing has demonstrated that Campylobacter hepaticus is responsible for spotty liver disease outbreaks among poultry. Furthermore, the practical has shown that Campylobacter hepaticus has genes that lead to niche adaptation, colonisation and virulence. This information means that further research is needed to minimise the risk of Campylobacter hepaticus outbreaks.

References

Joensen, K. G. et al. (2018) ‘Whole-genome sequencing of Campbylobacter jejuni isolated from Danish routine stool samples reveals surprising degree of clustering’, Clinical Microbiology and Infection, 24(2), pp. 201.e5-201.e8.

Practical manual: whole genome sequencing for identification and gene function prediction of bacterial genomes (2020).

Van, T. T. H. et al. (2017) ‘Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds’, Veterinary Microbiology, 207, pp. 226-230.

Van, T. T. H. et al. (2019) ‘Survival mechanisms of Campylobacter hepaticus identified by genomic analysis and comparative transcriptomic analysis of in vivo and in vitro derived bacteria’, Frontiers in Microbiology, 10, pp. 1-19.

Yang, C. et al. (2017) ‘Transmission of multidrug-resistant Mycobacterium tuberculosis in Shanghai, China: a retrospective observational study using whole-genome sequencing and epidemiological investigation’, The Lancet Infectious Diseases, 17(3), pp. 275-284.