Although cancer is a widespread disease that is vigorously studied and researched, we as humans have not yet developed a cure. Most health officials agree that the best way to stop cancer in a patient would be to try and stop it early on. While detecting cancer early might sound like an easy task it can be difficult. Whether it be the steep price or the sparse locations screenings and tests can be hard. But researchers across the globe have been striving for a better solution. One study by researchers at BioScentDx shows that Dogs can accurately detect early-onset cancer through smell. The researchers used a form of clicker training to teach for dogs to distinguish between normal blood serum and samples from patients with malignant lung cancer. After many trials and tests, the researchers concluded that the dogs had a 96.7 percent accuracy of identifying the lung cancer samples and a 97.5 percent accuracy of identifying the normal samples.
But how do the dogs do this? Researchers from many universities, including Stanford Med and BioScentDx, say it is from the very sensitive smell receptors that K9’s have. Dogs have 10,000 times more sensitive smell receptors than humans which would allow them to smell and identify various more biologic compounds such as cancers. Other researches have said that dogs are able to smell cancer due to the olfactory ability that they have. This allows them to detect very low concentrations of alkanes and aromatic compounds generated by malignant tumors through urine or in the breath of humans. The very thought that dogs could detect cancer dated back to 1989 when it was published in The Lancet Medical Journal.
If the research is deemed accurate by secondary trials and dogs are able to detect cancer, it could be very exciting for the medical community and for cancer research. The company BioScentDx has said that it would pave the way for further research along two paths, both of which could lead to new cancer-detection tools. One is using canine scent detection as a screening method for cancers, and the other would be to determine the reach of K9’s senses. The company plans to use canine sent detection to develop a non-invasive way of screening for cancer that would be less expensive as well as more accessible.
Overall, the use of cancer sniffing dogs in the medical and health science fields could be a huge discovery for the livelihood of many patients. If we already had the K9’s in action we could estimate that nearly ⅛ of all the people with cancer would have been screened earlier, stated the Center for Disease Control, which potentially could have saved hundred of thousands of lives. In the near future, many companies are planning on further testing canines to sniff cancer and other diseases too. And if it works, dogs could change the world for the betterment of society.
Comparative Analysis of Effectiveness of Apoptosis
As there are more than a million cases in the United States annually, and over 500,000 Americans die each year, cancer can be characterized as the continual unregulated rapid increase in the number of abnormal cells. The use of mouse model cancer cells serves an utmost significance in the field of research as the causal link to carcinogenesis, the initiation of cancer formation, and cancer genes can be used to test and develop new therapies. Chemotherapy is the most common approach when looking at cancer treatment, where cancer cells are killed using chemicals that are toxic to living cells. Antioxidants maintain homeostasis and the maintenance of cell integrity in human immune systems and have shown to increase the life quality of the patients. The purpose of this experiment was to determine which antioxidant will be the most effective at different volumes to promote apoptosis in mouse osteoblastic cancer cells (MC3T3). The hypothesis was that as the volume of antioxidant solution increases, then the apoptosis of MC3T3 will increase. The experiment was conducted as MC3T3 cells were first grown for 24 hours, then the antioxidants Lycopene, Resveratrol, and Vitamin C were then added to the cells. The cells were once again grown for 24 hours, and after the 24 hours, cell viability was measured through MTT assay. The results show, the cell viability of Lycopene at 100uL was 27%, it was the most effective in promoting apoptosis. The hypothesis was supported to the extent that Lycopene and Resveratrol were successful in promoting apoptosis at certain volumes. This experiment can be applied to help cancer patients by offering another treatment or in combination with chemotherapy to increase the life quality of the patient.
The purpose of this experiment is to determine which antioxidant will be the most effective at different volumes of the antioxidant, to promote apoptosis in mouse osteoblastic cancer cells.
Independent Variable – The volumes of the different antioxidants that will be induced into the cells
Dependent Variable – The cell viability of each antioxidant at different volumes, which will be measured using MTT assay
Control – Micro-wells not being induced with any treatments
Experimental Control – Etoposide, which is a chemotherapeutic agent proven to promote apoptosis and most prescribed chemotherapeutic agent in the world
Introduction & Background Research
Over 500,000 Americans die of cancer each year, as there are more than a million cases in the United States annually (Cooper, 2000). Cancer can be characterized as the continual unregulated rapid increase in the number of abnormal cells, leading to a group of cancer cells, which is a tumor (Cooper, 2000). Cancer is caused by carcinogens that start the growth of cancer cells; carcinogens are substances that promote or initiate the growth of cancer cells (Cooper, 2000). Cancer cells grow with continual proliferation, rather than healthy cells that respond appropriately to the signals that control normal cell behavior (Cooper, 2000). Tumors are a group of cancer cells that can be categorized into either benign or malignant (Cooper, 2000). A benign tumor is similar to a wart where it does not spread cancer to other tissues and other parts of the body and stays in a central location (Cooper, 2000).
On the other hand, a malignant tumor is capable of spreading to other tissue as well another parts of the body through lymphatic and circulatory systems (Cooper, 2000). Among the various unique types of cancer, they are classified as either sarcoma, carcinoma, lymphomas, or leukemias (Cooper, 2000). Carcinomas, which are the most common, and occurs in 90% of human cancer cases, are malignancies of epithelial cells, which are sheets of cells that line internal organs and cover the surface of the body (Cooper, 2000). Sarcomas are tumors that are solid in connective tissues, like bones, cartilage, and muscle, which rarely occur in humans, and lymphoma or leukemia rarely occurs with 8% human cancer cases, comes from the cells of the immune system and blood-forming cells (Cooper, 2000).
Metastasizing, which is the development of a malignant growth that is secondary to the primary site of cancer, which grows at a distance and spreads to other sites in the body (Macedo, Ladeira, Pinho, Saraiva, Bonito, Pinto, Goncalves, 2017). The third most frequent site of metastasis is the bone, which has a relative incidence percentage of 65-75% (Macedo et al., 2017). The three types of bone metastasis are classified as Osteolytic, Osteoblastic, or mixed (Macedo et al., 2017). Osteoblastic cancer is seen as the breakdown of the new bone similar to that of prostate cancer, small cell lung cancer, carcinoid, and Hodgkin lymphoma (Macedo et al., 2017). Bone metastasis is a sign that the disease has spread to other organs and predicts a short term probability of the disease in cancer patients (Macedo et al., 2017). Once cancer has spread to the bone, the chance of it being cured is low, though treatment can be applied to slow its growth because new drugs and treatments have shown a better quality of life and improved life expectancy (Macedo et al., 2017). Both Filipa Macedo and colleagues and Geoffrey Copper discuss the impact that cancer has left on society. Though they both discuss metastasis and how it leads to malignant tumors, Macedo looks at it only from bone metastasis, while Cooper analyzes metastasis through cancer in general. As they look at cancers through different outlooks, Macedo categorizes the four main types of cancers, and Coopers analyzes Sarcoma cancer, specifically bone cancer and the three types of bone cancers.
When looking at metastasis it is also essential to look at carcinogenesis, which is how cancer formation starts, and neoplastic development, which is the formation of diseases that conditioned to cause tumor growth, which occurs when oxidative stress, damage from cancer cells, go unrepaired, that among humans and animals, research has shown the degree of biological and genetic similarity (Balmain & Harris, 2000). Mice develop tumors in the same tissues and amazingly also in the same histopathological course as humans even though they are much smaller animals with a higher metabolic rate (Balmain & Harris, 200).Humans have similar characteristics such as anatomical, cellular, and molecular to mice, which have the utmost importance in function and critical properties in cancer (Tratar, Horvat, & Cemazar, 2018). The use of mouse models serves the utmost significance in the field of research as the causal link to carcinogenesis and cancer genes can be used to test and develop new therapies (Tratar et al. 2018). Both Balmain and colleagues and Tratar and colleagues discuss how mice cells are similar to human cells are similar biologically to the extent of their higher metabolic rate and other small factors. Tratar goes onto discuss how the use of mice cells can be useful in research to apply applications on to human cells. In contrast, Balmain looks more into the histopathology, examination of tissue at a microscopic level, similarities of the cell, such as the presence of Telomerase, an enzyme that helps maintain chromosome integrity in both human cells and mice cells.
As mice cells serve as useful models of human cancer cells, the use of mice cells can be used to test new treatments such as chemotherapy drugs and antioxidants. Chemotherapy is the most common approach when looking at cancer treatment, where cancer cells are killed using chemicals that are toxic to living cells (Thyagarajan & Sahu, 2018). As chemotherapy has shown to be effective, it has many consequences such as DNA damage and damage to DNA replication, and it induces cell death into living healthy cells (Thyagarajan & Sahu, 2018). As these effects of chemotherapy affect the quality of life of cancer patients, there is a need for alternative treatment. Antioxidants maintain homeostasis and the maintenance of cell integrity in human immune systems (Pham-Huy & Lien Ai et al., 2008). Many different types of antioxidants can be found in vegetables; they have been shown to delay or prevent oxidative stress (Pham-Huy & Lien Ai et al., 2008). Antioxidants also serve as an effective treatment for cancer as studies have shown an increase in apoptosis, programmed cell death, in different cancer cell lines, and inhibit cell proliferation (Pham-Huy & Lien Ai et al., 2008). Both Pham-Huy and colleagues and Thyagaran and Sahu show the effectiveness of antioxidants as an anticancer treatment. Though Thyagaran and Sahu examine the relationship between the effectiveness of chemotherapy and its effects on antioxidants efficacy. Pham-Huy, in contrast to Thyagaran and Sahu, looks at specific antioxidants and their effectiveness in different clinical studies. As there are many different types of antioxidants, such as Lycopene, Resveratrol, and Vitamin C (ascorbic acid), it is crucial to examine their effectiveness against cancer cells and their effectiveness when compared to chemotherapeutic drugs such as Etoposide.
Lycopene, which is mainly derived from tomatoes, tomato products, and other fruits, has shown to be a potent antioxidant and to reduce the incidence of cancer (Johary et al., 2012). Dietary carotenoids, a product of plants, such as Lycopene, have shown to be connected to the decreased risk of cancer and the maintenance of good health (Johary et al., 2012). Recent studies have shown Lycopene as an anticancer agent as its ability to inhibit metastasis, increasing the antioxidant response, preventing oxidative stress, and promoting apoptosis (van Breemen & Pajkovic, 2008). Lycopene has the potential, as a natural product, to be used in cancer treatment to increase the rate of apoptosis based on the studies that show Lycopene supplements when used against prostate cancer have shown reduced risk, anticancer activities, and chemoprevention, chemicals that prevent or slow development of cancer activities. Johary examines Lycopene as a dietary carotenoid and its many properties, which lead to beneficial effects such as preventing cancer. Van Breeman and Pajkovic look at the properties of Lycopene as an antioxidant and evaluates its efficacy by looking at clinical studies. Both authors view Lycopene from a different perspective, though they both establish it to be an effective antioxidant against cancer.
Resveratrol is a product containing high levels of naturally occurring phenols (organic products), which is derived from plant sources, berries, peanuts, and grapes (Bishayee, 2009). Resveratrol has shown to be effective against the three stages of carcinogenesis: initiation, promotion, and progression (Bishayee, 2009) Resveratrol has a robust anticancer property as it can inhibit proliferation in human tumor cells (Ko et al., 2017). As Resveratrol can stop carcinogenesis, it has brought much attention to the prevention of cancer, a therapeutic drug, treatment researchers (Bishayee, 2009). Resveratrol can make cancer cells sensitive to chemotherapeutic agents and reverse multidrug resistance to cancer cells when used with drugs that are used clinically because this shows its efficiency in fighting cancer cells (Bishayee, 2009). Reports have shown Resveratrol can make cancer cells sensitive to chemotherapeutic agents and reverse multidrug resistance to cancer cells when used with drugs that are used clinically. Ko and colleagues examine Resveratrol specifically when used for cancer treatment and look into studies that inhibit Resveratrol into variety of cancers. Bishayee also examines resveratrol when used for cancer treatment, but specifically looks into Rodent studies which can further be implicated. Both Bishayee and Ko and colleagues see resveratrol as an effective anti-cancer treatment as they both discuss the effectiveness even looking at different types of cancers.
Ascorbic acid romote cell proliferation and cell differentiation in cancer cells (Yang & Seo, 2013). Ascorbic acid, also known as Vitamin C, in recent studies, has shown its ability to promote cell proliferation and cell differentiation in cancer cells (Yang & Seo, 2013). Ascorbic acid has also been shown to increase the osteogenic formation of bone in bone cancer cells when treated with cell differentiation (Yang & Seo, 2013). Vitamin C also has been shown to increase the survival rate when inhibited with organisms with cancer cells and increase the survival rate of the mice (Yeom et al., 2009). The administration of ascorbic acid in cancer patients when they were in the terminal stage showed the increase in the quality of life because of this experiment shows the sufficiency of ascorbic acid when induced in sarcoma cells (Yeom et al., 2009).
Etoposide, an epipodophyllotoxin, a substance occurring naturally in Mayapple Plant, is structurally similar to another chemotherapeutic drug vincristine, a chemotherapeutic drug that is in the same group of drugs called alkaloids (Stadtmauer, 1989). Recent studies show the effectiveness of vincristine as an active agent against bone cancer, which is structurally similar to Etoposide (Stadtmauer, 1989). Higher doses of Etoposide will increase the rate of apoptosis in the cancer cells as shown to be an active agent when used at high dosages with other chemotherapeutic agents when applied to bone cancer (Stadtmauer, 1989). Etoposide has been shown to inhibit the growth of pancreatic cancer cells and induce apoptosis (Zhang & Zhang, 2013). Etoposide is a principal chemotherapeutic agent that has been shown to treat many types of cancers because, for more than two years, it remains the most prescribed drug for cancer treatment (Zhang & Zhang, 2013).
Research on antioxidants and their effects on cancer cells have shown that higher dosages increase the rate of apoptosis. Though they all increase apoptosis when the dosage is increased, it is also essential to discuss which treatment would be the most effective in comparison to Etoposide, which is a known chemotherapeutic agent. A study that examines the effects of Lycopene, Resveratrol and Vitamin C (ascorbic acid) on mouse osteoblastic cells (MC3T3) would enable us to see which one is the most effective compared to Etoposide.
As you increase the volume of the antioxidant solution in presence of mouse cancer cells, then the apoptosis of the mouse cancer cells will increase, because studies on antioxidants have shown to increase apoptosis in many different cancer cell lines.
Mouse Osteoblastic Cancer cell line (MC3T3) and other special instruments were provided by the University of Arkansas Little Rock. Throughout the whole experimentation all equipment was provided sterile and goggles, aprons, gloves, and other lab safety precautions were taken. An incubator was used throughout the experiment and was constantly at 37 C with 5% CO2 atmosphere.
To first begin collecting cells from the original medium in the original mammalian cell 1 culture flask, cells were washed with PBS (11.9 mM phosphates, pH 7.4, 13.7 mM 2 NaCl, 2.7 mM KCl). Then using Trypsin-EDTA (0.05% Trypsin/0.53 mM EDTA in 3 HBSS without sodium bicarbonate, calcium and magnesium), cells were dissociated and placed into a incubator for 5 minutes until cells detached. Then a centrifuge at 500 g for 4 5 minutes was used to separate both medium and cells. Cells were then resuspended into a 10 mL medium with 10% FBS which was supplemented with antibiotics penicillin 5 6 (500 units/ml) and streptomycin (500 units/ml). Cell density was then measured by 7 staining cells with Trypan blue dye by using a hemocytometer. Cells were then seeded 8 into a 96 well plate at 1.0 – 1.5 x 8000 cells per well with 100μl medium, where only 24 wells seeded. Cells were then left to be grown in the incubator for 24 hours.
1 A substance used to promote cell growth of microorganisms 2 Polyphosphate Buffer solution, used to maintain pH 3 is used to remove cells from original culture 4 A machine used to separate liquids or solids of different densities 5 Fetal Bovine Serum, used to increase within medium 6 Helps control bacterial contamination
Antioxidant solutions were prepared by either crushing Vitamin C pills or making solutions from gel tablets. 2 determinations of 10μl, 50μl, and 100μl of Vitamin C, Resveratrol, and Lycopene were made. Each antioxidant at each specific volume was placed into 2 wells. Etoposide at 10μl was also placed into wells. 2 wells were left with no treatment to serve as a control. Cells were then left to be grown in the incubator for 24 hours.
The method that was used to measure the cell viability in this experiment was MTT assay. MTT assay is the process by which analyzing the activity of mitochondrial-dehydrogenase as a way to the number of living cells. To first begin, the cells were washed with FBS twice that was medium free of any medium. After they were washed, 10μl of MTT solution (5 mg/ml) was added to the cell culture and then the microwell plate was left in the dark in the incubator for 4 hours because it is light sensitive. After the 4 hours, the medium was removed and the cells were lysed with 9 dimethyl sulfoxide, which helps dissolve MTT formazan which was produced mitochondrial succinate dehydrogenase. The cells were then placed into a microplate cell reader at 570nm to measure the absorbance of the purple color of each well produced by the MTT solution. Determinations were performed in duplicates for each treatment for better statistical analysis. The results of MTT assay were presented as a % showing the control values obtained from untreated cells. The cell viability was calculated using the % of dead cells = (A sample – A blank) /(A control – A blank) x 100.
7 Helps control bacterial contamination 8 Dye used to identify living tissue or cells 9 To undergo Lysis, breaking down of cell membrane
Data / Observations
Absorbance at 570 nm Trial #1
Absorbance at 570 nm Trial #2
10 μL Etoposide
Vitamin C (A)
Vitamin C (B)
Vitamin C (C)
Table 1. Absorbance Readings at two different determination of antioxidants at various volumes
Absorbance at 570 nm
10 μL Etoposide
Vitamin C (A)
Table 2.. Effects of Antioxidants at 10uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture
50μL of Antioxidants
Absorbance at 570 nm
10 μL Etoposide
Vitamin C (B)
Table 3.. Effects of Antioxidants at 50uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture.
100μL of Antioxidants
Absorbance at 570 nm
100 μL Etoposide
Vitamin C (C)
Table 4.. Effects of Antioxidants at 100uL on Cancer Viability. Shows the prominence of each antioxidants effect on the cancer cell culture.
Graph 1. Viability of Cancer Cells in 10uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture
Graph 2. Viability of Cancer Cells in 50uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture
Graph 3. Viability of Cancer Cells in 100uL of Antioxidant Solutions. Displays the different antioxidants’ effects on the cancer cell culture
Table 4. ANOVA Test Results. Shows the statistical variance of data and since the f-value is lower than the p-value in this case, the data is not statistically varied. Since the p-value is higher than 0.05 (> 0.05) it is not statistically significant.
Figure 1. MC3T3 cells after being detached and added to medium
Figure 2. 96-well plate. Contains only the cancer cells and antioxidants before growth
The expected result of apoptosis increasing when increased volumes of antioxidant the solution was true to a minimal extent. The results of some of the antioxidants at specific volumes had the opposite effect, where the cell viability of MC3T3 increased significantly. The cell viability of Vitamin C among the three different volumes, increased 243%, 298%, and 868% respectively. This result could be due to the fact that at a certain point of volume of the antioxidant solution, the cells will then feed on the Vitamin C increasing the viability significantly. Fernandes and colleagues looked at Vitamin C at different volumes and saw a similar result that at cell viability, was increased in cells treated with low volume of Vitamin C with respect to untreated bone cancer cells. Etoposide did not increase apoptosis significantly though 89% of cell viability was present after 10μL was seeded. Some antioxidants at specific volume were more effective in promoting apoptosis than the Etoposide. Lycopene at 100μL was the most effective in promoting apoptosis as cell viability was 27%, though at volume of 10μL and 50μL, it was 136% and 1162%. .The same effect that was on Vitamin C can be seen on Resveratrol as at a certain volume, the volume of the antioxidant will feed the cells until a certain volume has reached. For resveratrol, the opposite effect can be seen as at a lower volume of 10μL the cell viability was the lowest and as volume was increased the cell viability exponentially An ANOVA test was performed, and it ended with a p-value of 0.58602 and an f-value of .55892. This signifies that the data has an extreme variance between each treatment since they all had different results. Since the p-value is above 0.05, we have failed to reject the null hypothesis which is that there is no correlation between antioxidant treatment and cell viability. Within the experiment, there may have been some possible error as the vitamin C solution turned the well yellow while every other well had a more purple color. This may have been related to the vitamin’s natural color, but it could have influenced the results of the experiment. Due to time constraints, only 2 determinations were performed. Contamination could have occured as cells were being grown in an incubator with other organisms, or light contamination as MTT assay has to be done in a dark environment.
The hypothesis that as you increase the volume of the antioxidant solution in presence of mouse cancer cells, then the apoptosis of the mouse cancer cells will increase, was supported to the extent at which Resveratrol and Lycopene increased apoptosis when induced. Each antioxidant significantly demoted growth to a certain extent within the cancer cell apart from the Vitamin C solution. Furthermore, the polychemotherapy can be investigated by looking at not only chemotherapy but as well as a combined treatment of antioxidants and chemotherapy. Research can also be done on looking at the patient’s quality of life as they are being given normal chemotherapy and to also see if it is different as antioxidants are introduced.
This project required an immense amount of outside help from Dr. Nawab Ali and Mrs. Puja. The supervision of a qualified scientist of her expertise was necessary in order to follow procedures accordingly. Dr. Ali also gave great advice throughout the process. Without the assets and resources provided by the Dr. Ali’s lab, there would have been no way for this project to come about. Mrs. Becker and Mrs. Norris contributed a significant amount of help throughout the research and statistical analysis process.
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Cancer immunotherapy describes a compelling pathway in the treatment of malignant tumors, leveraging a patient’s adaptive immune system in combating their own unique cancers. Such therapies often utilize complex nanotechnology, relying upon synthetic particles and engineered carriers to induce a desired immune response in the tumor microenvironment (TME). The widely accepted paradigm of nanomedicine assumes its ability to selectively deliver cytotoxic agents to tumor sites, minimizing the system-adverse effects typically associated with other standards of care. This approach confers a modest improvement in patient survival outcomes and is the focus of a broad spectrum of ongoing research. A second category of treatment relies upon the specific molecular targeting of immune checkpoints, called immune-checkpoint inhibition (ICI). Here, dramatic improvements in patient outcomes have been observed, demonstrating unprecedented efficacy in small subsets. As a consequence, there is a strong emerging interest in understanding and enhancing nanomedical cancer immunotherapies. This report will summarize the modern scope of immunoengineering, proceed to an analysis of its primary challenges, and conclude on a discussion of future development.
Multiple forms of nanotherapeutics already see practical use and are readily available to cancer patients at the discretion of their medical caregivers. Further treatments are the focus of intense professional interest, with ongoing projects spanning early development to late-stage clinical trials. Immunoengineering refers to a fledgling field of interdisciplinary research combining expertise from materials science, bioengineering, nanotechnology, and immunology to influence the human immune system. In particular, it explores channels through which strong antitumor immune responses can be elicited through means unavailable to the conventional application of drugs in solution. The biological sciences work in conjunction with engineering methodologies in monitoring the diagnostics of biomarkers and enhancing the development of therapeutics. Biomaterials are employed in constructing targeted drug delivery solutions, focusing synthetic payloads on particular cell types in the TME and at specified anatomical locations.
Recent work in immunoengineering can be subdivided into five primary categories: widening the risk-to-safety therapeutic index, augmenting adoptive cell therapy, enhancing endogenous immune responses, improving mRNA and peptide vaccines, increasing the uptake of nucleic-acid based therapies, and improving perioperative immunotherapy. The therapeutic index is substantially bolstered by the intratumoral injection of immunotherapeutic particles, as matrices are localized and systemic side effects are highly reduced. Nanotechnology allows for the effective production and delivery of cultured T cells, which are further improved by particles that stimulate lymphocyte function and modify the TME for easier infiltration. Endogenous immune responses can be reinforced by the direct stimulation of antitumor immune cells, as well as the reprogramming or elimination of immunosuppressive macrophages. Vaccinations are enhanced by the co-delivery of multiple components in nanotechnology formulations and the specific targeting of lymph nodes. Nucleic-acid based therapies benefit from the use of targeted polymeric nanoparticles to transfect T cells, resulting in the expression of therapeutically significant proteins. Finally, macroscale delivery devices can promote the dispersal of T cells at tumor resection sites and the intraoperative local release of various classes of cancer immunotherapy. As such, modern immunoengineering already confers formidable advantages in cancer treatment.
Although immunoengineering presents various advantages in the permeability and retention of therapeutic agents at tumor sites, the primary barrier to both nanomedicines and immunotherapeutic treatments still lies in the tumor microenvironment. Local changes induced by cancer cells inhibit effective delivery and compromise efficacy, even when therapeutic molecules are able to accumulate in the TME. These changes are pathophysiological and primarily manifest in the form of abnormalities in angiogenic and fibrotic signaling. The TME of primary tumors and any metastases derive their treatment resistance by directly and indirectly inducing immunosuppression through pathological angiogenesis and desmoplasia. Indirect immunosuppression occurs as oxygen delivery is inhibited, resulting in hypoxia and increased acidity. This dysfunctional growth in tumor vasculature varies substantially and explains high levels of spatial intratumor and intertumor variance in drug distribution, even between patients with tumors of the same type and between multiple tumors in the same patient.
The microenvironment surrounding various tumors can be broadly attributed to three main, cycling immune phenotypes, which significantly influence sensitivity to immunotherapies and nanomedicines. First, the immune-desert phenotype is characterized by a lack of antitumor immune cells, reduced response to antigen presentation, and limited T cell priming. Local hypoxia induces angiogenic growth factors that promote immunological ignorance to antigens and increase the expression of chemokines that promote tumor-supporting immune regulatory cells. Second, the immune-excluded phenotype localizes immune cells to the periphery of the tumor microenvironment, inhibiting their further penetration through immature blood vessels and extravascular stroma. This dysfunctional growth stems from the expression of a transforming growth factor, resulting in an excessive extracellular matrix and a high density of cancer-associated fibroblasts. Although more susceptible to immunotherapies through reactivation of surrounding T cells, desmoplasia and angiogenesis prevent cytotoxic lymphocytes from migrating to cancer cells through a variety of motility inhibiting factors. Lastly, the inflamed phenotype contains immune cells within the parenchyma, indicative of a failed immune response. With T cells already within the TME, this phenotype is most vulnerable to immunotherapy but often contains numerous hypoxia-suppressed immune cells. The activation of certain growth factors recruits more immunosuppressive cells, as well as upregulating the expression of immune checkpoint molecules. Although preliminary strategies exist to “normalize” the TME, they represent the greatest current barrier to effective immunotherapeutic treatment.
As a burgeoning field, research opportunities in immunoengineering are extensive. In particular, the avenues through which further progress can be made are multiform. While novel therapies are a constant focus, improvements in bioengineering and materials science constitute the body of advancement in nanomedical cancer immunotherapy. Uniquely, quantitative and materials-based approaches are critical in the design of therapeutic agents, aiming to enhance their delivery, specificity, and efficacy in the TME. Another primary point of emphasis, however, is the potential for the synthetic manufacturing of immune cells and tissue. Technological advancements that support the large-scale production of primary, secondary, and tertiary lymphoid organs naturally enable the concomitant growth of immune-cell based therapeutics. Although many aspects of the immune system remain the subject of ongoing research, certain elements can be constructed under the constraints of modern bioengineering systems.
Examining primary lymphoid organs, the bone marrow and the thymus constitute the most compelling targets for artificial production. As the primary site of development for all hematopoietic cells and the main location for B cell maturation, the construction of bone marrow is critical to facilitating further growth in cancer immunotherapy. The thymus follows closely after, as it allows for the generation of self-tolerant T cells essential for adaptive immunity. In engineering secondary lymphoid organs, the synthesis of activated T and B cells represent attractive challenges for future development. Artificially cultured T cells are a necessary component of adoptive T cell therapy, promising revolutionary applications in immunotherapy once fundamental roadblocks in inactivation and dysfunction are resolved. Activated B cells are inherently important to immune health, differentiating into antibody-secreting cells such as plasma cells and memory B cells. Challenges here lie in reconstructing the germinal center, as well as several less-understood components of B cell activation. Finally, ectopic tertiary structures present inherent difficulties in synthetic manufacture due to their complexity, variance, and unclear nature. Although suspected to be immune inductive sites for protective immunity in infectious diseases, current literature continues to investigate the exact function of tertiary lymphoid structures. Under the existing body of research, engineered models will have to carefully delineate their immunological purpose. Further investigation on all levels of lymphoid organization is necessary to facilitate the scalable ex vivo generation of immune cells and tissue.
Immuno-engineering represents an attractive avenue for cancer treatment with incredible potential for future advancement. As a field in its infancy, the breadth of current research is naturally limited, yet the advantages of immuno-therapeutic treatment are increasingly clear as larger aggregates of clinical data are collected. Future development may result in highly effective remediation for a wide array of cancer forms, especially if key challenges in the tumor microenvironment are surpassed and innovative engineering strategies allow for the large-scale production of immune organs.
Martin, J. D., Cabral, H., Stylianopoulos, T., & Jain, R. K. (2020). Improving cancer immunotherapy using nanomedicines: Progress, opportunities and challenges. Nature Reviews Clinical Oncology, 17(4), 251–266. https://doi.org/10.1038/s41571-019-0308-zWang, D., Wang, T., Yu, H., Feng, B., Zhou, L., Zhou, F., Hou, B., Zhang, H., Luo, M., & Li, Y. (2019). Engineering nanoparticles to locally activate T cells in the tumor microenvironment. Science Immunology, 4(37). https://doi.org/10.1126/sciimmunol.aau6584
The purpose of this proposal is to establish a systematic method of testing and tracking to prevent another pandemic. This method depends on the creation of a global database and the use of generic viral testing, antigen testing, and data analysis prediction models. Essentially, this will (a) detect whether a person has contracted a specific disease or virus, (b) detect the severity of the disease within an infected person, (c) provide data for further research on the vaccine or treatment of certain diseases, analyze (d) percentage of population affected by a certain disease and (e) predict which part of the world may experience an outbreak of a certain disease.
The COVID-19 global pandemic has impacted the world, by causing numerous deaths, negatively impacting economies, and bringing unemployment rates to a historical high. Implementing a standard method of testing at various ports of entry, such as border control, with testing for different infectious diseases will help prevent the recurrence of another pandemic.
The subject’s DNA sample and general information (ie. recent places visited, and if they have been recently infected with a contagious disease) would be collected. The DNA sample would be sent to the cloud, a database with DNA samples (from various countries organizations and companies) of different contagious diseases. The cloud would have DNA samples of the specific strains of each disease and the different densities for each disease. If the subject is tested positive for a specific disease, the DNA sample will also be matched with a specific density. Based on the results of this, the viral exposure density of the initial stage will determine the severity of the disease within a person. The individual will receive a specific course of treatment. If the positively-tested individual recovers, they may be randomly asked to take an antibody test to provide further data about the immunity against specific diseases.
Tracking and Predicting
By constantly analyzing the data, researchers can track the diseases that are affecting the majority of the population. The general information would help researchers in using data analysis prediction models to predict where a specific outbreak could occur.
Potential Pitfalls and Solutions
Privacy is a major issue, as people may not be willing to voluntarily share their data. A potential solution includes the creation of a policy to ensure the security of people’s data. Additionally, genomic sequencing is an expensive method. Alternative methods such as PCR and qPCR can be used instead. However, these methods come with some disadvantages, as PCR may produce false negative and positive results. PCR, qPCR, and genomic sequencing can each be tested individually to decide which is more beneficial.
Although the world has been impacted by COVID-19 global pandemic, this does not mean that COVID-19 is the only disease that can cause a pandemic in the future. Any other contagious disease without a vaccine can potentially cause a pandemic. A systematic method of testing and tracking to prevent outbreaks will decrease the chances of another pandemic.
Hasman, H., Saputra, D., Sicheritz-Ponten, T., Lund, O., Svendsen, C. A., Frimodt-Møller, N., & Aarestrup, F. M. (2014, January 1). Rapid Whole-Genome Sequencing for Detection and Characterization of Microorganisms Directly from Clinical Samples. https://jcm.asm.org/content/52/1/139.
National Laboratory of Enteric Pathogens, Bureau of Microbiology, Laboratory Centre for Disease Control. (1991). The polymerase chain reaction: An overview and development of diagnostic PCR protocols at the LCDC. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327995/.
Although the advent of “Phage therapy” in the early 20th century by the French-Canadian Microbiologist, Felix d’ Herelle, it was met with great enthusiasm. However, it was soon forgotten with the introduction of antibiotics (Altamirano & Barr, 2019) and the little amount of information available on phages at that time. The continuous use and often misuse of antibiotics, has resulted in the formation of antibiotic-resistant bacteria carrying resistance genes (Lin, Koskella, & Lin, 2017) which encode proteins that annul the effect of these antibacterial agents. The alarming increase in antibiotic resistance has led to a renewed interest in phage therapy; especially the therapeutic use of phages to treat bacterial infections. This report, therefore, aims at discussing the uses and benefits of bacteriophages as an alternative to antibiotics, antibiotic resistance and strategies to combat it, the combination of phages and antibiotics, advantages of phages over antibiotics and the challenges and opportunities of phage therapy.
1.1 DISCOVERY OF PHAGES
Phages were discovered independently by Fredrick Twort in 1917 and Felix d’Herelle in 1919. The term “BACTERIOPHAGE” which literally means “BACTERIUM-EATER” was coined by Felix after he successfully treated patients infected with Shigella dysenteriae using what he called “ an invincible, antagonistic microbe of the dysentery bacillus” (Dublanchet & Bourne, 2007, pp. 15-18).
1.2 BIOLOGY OF PHAGES
Bacteriophages are viruses that infect and replicate only inside bacteria. They are composed of a genetic material which is either DNA or RNA and an outer protein layer called CAPSID. Most, if not all, phages are infectious only to bacteria that have complementary receptors to theirs (Lin et al., 2017). The phage binds irreversibly to these receptors using specialized structures such as tail fibres and base pins (found in tailed phages, for example, T4 phages that infect Escherichia coli) or other structures. Thereafter, the genetic material is injected into the target pathogen through an “Injection mechanism”.
Based on the life cycle of phages, the genetic material can replicate using the host cellular machinery to produce multiple progenies which lyse the cell, using phage-derived lytic proteins, and are released to repeat the cycle (lytic/virulent phages).
Alternatively, the genome can be integrated with the host chromosome forming a “prophage” that enters a latent phase within the host. Consequently, the prophage replicates together with the host’s chromosome thereby leading to the vertical transfer of the phage (genome), that is, from mother to daughter cell (lysogenic /temperate phages) (Lin et al., 2017), some of which excise from the bacterial chromosome due to environmental or physiological stressors and later undergo the lytic cycle (Kortright, Chan, Koff, & Turner, 2019). The most common lytic phages associated with human pathogens and gut microflora are in the orders Caudovirales, “tailed phages” which contain double-stranded DNA genomes, and Microvirales, which are tailless, single-stranded DNA viruses (Lin et al., 2017).
Bacteria have evolved anti-phage systems to defend themselves against viral penetration. As listed by Dy, Richter, Salmond, and Fineran (2014), the systems include adsorption, injection blocking, inhibition, toxin-antitoxin and the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR Associated System (CRISPR/Cas) (Dy et al., 2014; Lin et al., 2017).
However, Safari et al. (2020) reported that phages have also co-evolved to resist these systems, by assessing anti-CRISPR proteins and evading Restriction modification (R/E) systems.
1.3. DESCRIPTION OF PHAGE THERAPY
The extensive work of Felix on phages as antibacterial agents led to a phenomenon called “phage therapy”. This can be defined as the use of specific viruses to treat bacterial infections with the aim of lysing the bacterial cell. Lytic phages are widely used against the lysogenic phages and this is attributed to the ability of these phages to directly kill the bacteria. A disadvantage to the use of lysogenic phages is their inherent ability to mediate gene transfer between bacteria by specialized transduction (Monteiro, Pires, Costa, & Azeredo, 2019). The hindrances faced by many scientists during the earliest trials of phage therapy such as the limited stability of the viruses in solution, a significant drop in phage titre during storage and processing (Malik et al., 2017), contamination from bacterial antigen and delivery of phages to the site of infection (Lin et al., 2017), led to the wide dismissal of this therapeutic method. Moreover, a better understanding of the mechanism of action of antibiotics in the 1940s made antibiotic therapy gain more attention and led to a significant decrease in the interest of phages by most western countries. Exceptions to these include countries in Europe, e.g. Georgia, Poland etc. where phage therapy has been continually practised for decades and used to treat infections caused by most clinical antibiotic-resistant pathogens (Abedon, Garcia, Mullany, & Aminov, 2017; Lin et al., 2017).
2. ANTIBIOTICS AND ANTIBIOTIC RESISTANCE
Antibiotics are a type of antimicrobial agent. They are produced by microorganisms to inhibit the growth and replication of other microbes and are most effective against bacterial infections. They are recognized as one of the most successful forms of chemotherapy developed in the 20th century (Banin, Hughes, & Kuipers, 2017) and the entire history of Medicine at large. Since the introduction of antibiotics over 70 years ago, they have been used extensively in all fields of medicine and allowed great advancements in Medicine, for example, the use of antibiotics in organ transplantation, Chemotherapy etc. (Altamirano & Barr, 2019).
2.1. DISCOVERY OF ANTIBIOTICS
The discovery of antibiotics is credited to two major scientists which are Alexander Fleming, a physician cum Microbiologist and Paul Ehrlich, a German Scientist. Ehrlich envisioned a “magic bullet” which would selectively destroy a disease-causing microbe without harming the host. In 1909, he came about a compound called ARSPHENAMINE (an organoarsenic derivative) that cured syphilis infected rabbits (Aminov, 2010). The compound marketed as “Salvarsan”, exhibited the potential of curing Syphilis infected patients.
However due to its severe side-effects, from rashes to liver damage, an alternative compound “Neosalvarsan” was developed and preferably used as it was less toxic and more soluble (Kon & Rai, 2016). These two compounds became very successful and were frequently recommended until the introduction of Penicillin in 1940. Penicillin known as the first natural antibiotic produced (Mater methods, 2018) was discovered accidentally by Alexander Fleming in 1928. During one of his studies, he noticed a zone of inhibition of growth on agar plates which he initially thought to be contaminated with the Penicillium mould (Kon & Rai, 2016). He later identified the substance like Penicillin. The isolation of penicillin from the surface of agar plates made it easier, more reliable and required fewer resources as compared to the animal model testing methods such as those used by Ehrlich (Aminov, 2010). As a result, this method became widely used to produce penicillin in large quantities and was made available for use on humans in 1940. Nevertheless, more efficient technologies have been used and are still in use to develop drugs effective against microorganisms. Following the evolutionary trend, microbes, especially bacteria, have evolved to resist the effects of these antimicrobial agents; this would become known as “antibiotic/antimicrobial resistance”.
2.2. ANTIBIOTIC RESISTANCE
Antibiotic resistance can be simply defined, as the ability of bacteria to evade antibiotics designed to kill them. Also known as antimicrobial resistance (AMR), it is “the resistance of microorganisms to antimicrobial agents which they were first sensitive to” (Kon & Rai, 2016). Generally, these bacteria have evolved to produce resistance genes as well as novel mechanisms which govern a higher Minimum Inhibitory Concentration (MIC) than the original wild strain of the organism (Acar & Rostol, 2001).
2.3. RESISTANCE: NATURAL OR MAN-MADE?
Bacterial resistance to antibiotics can either be a natural (innate) attribute or man-made (acquired) mechanism (Acar & Rostol, 2001). The latter includes anthropogenic activities which are majorly the clinical and industrial use of antibiotics (Altamirano & Barr, 2019). From the use of antibiotics in agriculture to enhance the growth of livestock, treating animals, or to increase the nutrients in the soil, to prolonged use for treating infectious diseases of humans (Ventola, 2015). In contrast, natural resistance arises due to horizontal transfer of genes; the transfer or movement of genes through the 3 stages “transformation, transduction and conduction” (Altamirano & Barr, 2019). In addition, Transposons that transmit mobile integrons allow pathogens to share resistance mechanisms. This attribute is very beneficial to organisms that are capable of resisting only one type of antibiotic. With this, they can acquire numerous antibiotics from transposons that transfer different antibiotic resistance genes (Kon & Rai, 2016). Furthermore, innate resistance is also a result of the spontaneous mutations that occur in chromosomal genes as an answer to selection pressure exerted by antibiotics, synthetics, antiseptics and other drugs (Acar & Rostol, 2001; Altamirano & Barr, 2019). According to Acar and Rostol (2001), no strict separation exists between chromosomal and transferable resistance. These attributes are used solely as an epidemiological tool.
The different events that occur in a bacterial life leading to resistance are summarized in the table below:
TABLE : EVENTS LEADING TO RESISTANCE IN BACTERIA
SOURCE: Acar, J. and Röstel, B. (2001). Why and How Resistance Develops. Antimicrobial Resistance: An Overview, 20(3), 801.
Microorganisms utilize numerous resistance mechanisms. As listed by Kon and Rai (2016), they are;
1) Porin modification.
2) Efflux pumps.
3) PBP modification.
4) Enzymatic inhibition.
2.4. EXTENT AND CONSEQUENCES
The excessive use of antibiotics has resulted in the emergence of Multidrug-resistant organisms (MDR) also known as “superbugs”. Other examples of resistant pathogens are Extensively drug-resistant (XDR) and Pan drug-resistant (PDR) pathogens (Altamirano & Barr, 2019). Many bacteria have been classified as presenting serious economic, societal and health threats by the Centre of Disease Control (CDC) (Ventola, 2015) particularly the ESKAPE pathogens; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumonia, Actinobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp which cause serious nosocomial infections (Lin et al., 2017) and with the characteristic ability to evade the biocidal activity of antibiotics using highly developed resistance mechanisms (Altamirano & Barr, 2019).
The burden of antimicrobial resistance has led to an increased raging to return to the “pre-antibiotic era” (Lin et al., 2017) that is, the treatment methods used before the introduction of antibiotics. Public health organizations such as CDC and WHO has estimated that a total of 2 million illnesses are caused by MDR pathogens with at least 23,000 deaths per year (Lin et al., 2017). Among the deadliest MDRs is the Methicillin-Resistant Staphylococcus aureus (MRSA) which alone accounts for more deaths yearly than HIV/AIDS, Parkinson’s disease and homicide combined (Ventola, 2015; Lin et al., 2017). According to Altamirano and Barr (2019), a review carried out on AMR predicts that by 2050 an estimated cost of 10 trillion USD would have been spent on treatment with over 10 million deaths occurring. However, these statistics do not fully encompass the extent of AMR as the economic burden is hard to quantify. In addition, several consequences of AMR must be taken into account such as reduced possibilities of treating infections due to the increasing amounts of less efficacious agents (Friedman, Temkin, & Carmeli, 2015), loss of productivity, lower economic output and increased costs associated with highly expensive antibiotics (Friedman et al., 2015; Prestinaci, Pezzotti, & Pantosti, 2015). So a reversal to older treatment techniques such as disinfection, amputation, isolation etc. (Michael, Dominey-Howes, & Labbate, 2014) which are not only less effective, but also have a longer time of action, may lead to prolonged infections and finally, death.
2.5. STRATEGIES TO COMBAT ANTIBIOTIC RESISTANCE
Antibiotic resistance is caused mainly by the excessive use and/or misuse of antibiotics, in treating viral and fungal infections. If any strategy is to be utilized, “it has to be multidimensional, multidisciplinary and global” (Altamirano & Barr, 2019, p. 5). Some of them are policies promulgated by the CDC, for example, prohibiting the unnecessary use of antibiotics on agriculture and livestock (Singhai, 2016) and creating adequate awareness to the general public on the health detriments of self-medication and improper dosing. In addition, stewardship programs have been provided to educate clinicians on following evidence based guidelines in prescribing antibiotics for the right infections after proper diagnosis (Manning, Pfeiffer, & Larson, 2016). Also, antibiotic combination therapy is an emerging alternative which offers great effects on pathogens as opposed to the individual effect of each antibiotic (Maheshwari, 2004). Although these strategies do not completely eradicate the rise of antibiotic resistant pathogens, they slow it down. It is of great importance to note that old therapeutic methods, new methods, and the neglected ones such as phage therapy are also encouraged.
3. BACTERIOPHAGES AS THERAPEUTIC AGENTS
Phage therapy is an age long phenomenon, although introduced over a century ago, it was abandoned after the emergence of antibiotic therapy which became more successful. However, the rise in antibiotic resistance has led to a renewed interest in phages as a promising therapeutic agent. This interest is attributed to a better understanding of the mechanism of action of phages, their biology and genetics (Altamirano & Barr, 2019). An ideal anti-bacterial drug should exhibit a wide range of activity, lack a specific target that would limit its application i.e. it should be multidimensional, remain viable when administered orally, maintain activity in biofilms, accumulate in macro-forms, penetrate through bacteria cell walls and be usable on sensitive patients (Guo et al., 2020). Phages, however, are natural anti-bacterial agents. Due to their specificity, they possess a narrow range of bactericidal activity. Thus far, no adverse effects have been recorded on the oral administration of phages (Guo et al., 2020). Phages also lack the ability to accumulate in macro-forms, however, preparations of phages called “phage cocktails” are used to target multiple receptors on the host cell surface thereby expanding the bactericidal spectrum. Notwithstanding, more information about the host receptors for each phages should be established as well as the use of combination therapy to limit the advent of phage resistant hosts (Altamirano and Barr, 2019).
Lytic phages are strictly used for phage therapy. Temperate phages are not advised due to the high risk of lysogenic conversion which is one of the ways which bacteria acquire resistant traits and genes that increases their virulence (Altamirano & Barr, 2019). These phages can be administered through intra-peritoneal, intra-venous, oral routes and even sprayed on the site of infection (Sagor et al., 2005). Additionally, lytic phages have been shown to be good bio-control agents against food borne pathogens and have several advantages over sanitizers, detergents etc. (Sagor et al., 2005). They have been used successfully for both therapeutic and prophylactic purposes in surgery and treating gastro-enteric infections especially those caused by Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa (Lin et al., 2017). Also, “BIOPHAGE-PA; a preparation of phages that target multidrug resistant Pseudomonas aeruginosa has been used to treat patients with chronic otitis” (Guo et al., 2020).
Research has been, and still is, carried out to determine the effectiveness of phages many of which were done in countries that embraced phage therapy before it was dismissed. Moreover, the first test to determine the safety of administering phages was carried out in Switzerland in 2005 (Altamirano & Barr, 2019; Guo et al., 2020). This trial was to determine the effect of a T4 phage targeted against Escherichia coli for the treatment of diarrhoea. No adverse effect was recorded neither was T4 specific antibodies found in the serum of the volunteers (Guo et al., 2020), phages did not multiply in the gut of the individuals and only a small fraction of phages was recovered from the faeces of the individuals (Altamirano and Barr, 2019). With the innovations on technology, the genetics of phages and how to genetically engineer them to deliver CRISPR/Cas programmed to destroy antibiotic resistant genes and plasmids in bacteria (Yosef, Manor, Kiro, & Qimron, 2015; Lin et al., 2017) are in study. Research is being conductedon the efficacy, safety, route of administration, side effects etc. of phage therapy. There are also arising complications which limits the scope of phage therapy such as liberation of endotoxins due to the breakdown of bacterial cell wall, phage resistance, limited host range (Guo et al., 2020), susceptibility to host restriction enzymes etc. (Sagor et al., 2005).
Phages are abundant in nature and are natural bacteria pathogens., making it an effective alternative to antibiotic therapy. Moreover, the low rate of antibiotic recovery called the “dry pipeline” aggravates the problem of antibiotic resistance (Altamirano & Barr, 2019), phage therapy has proven to be the best alternative yet.
3.1. COMPARING ANTIBIOTICS TO PHAGE THERAPY
Phages and antibiotics are regarded as bactericidal agents, the former being a natural killer of bacteria and the latter synthetic or semi-synthetic compounds, however, involving lysis and inhibition respectively (Lin et al., 2017). The following points elaborate on some of the differences between both antibacterial agents.
The major difference between phages and antibiotics is the specificity of phages to the host which is due to the specific receptors found on their envelopes or capsids. Unlike antibiotics with a much wider spectrum, they are not known to cause side effects and or secondary infections (Romero-calle, Benevides, Goes-Neto, & Billington, 2019) such as mucosa candidiasis, antibiotic-associated diarrhoea, pseudomembranous colitis caused by Clostridium difficile (Altamirano & Barr, 2019; Lin et al., 2017). Although the specificity to strain and species is an added advantage, it comes with its drawbacks. For instance, the fact that phage therapy aims directly at a particular pathogen limits its effect on infections (Altamirano & Barr, 2019) caused by more than one pathogen (Lin et al., 2017). Notwithstanding, this can be sorted using phage cocktails and accurate diagnosis of the causative agent. Latz et al. (2016) reported that phages that target antibiotic-resistant bacteria are more likely to be found in areas with high numbers of individuals infected with resistant bacteria. This provides an easy way to access phages infective to a particular resistant pathogen (Lin et al., 2017).
Phage therapy has recently garnered attention by western medicine and while much of the information on its safety is new and underdeveloped (Lin et al., 2017), oral administration of phages has been proven to be safe. Antibiotic therapy, on the other hand, is very well documented to cause anaphylaxis and other hypersensitivity reactions, nephro-toxicity, toxic necrolysis and more. especially in immunocompromised patients. This is accounted for by the synthetic nature of antibiotics, particularly the beta lactams which are notorious triggers of anaphylactic reactions (Altamirano & Barr, 2019). While the pros of phage therapy significantly outweigh the cons, there are hypothetical cases whereby the administration of phages worsens the patient’s condition especially if they are immunodeficient (Lin et al., 2017). Scientists still argue the possibility of this as no evidence has been reported to support this claim.
3.1.3. BIOFILM PENETRATION:
Biofilms are made up of a matrix of polysaccharide secreted by bacteria to serve as a barrier against external forces such as resisting the penetration of antibiotics. Normally, large doses of antibiotics are required to penetrate the biofilm. This results in regrowth of the pathogen after antibiotic therapy is stopped and also poses serious health risks (Lin et al., 2017). In contrast, phages are equipped with enzymes on their capsid (Extracellular Polymeric Substance) that degrades extracellular polymers (biofilms) (Lin et al., 2017) thereby granting them the access to the target bacteria. Upon the release of progeny virions, the biofilm is gradually broken down as they infect and lyse each bacteria cell (Kon & Rai, 2016). Parasion, Kwiatek, Gryko, Mizak, and Malm (2013) reported that the in-vitro application of phages on the colonies of Pseudomonas aeruginosa not only degraded existing biofilm but also prevented additional biofilm formation. This suggests that phage therapy can also be used to reduce infections caused by biofilm-coated pathogens on the surface of medical devices (Lin et al., 2017).
3.1.4. EFFECT OF BACTERIAL RESISTANCE:
It is widely acknowledged that pathogens are evolving to resist most of the antibiotics available. Although phages are not an exception to this, they are not as worrisome as with antibiotics (Kon & Rai, 2016). The best- known anti-phage mechanisms include but is not limited to prevention of viral DNA entry, eluding viral receptors, CRISPR/Cas systems that recognize and degrade previously encountered DNA (Altamirano & Barr, 2019). Besides, most of the bacteria resistant mechanisms can be avoided using phage cocktails or a combination of phage and antibiotic therapy called “combination therapy” (Golkar, 2014).
3.1.5. RELATIONSHIP WITH THE IMMUNE SYSTEM
Phages have the distinct ability to spread systematically and self-replicate at the site of infection, however, they do not completely eradicate the infection. This is because the complete removal of the pathogens results in the termination of the viral replicative cycle (Altamirano & Barr, 2019). This means that the immune system is needed to remove lingering bacterial populations in order for phage therapy to be effective—a collaboration termed “Immuno-phage synergy” (Altamirano & Barr, 2019). This is a major disadvantage of phage therapy as it presents serious health threats to immunocompromised patients. In contrast, antibiotics work independent of the patient’s immune system and are therefore preferred in some cases. Further research on the immune-phage phenomenon is still required especially with clinical trials as regards human health, as much of the current research is limited to animal models.
4. APPROACHES TO PHAGE THERAPY DELIVERY
Generally, there are two known types of phage therapy based on the spectrum of activity and host range the phages possess, they are mono-phage and poly-phage therapy. The former involves the use of phages with very narrow host range, that is, specific to single or a few bacteria strain(s) within a species (Chan & Abedon, 2012). It, however, has its own limitations; the evolutionary changes that occur between phages, although an advantage over antibiotics, does not decrease the possibilities of bacteria resisting phage attacks (Altamirano & Barr, 2019).
Moreover, due to the narrow spectrum, there is a constant need to match the phages with the pathogens to determine their suitability for clinical trials (Altamirano & Barr, 2019; Chan & Abedon, 2012). To address these limitations, poly-phage therapy such as “phage cocktails” are used—this involves the combination of different phage types, usually mono-phages, to improve the phage spectrum of activity.
4.1. PHAGE COCKTAILS
Phage cocktails are preparation of phages directed towards different strains of bacteria and ultimately different species. Some drawbacks of it are the potential of the mixed phages to co-infect a bacterium (Chan & Abedon, 2012) and a longer purification process (Altamirano & Barr, 2019). However, phage cocktails have been used to successfully treat diarrhoeic and pyogenic infections (Sarker et al., 2012). Phage therapy can be delivered through different routes, the common ones being oral and parental routes. Others are topical, inhalation and otic routes.
4.2. ORAL PHAGE DELIVERY
This is the most studied route of phage therapy delivery, proven to be safe and successfully used to treat gastrointestinal tract infections (Ryan, Gorman, Donnelly, & Gilmore, 2011) mainly caused by MDR pathogens (Zelasko, Gorski, & Dabrowska, 2017). The acidity of the stomach greatly limit phages by deactivating them, however, it can be prevented by encapsulating them in a liposome or mannitol-alginate macrospheres to enhance entry and shield them from neutralizing antibodies (Singla, Harjai, Katare, & Chhibber, 2016).
4.3. TOPICAL PHAGE DELIVERY
In recent years, attention has been directed towards the use of phages in wound infections particularly caused by MDR bacteria (Chang, Morales, Okamoto, & Chan, 2020). In a study conducted by Ryan et al. (2011) to test the efficacy of phage therapy on wound infected by Salmonella spp and Campylobacter jejuni, phage cocktails were cultured, Salmonella enterica serovar enteritis phage type 4 strain P125589 was inoculated on the wound and spread around the wound surface using a glass spreader. The results showed that the efficacy of the therapy depended on the MOI ratio of phage particles to bacteria cells, the higher the number of phage particles the lower the bacteria concentration (Ryan et al., 2011). Furthermore, the phages can be administered in: liquid forms such as phosphate-buffered solutions (PBS) (dripped or sprayed on the infection site) and semisolid forms such as hydrogels (agarose), organogels, creams etc. (Chang et al., 2020) which are more easily applied and never runoff.
4.4. INHALATION OF BACTERIOPHAGES
Respiratory tract infections e.g. tuberculosis and chronic pneumonia are among the leading causes of death and public health decline (Bodier-Montagutelli et al., 2017). The recent advances in technology provides an opportunity for further research into the use of phage inhalation in treating lung infections. One method is aerosolisation using the nebulizer (Malik et al., 2017) whereby the liquid cocktails are inhaled directly into the lungs; this provides a fast action rate and maintains the phage titre (Bodier-Montagutelli et al., 2017). In a study to determine the efficacy of phage therapy administered by inhalation targeting Pseudomonas aeruginosa, ten weeks old mice infected with bioluminescent P. aeroginosa suspended in 50ml PBS were used (Ryan et al., 2011). This was followed by the phage administration, using a nebulizer, after two hours. Results reported showed that 90% of the mice survived with a very remarkable drop in bacteria count (Ryan et al., 2011). However, these studies are not sufficient to correctly dictate the efficiency of phage therapy. Further studies are required in both infected and uninfected models to aid clinical trials in humans (Bodier-Montagutelli et al., 2017) and to determine the best route and regimen for phage therapy.
5. PHAGE AND ANTIBIOTICS COMBINED
With an increase in antimicrobial resistance and MDR bacteria, different alternatives are sought-after to fight against these pathogens, one of which is phage therapy. The efficacy of phage therapy is still questioned by scientists as bacteriophages are also prone to resistance by bacteria. However, to avoid replacing antibiotics, phages and antibiotics can be combined to provide an effect greater than the sum of their independent effects (Tagliaferri, Jansen, & Horz, 2019). Furthermore, combining different therapeutic methods has proven to be a great strategy against infections caused by MDR and opportunistic pathogens (Altamirano & Barr, 2019). Multidrug (combinational) therapy have been successfully used in treating HIV (Highly Active Anti-Retroviral Therapy), malaria and tuberculosis treatments (Altamirano & Barr, 2019). In light of this, Comeau, Tétart, Trojet, Prère, and Krisch (2007) reported that in the presence of sub-lethal concentrations of antibiotics phage production is increased leading to a remarkable drop in bacterial cells; a phenomenon called “Phage Antibiotic Synergy”.
5.1. PHAGE ANTIBIOTIC SYNERGY
Phage antibiotic synergy (PAS) is a phenomenon whereby “sub-lethal doses of certain antibiotics substantially stimulate the host cell’s production of lytic phages” (Altamirano & Barr, 2019; Kim et al., 2018). The PAS is one of the mechanisms which enables phages to adapt to and benefit from their environment: the presence of small concentrations of antibiotics in the environment poses a threat to bacterial cells, subsequently, phages take advantage of this “stressed state” of the cells to propagate efficiently (Comeau et al., 2007). This process would help to reduce antibiotic use and resistance levels. To show PAS, Oechslin et al. (2016) combined 10^8 PFU/mL of phage cocktail with 2.5 MIC of 0.9μg/mL of Ciproflaxin. The antibiotic was highly synergistic with the phages as indicated by ≥ log 3 CFU/ml decrease in bacteria count (Pseudomonas aeruginosa) after 24 hours. The synergistic activity is however dependent on the antibiotic used (Segall, Roach, & Strathdee, 2019). The PAS effects, higher burst size and plaque assays, observed when a combination of siphovirus and ciproflaxin was used against Escherichia coli isolated from urine samples, were not detected with other antibiotics such as tetracyclin (Tagliaferri et al., 2019). Furthermore, Ryan, Alkawareek, Donnelly, and Gilmore (2012) reported complementary results using phage T4 and a low concentration of cefotaxime against Escherichia coli strain 11303. Besides the expected results, the bacteria biofilms were significantly reduced compared to using either treatments (Tagliaferri et al., 2019; Ryan et al., 2012).
The effects of PAS has further been studied using different combinations of antibiotics and phages to fight against highly resistant, notably the ESKAPE, pathogens. More studies are still required as this is a relatively new phenomenon first discovered in 2007 and a majority of the findings are speculative. Nevertheless, the encouraging results obtained so far proves that Phage/antibiotic combination would likely provide the remedy for infections caused by MDR pathogens.
6.OPPORTUNITIES AND CHALLENGES OF PHAGE THERAPY
Phage therapy has a wide range of advantages sometimes regarded as phage properties. However, it has minor disadvantages that can readily be resolved. Although few, the cons should not be overlooked as phages are susceptible to being resisted by bacteria. Some of the opportunities for phage therapy includes:
Phage therapy can potentially limit the dependence on antibiotics which would reduce resistance,
Only a single dose of phage therapy may be required to produce an effect as they are live agents and replicate within the bacterial cell, achieving “active therapy” and auto-dosing (Loc-Carrillo & Abedon, 2011),
The low cost of isolation; phages are abundant in nature and easily isolated (Loc-Carrillo & Abedon, 2011),
Phages are versatile; they can be applied in liquid forms, creams gels, etc. and also combined with antibiotics (Chang et al., 2020).
According to Oliveira, Sillankorva, Merabishvili, Kluskens, and Azeredo (2015), the potential challenges of phage therapy and its development are divided into two factors namely
A) Intrinsic factors include:
Specificity/narrow host range of the phages (Loc-Carrillo & Abedon, 2011; Wu et al., 2013).
Phage type: the phages used are “obligately lytic” because temperate phages can convert phage-sensitive bacteria into insensitive ones thereby conferring superinfection immunity on the bacteria (Loc-Carrillo & Abedon, 2011; Wu et al., 2013) and also possibly transfer toxin-encoding genes among bacteria (transduction) (Sulakvelidze, 2011).
The inherent ability to induce resistance.
B) Extrinsic factors include:
Incomplete removal of contaminations due to insufficient purification methods of phage preparations (Pires, Costa, Pinto, Meneses, & Azeredo, 2020; Sulakvelidze, 2011),
Development of phage neutralizing antibodies, although the kinetics of phages are faster than the production of antibodies (Oliveira et al., 2015),
Treatment of intracellular pathogens; these pathogens multiply within cells and are shielded from external forces including phages (Oliveira et al., 2015).
Other major limitations include: The accessibility of phages (Altamirano & Barr, 2019), Lack of awareness, and regulatory constrains: although phage therapy is accepted as an alternative treatment, it is not approved for use in Europe and USA mainly because of the uncertainty surrounding its production and use (HealthLine, 2019).
Figure 3: Factors that limit phage therapy.
SOURCE: Oliveira, H., Sillankorva S., Merabishvili, M., Kluskens,L.D. and Azeredo J. (2015).
In an era of antibiotic resistance, phage therapy has proven to be an efficient and effective alternative to antibiotics. Bacteriophages are natural predators of bacteria and therefore possess properties better suited as an antimicrobial agent compared to the chemical counterpart. Phage therapy can be administered to the site of infection either directly (locally) or through the bloodstream. Furthermore, phages can be combined with antibiotics to produce a better therapeutic effect than when used independently. Although the transfer of phage therapy from the laboratory to the bedside may seem challenging due to limited experiments and studies, if explored effectively it would, no doubt, put an end to the dependence on antibiotics as well as cure infections caused by MDR pathogens.
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Humans are able to isolate their DNA through a series of different techniques such as using centripetal force and alternating temperatures in order to later use the isolated DNA in a Polymerase Chain Reaction (PCR) experiment. In this article, I am going to discuss how I used my remaining products from my PCR experiment to discover my genotype.
The process I used to deductively discover what my genotype was is called Gel Electrophoresis. Gel Electrophoresis is a method that allows individuals to abstract and analyze a variety of macromolecules depending on their size or charge. In simpler terms, Gel Electrophoresis is used to separate DNA fragments such as DNA, RNA, and proteins based on individual size or charge. First, DNA samples are loaded into small slits at the end of a gel also known as wells or indentations. The gel electrophoresis is utilized by researchers so that they can visually see and analyze the separation of fragments. Furthermore, there is an electrical component that is used in the electrophoresis process, when applied, the electrical current pulls the DNA samples through the gel. Because DNA fragments are negatively charged, they move towards the positive electrode. Lastly, depending on the size and charge of the macromolecules, the molecules will migrate in different directions and at different speeds which help researchers to deduce various conclusions.
Figure 1: Figure 1 illustrates how Gel Electrophoresis results are analyzed. More, there is a graph that represents the relationship between DNA Fragment Size and Distance that were concluded from the results seen at left.
Although Gel Electrophoresis may sound uncommon, it is becoming a commonly used method for medical fields and other scientific experiments. It is most frequently utilized in forensics, molecular biology, genetics, microbiology, and biochemistry. For instance, in forensics, it can be used in DNA fingerprinting. Forensic scientists are able to compare samples of DNA and obtain prints by using this method. After the Gel Electrophoresis lab is completed, it will appear in the bands where it will form a pattern outlining a fingerprint. It could be used in many different ways when testing antibiotics and vaccines. One way they can be tested is for their purity by applying electrophoresis to a solution in the form of a paper strip impregnated with the antibiotic or vaccine. Researchers can differentiate between the antibiotic or vaccine and determine any impurities. They can also determine how concentrated an antibiotic is, which is crucial for applying accurate dosages. Of course, Gel Electrophoresis is not just limited to the things I listed, it can be used in numerous ways. This process has helped people in many ways such as learning more about their genetics and has helped conclude various diagnostics as well.
Figure 2: In Figure 2, an example of an Electrophersis tank, the tool used to separate the DNA fragments, is shown.
In my high school Forensics Science course, we utilized Gel Electrophoresis to analyze the separation of PCR products. Firstly, we began by preparing the agarose gel with SYBR Safe stain. Agarose is a jello-like component that helps separate DNA fragments based on size. In the ends of the gel, where the wells are located our groups filled them with our PCR products from our last lab. Moreover, the gel is produced through a combination of agarose molecules that are held together by hydrogen bonds. In order to prepare the gel for the lab, we had to mix agarose powder with 1X TBE buffer in a 250 mL flask. Then, we had to dissolve the powder by heating the solution to its boiling point. We achieved this by simply microwaving the solution on high for approximately one minute. Once heated, we mixed the solution with 15-second intervals in between until the solution appeared dissolved (the solution should be clear almost like water). After, we had the agarose cool to around 60 degrees celsius by diligently swirling the flask in order to prompt the dissipation of heat. While the agarose cooled, we sealed the ends of the gel-casing tray using rubber end caps, and then placed the “comb” also known as gel cassette in the appropriate notch. Next, we added SYBR Safe, a cyanine dye used as a nucleic acid stain that binds to DNA and absorbs blue light and illuminates green light. Once added to the agarose solution, we mixed them together by swirling the flask. Then, we poured the cooled agarose solution into the prepared gel gasting tray which solidified well after 20 minutes Lastly, we removed the end caps from the comb and were left with our ready to go Agarose Gel Electrophoresis lab.
At this point, my forensics class was ready to begin the separation of PCR products by using Gel Electrophoresis. We placed the gel onto the tray or the electrophoresis chamber and covered the gel with 1X TBE electrophoresis buffer. We used the typical M12 EDVOTEX Model which requires a volume up to 400 mL of buffer. However, in many cases people can also use an MG and M12 Model, a newer model that requires approximately 300 mL of buffer, or an M36 Model which requires at least a volume of 1000 mL. Next, we took our PCR products, our isolated and amplified DNA, and injected 25 µL of it using a P200 micropipette into the wells of electrophoresis tank.
Figure 3: Figure 3 demonstrates the steps to prepare the Gel Electrophoresis tank to be utilized in an experiment.
Once the electrophoresis completed, we removed the gel and casting tray from the electrophoresis chamber. Then we slid the gel off the casting onto a tray. By using a transilluminator, we are able to visually see our separation of PCR product results. The DNA illuminated as bright green bands on the dim background. After observing our results, the class photographed them in order to further analyze them. Lastly, we removed and disposed of the gel and cleaned al of our stations and the equipment carefully.
Figure 4: An example of how the products from my PCR experiment transitioned into being used in the Gel Electrophoresis lab. 1) DNA is extracted 2) Isolation and amplification of DNA 3) DNA added to the gel wells 4) Electric current applied to the gel 5) DNA bands are separated by size 6) DNA bands are stained.
While my class analyzed their individual band of DNA, we learned how to differentiate the different genotypes, heterozygous, and homozygous by analyzing their different individual traits. For instance, those with homozygous DNA will have a thicker band while those who have heterozygous DNA will have a relatively thinner, straight, ad more structural line. By comparing the genotype traits with my sample, it was most similar to homozygous traits. Thus, I was able to infer that my genotype is in fact heterozygous. The reason for my genotype being heterozygous means that both my parents, mother, and father, carry two different DNA strands. Due to the process of child making, I obtained one of the two DNA strands from each parent thus creating who and what I look like today.
Figure 5: This is what a final product of Gel Electrophoresis would look like. These are a set of different DNA bands. By looking at the visual, some of the DNA ladders can be seen with similar lines as a neighboring one. This means that they are similar in their DNA structures.
Edvotek. “Module I: Isolation of DNA from Human Cheek Cells.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Edvotek. “Module II: Amplification of the D1S80 locus.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Enns, Nick. “PCR Notes.” Forensics I, 24 Feb. 2020. Pioneer Valley High School.
Enns, Nick. “DNA Isolation Notes.” Forensics I, 30 Jan. 2020. Pioneer Valley High School.
During the lockdown, many museums and festivals have chosen to continue their yearly programs in a digital format. Some of the most famous events in the world of academia, such as the Hay Festival, have become free and available online, creating countless opportunities for individuals from all over the world to come together and learn. When I heard about the Hay Festival digital program, I was extremely excited and delved in and booked dozens of lectures with famous names such as Laura Bates, Gloria Steinam, and Stephen Fry. Although all of them were incredible, the one that stood out to me was “The Drugs that Don’t Work” with Dame Sally Davies.
Sally Davies was the Chief Medical Officer for England as well as the Chief Scientific Adviser at the Department of Health. She started her career as a clinical practitioner and has pioneered research and advancements in many medical fields. The particular lecture I watched was based on her book of the same title, “The Drugs that Don’t Work”. The book is based on antimicrobial resistance, a deadly phenomenon afflicting the world. Antimicrobial resistance, or AMR, is when microorganisms change or mutate (change in the base sequence of the DNA) after being exposed to an antimicrobial drug, such as an antibiotic or anti-malaria. This allows the microorganisms to become immune to the effects of the drugs and “superbugs”, such as MRSA, can develop.
MRSA, or Methicillin Staphylococcus Aureus, is a “superbug” which can cause infections in various areas of the body. And the main issue faced with MRSA is that it is resistant to most common antibiotics which allows it to spread rapidly in hospitals through contact, causing infections ranging from mild skin ones to life-threatening ones such as lung, blood, and surgical wound infections. For a while, it was a major problem in British hospitals, and many surgical patients were advised to bring their own hand towels to use for washing to ensure the towel wouldn’t drape along the floor and potentially have contact with the bacteria. It is estimated that 2% of the population will carry MRSA and it can be transmitted through touching someone with it or an object that a carrier has held. In hospitals now they still test you for MRSA before any operation and you will likely have a swab taken from the skin in your armpit, groin or nose to test for it.
One of the things that were most poignant to me about her lecture was just how often antimicrobials are used in the farming and aquaculture industries. Many farmers use antibiotics to promote growth in plants and reduce the risk of infections. The antibiotics are a cheaper alternative to implementing infection control and hygiene measures so have been used excessively and unnecessarily. Not only this, but they are also used in aquaculture, the breeding, and farming of fish, to prevent and treat bacterial disease. These same antibiotics are used to treat human pathogens as well, so as they are used so frequently in farming industries, it allows them to adapt their DNA coding and become resistant to treatment. This is detrimental to human health and Sally Davies said that every year, more people are now dying of the effects of AMR than cancer.
Are you shocked that you do not have much knowledge on an issue that could prove so deadly to the human race? Dame Sally Davies said that one of the greatest downfalls is the lack of education for the public on AMR. Although some people in the UK may be accustomed to the little “AMR song” they play in general practitioner surgeries, we have nowhere near enough exposure to the true risks of antimicrobial resistance. Sally Davies even went as far as to state that the effects of AMR could reduce the global GDP, gross domestic product, by 3.5% which is a significant loss.
In order to reduce the increase of antimicrobial resistance, there are a few measures that everyone can take. For example, it is imperative that you do not share prescriptions with others as If that person has a viral infection, and you are giving them antibiotics, the medication will not work and will increase the AMR in the microbiome in their gut. Misusing medication is one of the most dangerous things you can do, even if you are not talking about antimicrobial resistance. Furthermore, do not use any leftover prescriptions or self prescribe if you have not sought the advice of a registered doctor. Although you might think that there is no harm in trialing a medication, especially if you are experiencing similar symptoms as before, it is incredibly unwise to do this without going to your doctor first.
This lecture was informative and shed light on a complex issue that may prove very dangerous in our future. Antimicrobial resistance needs to be addressed and it is important that the public is educated on it and how they can reduce their risk.
I learned about PCR in my junior year in my Forensics course. As the day came closer where we conducted a lab using PCR, I came to realize how difficult and complex this lab was going to be. Before learning the concepts of PCR, I had questions such as “what is PCR, and how does that relate to replicating one’s DNA?” “What is PCR used for, and what is its relevance in the world?” Questions like these had me on the edge of my seat, eager to learn more about it.
Polymerase Chain Reaction, or PCR, is a process that allows an individual to replicate many copies of a precise segment of their DNA through a series of calculated procedures. Ever since it was first invented in 1984 by Dr. Kary Mullis, It has become revolutionary in the science of DNA fingerprinting due to its feasibility to amplify DNA. To begin replicating DNA, there needs to be a sample of DNA to use. A DNA sample can be obtained in many ways: blood, semen, feces, hair, saliva, tissue, and cells. For our high school-based experiment, our class used the DNA within our saliva. Moreover, Taq Polymerase is fundamental to the experiment because it helps in the DNA replicating process. Although DNA polymerase is typically utilized in PCR, our class used Taq polymerase because it can withstand much higher temperatures and denaturing conditions. It is also much easier to isolate. Taq DNA Polymerase is a naturally occurring set of proteins that copy a cell’s DNA before dividing itself into two. When the DNA Polymerase finds itself bumping into a primer that is base-paired with a longer piece of DNA, it will attach itself to the end of the primer and then add nucleotides. Because primers are mostly man-made and created within a laboratory, they can have as many nucleotides, molecules consisting of a nucleoside and a phosphate group, as one wishes.
FIGURE 1: A representation of DNA Polymerase function in PCR.
Now the question that still stands is, “What is its relevance in the world?” PCR can be used for a variety of purposes, but it is most frequently used in Forensics analyses and Medical diagnostics. By examining several different STRS from a particular individual, examiners can obtain a unique trait that is unlike anyone else’s, and they will be able to use that information to determine many different factors of that person. For instance, in Forensics, PCR can be used to match a suspect’s DNA to one that was found at a crime scene. Forensic scientists could take the DNA sample found at the crime scene and from suspects of the crime, replicate the DNA samples using the PCR process, and compare and contrast the samples by examining highly polymorphic DNA regions. They then would be able to determine who may have been responsible for the crime. Further, many specialists use the PCR process every day to diagnose individuals with a disease. Through PCR, specialists can examine any inconsistencies or mutations in the replicated DNA sequences, and determine whether or not a person has a particular disease. They can also use it to identify bacteria or viruses.
To begin the experiment, we had to extract our DNA from our saliva. We did this by rinsing our mouths with saline, salt water, and spitting it into a cup. The saline solution helps neutralize the DNA charge and remove proteins that are potentially bound to the DNA. This allowed us to have pure DNA to use for the experiment. Now that we had our pure DNA, we had to isolate the DNA from the human cheek cells. It is important to have freshly isolated DNA because it will provide pristine amplification results compared to much older and degraded pieces of DNA. To begin the isolation process, my classmates and I took our DNA solution and transferred 1.5 mL of DNA solution into a small tube. To transfer our solutions, we used a p1000 micropipette to extract a precise amount of DNA solution from the cup and transferred the amount into the tube. The reason we transferred our solution into a small tube is so that it could fit into a centrifuge. The centrifuge spins the solution at high speeds for approximately two minutes to separate the components of the solution with centripetal force.
After the class completed their first centrifuge stage, they took their tubes containing the potentially separated solution and resuspended the cheek cells in a 140 µL lysis buffer, a solution used to break open cells. This was accomplished by pipetting up and down with a p200 micropipette and by vortexing vigorously. Next, we placed our tubes into a water bath float. We incubated the tubes containing the solutions in a 55 degrees Celsius water bath for 5 minutes. After 5 minutes, we took our tubes and flicked the tube vigorously for about 20 seconds.
We then put the tubes in the bath to incubate again in 99 degrees celsius water for 5 minutes instead. After the 5 minutes, we placed them into the centrifuge once more for 2 minutes at full speed. Lastly, we took our tubes and distributed 80 µl of supernatant using a p20 micropipette into a separate tube. Now that our DNA was successfully isolated, it was time to move onto the amplification stage in the PCR process.
We began the next stage by taking a fresh 0.2 mL PCR tube and added a 20 µL D1S80 primer mix (yellow), 5 µL extracted DNA (red), and a PCR EdvoBead PLUS which provides reagents for approximately 25 PCR reactions. PCR Beads have been optimized for PCR reactions and contain buffer, nucleotides, and Taq DNA Polymerase. Last, we mixed together our solutions. Our final mixture was a beautiful light orange color, and now that we finally had our solution that contains positive control primers, template DNA, and PCR components, we were ready to begin the PCR amplification of our DNA.
Figure 2: A visual representation of the steps to isolate DNA.
There are three major steps in replicating DNA in the PCR process. Step one of the PCR process is denaturation. Breaking open of the cells or denaturing provides a single-stranded template for the subsequent step. To break open the cells, the tube is heated at 94 degrees celsius for 15 seconds. The initial denaturation will start at 94 degrees celsius for 30 seconds instead. Step two in the process is annealing. Also known as the cooling process, annealing allows the reaction to cool at 65 degrees celsius for 30 seconds, so the primers can bind to the complementary sequences on the single-stranded template DNA. The last major step in PCR is an extension, sometimes referred to as elongation. In this process, the reaction is placed in raised temperatures of 72 degrees celsius for 40 seconds. Each step of the process will go through 32 cycles repeating these steps chronologically over a course of 2 hours. Lastly, the solution will sit for its final extension at 72 degrees celsius for 30 seconds. After we had finished amplifying the DNA, we had over a million copies of specific DNA sequences in the palms of our hands. Although this may seem like an enormous amount of DNA, it can hardly be seen in the solution.
Figure 3: Illustration of the three major steps in PCR: denaturation, annealing, and elongation.
Now that we have completed the PCR process, we were able to use our DNA separations for another lab. In the next article, I will explain how I used my separation PCR products in Gel Electrophoresis and was able to determine my genotype.
Edvotek. “Module I: Isolation of DNA from Human Cheek Cells.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Edvotek. “Module II: Amplification of the D1S80 locus.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Enns, Nick. “PCR Notes.” Forensics I, 24 Feb. 2020. Pioneer Valley High School.
Enns, Nick. “DNA Isolation Notes.” Forensics I, 30 Jan. 2020. Pioneer Valley High School.