Abstract

Every person has two kidney organs responsible for filtering and cleaning fluid waste from the blood. Kidney disease occurs when one or both kidneys lose their ability to function properly. Patients are either placed on dialysis or receive kidney organ transplants in order to maintain their lives. We discuss in this brief review what some of the causes of kidney failure are and how to potentially prevent kidney failure. The process of receiving a kidney is also discussed along with the risks involved with such a procedure. While there are many precautionary measures taken before a patient receives a kidney, there have been significant improvements in the lives of patients once they receive a kidney. Survival rates have significantly increased with the advancement of medical procedures and treatments. Nevertheless, there are areas that can be further developed to increase the number of patients eligible for kidney transplants and to prevent deleterious side effects of drugs.

Introduction

A kidney transplant is a surgical procedure in which a diseased kidney is replaced with a healthy kidney from a donor. The kidneys are a pair of organs located on each side of the lower abdomen that function to remove waste and excess fluid from the body by producing urine. Although humans have two kidneys, they can survive with only one functioning kidney. As shown in Figure 1, kidney failure (end stage kidney renal disease) can result if a kidney loses its filtration ability and allows certain wastes and fluids to gather in the body. People that are diagnosed with end stage renal disease must have a kidney transplant or undergo dialysis, where a machine mechanically removes fluid wastes from the body, replacing the function of the diseased kidney(s), in order to survive (Mayo, 2022). Examples of kidney diseases include lupus nephritis and kidney cancer, all requiring a kidney transplant to prolong the survival of the patient for as long as possible (Rodgers, 2022). Patients who are eligible for a kidney transplant surgery have a higher chance of a longer life expectancy and overall better quality of life. A donated kidney can be transplanted from a relative, an unrelated donor, or a deceased donor. Based on the statistics of current surgeries, patients who receive a kidney from a live donor have experienced more benefits than patients who have received a kidney from a deceased donor who has passed (Thongprayoon et al., 2020).

The history of kidney transplants comes with many unsuccessful attempts of experimentation, but has ultimately persevered past these setbacks. The first ever human kidney transplant was performed in 1936 as an allograft (human-to-human). The transplanted kidney left an area of the ureter unattached and because organ rejection was not yet understood, the transplant ultimately failed (Turner, 2018). After improving on the methods of transplantation, the first successful human-to-human kidney transplant took place in 1954 between identical twins (Williams, 2008). Based on prior failed kidney transplant surgeries, the medical team speculated the fact that a kidney transplant with identical twins may be successful. Amazingly, the kidney was able to adjust to the body of the recipient because the organ did not appear “foreign” when transplanted into the body due to the fact that the identical twins shared many similar traits. Both patients survived for nine years without immunosuppressive therapy (to be discussed later) after undergoing this surgery (Dziewanowski et al., 2011). Shortly after, the first successful kidney transplant between unrelated individuals took place in 1962. These monumental results would soon become a breakthrough in medicine offering people a solution to such a chronic disease.

Kidney transplantation has now become an option to increase the rate of survival and life expectancy for kidney disease patients. Although there are advantages to having the procedure performed on patients, there are prerequisites, limitations and risks involved in undergoing such a procedure. Patients are first put on a waiting list as a candidate to receive a kidney from donors, but a shortage of organs available for donation makes it difficult for this process to occur quickly. During this waiting period, patients must be placed on dialysis until they are eligible to receive a kidney organ. Nonetheless, this operation is not fit for everyone. For example, people of older age with a history of cardiovascular conditions do not have the option to have a kidney transplant. Patients may also not be eligible for a kidney transplant if they have a history with drug or alcohol usage, an inability to take post-surgery medications, or no health insurance (Rodgers, 2018). Although there are many advantages to kidney transplantations such as fewer food restrictions and higher energy levels for work or travel, there are some considerations to having the operation done. The risks of a kidney transplant include possible infection, damage, or bleeding in the surrounding organs in the lower abdomen. After the surgery, the patient will be required to take strong medications for the rest of their lifetime to prevent organ rejection by lowering their immune system (Hendry & Robb, 2020). These medications come with many side effects such as a higher risk of infections and diseases like cancer. An example of a significant risk factor is the development of diabetes mellitus, which has been shown to be acquired by almost 70% of patients with a kidney transplant post transplantation (Peev et al., 2014). It is crucial for the patient to consider all of these factors before deciding whether the surgical procedure is the best option.

II. The Importance of Kidney Transplants

Kidney transplant surgeries are becoming more common as the number of kidney failures have dramatically increased over the years (Figure 2). This can be a result of common diseases such as diabetes, high blood pressure, and cardiovascular disease which all increase the likelihood of developing chronic kidney failure. Each single kidney is made up of over one million small filtering units known as nephrons. Any condition, such as those previously listed, that may damage or cause potential harm to the nephrons can result in kidney disease (Digital, 2020). There are approximately 1 in every 3 adults in the world that are diagnosed with a type of diabetes with 1 in every 3 diabetic also developing chronic kidney disease (Walensky, 2021). High levels of blood glucose that are a result of the diagnosis of diabetes damages blood vessels in the kidneys which clusters and limits the kidneys’ abilities to filter waste. Furthermore, hypertension and obesity rates are increasing and have also been causes of kidney failures. Roughly 23% of patients who are diagnosed with kidney disease are also obese, demonstrating the close gap between the two conditions (Tran et al., 2016). Being overweight forces the kidneys to perform extra work to maintain balance by filtering waste and removing excess fluids that may come in addition to the supplemental body fat (Kovesdy et al., 2017).

Although kidney transplantation is the most prominent option for prolonging the lifespan of a patient with a diseased kidney, the procedure is not as simple as it may seem. In the United States, there are over 3,000 new patients added to the kidney organ transplant list each month, making it extremely difficult to find and receive a kidney in a short period of time. There are at least thirteen people who die each day waiting for a kidney, and others are put on dialysis to rely on the machine until they find a matching donor (UNOS, 2016). There were more than 20,000 donor organs available for transplant in the United States in 2016, but more than 100,000 patients were on the waiting list to receive a transplant. Given the stringent requirements to qualify a patient to receive a kidney, not all donor kidneys can be transplanted thus increasing the number of donor organs at about 8% per year (USRD, 2020). As shown in Figure 2, the annual need for kidney transplants is significantly greater than any other organ transplant. Recent statistics show that there has been a record in both the number of total kidney transplant surgeries and also the number of lives saved in 2021. The number of living donors had substantially decreased in 2020 due to the outbreak of the COVID pandemic and although the number of living donors was slightly higher in 2021, the number was still significantly less as compared to previous years. There were more than 6,000 organ transplants performed in 2021, which was approximately 14% higher than in 2020. Note that from 2010 to 2020 the total number of transplants performed was about 7,000 (Figure 2). Because of the result of the recent pandemic, more kidneys have been donated from deceased donors in order to maintain the consistency of survival rates (UNOS, 2022).

Kidney transplants have significantly shown beneficial outcomes and significant improvements in the lives of patients. Because of how common kidney disease has become, the need for transplants only continues to grow. A living donor kidney survives on average anywhere from 12-20 years, whereas a diseased kidney organ survives around 8-12 years (BIDIC, 2022). Short term rejections (within 3 months of surgery) occured in about 17.3% of patients 10 years ago, but have significantly decreased to around 4% as of current statistics (Lee et al., 2021). Survival expectancy rates of kidney transplants ranged around 75% around 10 years ago (Walensky, 2021), but has risen up to around 94-97% as of 2022 (BIDIC, 2022). This is mainly due to the increasing number of successful surgeries every year. The improvement of national transplant statistics comes from the benefit of developing medicine and research.

The most common age group to develop both kidney diseases and receive transplants is between 50-64. Although more than 45,000 individuals in this age group were on waiting lists for a kidney in 2021, current improvements in medicine and transplant surgery have nevertheless given these people the potential to live longer with less complications (Elflein, 2021). Unfortunately, there are many food restrictions and physical limitations that all kidney disease patients endure while living on dialysis. Having a kidney transplant relieves these individuals from the restrictions put on their daily life, which thus allows them to live healthier and more comfortably (Lee et al., 2021). Kidney transplants help to save the lives of over 20,000 individuals (i.e. actual living recipients of kidneys and patients who match for a transplant) every year (UNOS, 2022).

II. The Types of Kidney Transplant Rejections

The immune system protects the body by identifying cell markers and recognizing the cells as being healthy or foreign. Lacking these markers sometimes prompts immune cells to attack the cells to destroy them. Individuals have a unique and different collection of cells in their immune system. Foreign cells are removed from the body in order to provide protection from possible infections, illnesses, or diseases (Waltzer, 2019). The most common examples of foreign invaders include bacteria, viruses, parasites, and fungi that the immune system works to remove from the body. Foreign cells can also be healthy cells but from different individuals. Receiving a kidney transplant is seen as foreign by the immune system because the entrance of new antigens (cell markers on donor cells) prompt an immune response in the recipient. The immune system then works to reject the kidney by removing cells and antibodies that may cause potential harm. Transplant recipients therefore take immunosuppressive (anti-rejection) drugs to prevent damage to the kidney. It is extremely crucial to take these medications at around the same time every day in order to avoid kidney rejection (Hendry & Robb, 2020).

One of the three main types of kidney rejection is hyperacute rejection, which occurs within the first couple minutes of the surgery. This occurrence is irreversible and immediately results in the loss of the transplanted kidney and must be removed from the body promptly. Hyperacute rejection is diagnosed when the patient experiences immediate symptoms after surgery. Diagnosing this type of rejection is not too difficult, and even a simple urine test can diagnose or predict a rejection. Hyperacute rejection is caused by already existing antibodies that recognize the foreign kidney and cells from the donor and act immediately to reject this outlier. Fortunately, hyperacute rejection has now become extremely rare due to a special test called a crossmatch, which is completed before the transplant takes place. Instead of having the patient undergo the entire procedure leading to the surgery just to find out there is no match present, there are now prerequisites that are done which can determine superficially that there will be no severe reaction with the kidney. The tissue cross match is used to check how the immune system of the recipient may act to the placement of the new donated kidney (KHSCN, 2019). A positive result of this crossmatch indicates that the antibodies of the recipient will attack those of the donor, thus making the kidney unsuitable for the transplant process. A negative result of a crossmatch implies that there is no reaction between the antibodies of the recipient and the donor, thus making the kidney suitable for the transplant (Smoot, 2021).

Acute rejection is the second and most common type of rejection that is prone to happen anytime, but most frequently occurs around the first three months post transplant surgery. About 15-25% of recipients who experience this type of rejection within their first three months of surgery (Czech, 2022). This is usually a significant cause of allograft dysfunction, which causes an increase in serum creatinine level and correlates to a decrease in glomerular filtration rates (Goldenberg et al., 2016). Symptoms that prompt the doctor to perform tests to further observe this condition include an elevated temperature, tenderness or pain over the kidney transplant, a rapid increase in blood pressure or body weight, or a sudden decrease in urine output (Czech, 2022). Although this rejection is possible to be reversed in most cases when treated early, it may also result in a negative impact on the long-term graft survival overall. There are many instances where failed kidneys are not able to regain their functions even with the use of maximal antirejection therapy (Goldenberg et al., 2016).

Lastly, chronic rejections occur when any of the alternate rejection processes never completely reconcile and they continue to occur over long periods of time. This type of rejection can also be a result of when immunosuppressants stop regulation in the immune system due to their side effects. Chronic rejections typically occur in the first six months after surgery. Transplanted kidneys that have chronic rejection are found to have developed scarring of the tissue and damage to the blood vessels (Punch, 2022). Chronic rejection most commonly occurs among patients that have not received a sufficient amount of immunosuppression or medication following the transplant surgery (Henry & Robb, 2020). Symptoms that most likely indicate the presence of a chronic rejection in a kidney transplant are similar to those for the acute rejection, but come at later stages because of the different time periods the varying types of rejections occur at. Because there are no early symptoms for chronic rejections, this type of rejection is also commonly diagnosed by changes in laboratory tests or possible kidney biopsies. Although there is no medication to reverse chronic rejection, the kidney is generally able to last for months after the diagnosis is made by the doctor (Waltzer, 2019).

IV. How to Have a Successful Kidney Transplant Surgery

As previously mentioned, there are many different symptoms that may indicate possible kidney failure and prompt a doctor to run a diagnostic test on the patient. However in order to prevent the occurrence of these symptoms of failure as best as possible, it is crucial that the kidney from the donor and the patient are the right match. The three main blood tests that are used to check this are blood typing, tissue typing, and cross matching (Center, 2022). Blood typing is the first test done to determine if the kidney donor and the recipient have compatible blood types, indicating whether or not the kidney can be potentially transplanted. The next test after passing the initial blood typing exam is the tissue typing test, which is accomplished through routine testing and short turn around testing (STAT). Blood is drawn from the inside of the cheeks to determine if the potential donor and the recipient have a compatible HLA or tissue type (Center, 2022). As briefly mentioned above, cross matching is the ultimate step before the kidney officially becomes approved to be a donor for the recipient. Samples of blood are taken from both the donor and the recipient, then the blood cells of the donor are mixed with the serum of the recipient. A negative crossmatch result indicates that there is no presence of antibodies attacking the donor while a positive result shows that the kidney is not suitable for donation (Smoot, 2021).

However in order to prevent rejection of the kidney after the transplant surgery is completed, doctors prescribe immunosuppressants to help the newly transferred kidney survive in the new recipient’s body. The immunosuppressants must be taken every day for the rest of the recipients’ lives to prevent harmful interactions between foreign antibodies that may cause serious damage. The dosage of the medication is adjusted because of the many side effects that also commonly occur among patients as a result of the immunosuppressants. The most common side effects are constant migraines, hair loss, high blood pressure, or nausea, which can all become a consequence of taking the medication every day (Kalluri, 2012). Prednisone, tacrolimus, cyclosporine, mycophenolate mofetil, imuran, rapamune are among the most commonly prescribed immunosuppressants. Because there is such a wide variety, most patients take a combination of around three of these listed drugs (Trotta, 2021). About 6 months to a year after the kidney transplant surgery is administered, the dosage of the immunosuppressants are usually lowered in order to prevent the presence of the common side effects.

Conclusion

Despite all the information that has been gathered on this topic of kidney transplants, there are still a few questions that have been left unclarified. For example, why do women have a slower overall progression to end stage renal kidney disease than men? There are always biological and systematic factors to consider, but it is unclear whether the rates of progression are due to different accesses to care or possibly the true differences in the severity of their diseases. Furthermore, why are there always more women donating their kidneys than men? This question also lacks a clear answer and there are so many unique possibilities ranging from risks of kidney disease, personal requests, cultural factors, and much more. There is always a vast difference between genders regarding their access to care for their conditions, but there is not necessarily enough data obtained to recognize the range of these differences.

Chronic kidney disease and end stage renal disease are both extremely common, but medicine has shown progress in decreasing the side effects as a result of these procedures. However, there is still plenty of work to be researched and done. Improvement on the medical and treatment front such as ongoing clinical trials for different cell therapies to minimize the side effects would be an example of a beneficial approach to take the next step in the research. Due to the deleterious side effects of immunosuppressant drugs, more focus has been drawn to immunotherapeutics and cellular therapies to use the body’s own immune system to suppress activated immune cells that target the transplanted organ. There are ongoing clinical trials as well as numerous research projects all aiming to develop breakthrough cellular therapies that are more natural to the body and potentially safer options than drugs.

Works Cited

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About the author

Emily Kim

Emily is currently a junior in the Academy for Medical Science and Technology at the Bergen County Academies High School. She is also a competitive nationally ranked swimmer and is looking to pursue medicine in the future.

The post Kidney Diseases and Transplants: Diagnosis, Procedure, and Prolongation appeared first on Exploratio Journal.

Abstract

From past times to recent times antibiotic resistance has emerged as a global threat. Antibiotic resistance has mainly affected the progress in health care, food industries and ultimately life expectancy. Almost all regions of the world are suffering from these antibiotic resistant diseases. This is mainly due to the movement of people, animals and goods across borders and countries. The emergence of different species of bacterium like Staphylococcus aureus, Enterococcus faecium, Streptococcus pyogenes, Staphylococcus pneumonia and more by adapting various defense mechanisms, has lead to the development of resistance towards antibiotics like Penicillin and Methicillin. This resistance is mainly caused by the presence of two important genes called mec-A that codes the PBP2a protein. mec-A and PBP2a are known to confer resistance, but the mechanism of resistance remains unclear. Further study of the mechanism has the potential to develop a new generation of antibiotics. Here, we have investigated the structural biology basis of PBP2a antibiotic resistance and the contribution of the genetic background of resistant S. aureus strains. It has been found that PBP2a confers resistance through its Ser403 residue in its active site, but that PBP2a is not sufficient to drive resistance. Genetic studies have identified an additional gene responsible for conferring PBP2a-based resistance, that is the bla gene that codes a beta lactamase. The presence of bla genes along with mec-A is important for effective activity of the PBP2a protein, as the presence of mec-A alone showed insufficient activity of the PBP2a protein. Western blot analysis of PBP2a expression in different S. aureus strains determined, that the concentration of PBP2a protein was high in cells containing plasmids that carried both mec-A and bla genes and low concentration of PBP2a was found in cells containing plasmids carrying only the mec-A gene.

Introduction

Antibiotic resistance is a property where a bacteria becomes resistant to antibiotics that are designed to kill them. Bacteria can escape inhibitory effects of drugs by acquiring certain mechanisms of resistance. There are several problems that are caused due to the persistent increase in the antibacterial resistance in bacteria. Antibiotic resistance occurs naturally by random mutations (Julin Davis and Dorothy Davis, 2010) through the process of natural selection and also can occur by selective pressure on the bacterial population (Alfredo Tello, et al. 2012). If a resistant gene is generated, it can be transferred from one strain to another by exchange of plasmids through horizontal gene transfer (HGT). During this process of acquiring resistance the antibiotics act as environmental pressure, due to which bacteria undergo certain mutations for its survival. When such strains proliferate, they produce progeny that are resistant towards various antibiotics by evolving certain resistance mechanisms.

Over usage of antibiotics, usage of broad spectrum antibiotics, incorrect diagnostics, unwanted prescription, wrong usage of antibiotics by patients and use of antibiotics in cattle feed for enhancing early growth, are mainly responsible for antibiotic resistance. It was discovered that the lexA gene is mostly responsible for these mutations (Charlie Ye Mo, 2016). A bacteria called Staphylococcus aureus is one of the major resistant pathogens, which is responsible for causing various diseases. Later on it became known as MRSA that was first detected in Britain in 1961, which is resistant to the methicillin antibiotic. Later in 1991, 4% of fatal cases of infections were recorded and rose to 37% by 1999 in the UK. In the USA 50% of the Staphylococcus aureus were resistant to tetracycline, penicillin, methicillin and erythromycin. Vancomycin used to be an effective drug at that period for Staphylococcus aureus but in the late 1990’s intermediate strains were detected with moderate resistance to Vancomycin at 4 μg/ml, which was named Vancomycin intermediate Staphylococcus aureus. In Japan in 1996 the first case was identified with a strain which is resistant to Vamcomycin at >16 μg/ml and the second case was found in the USA in 2002 (Susana Gardete and Alexander Tomasz, 2014 july).

In the late 1990’s new antibiotics like oxazolidinone and linezolid were found to be effective against MRSA. In 2003 Staphylococcus aureus resistance to linezolid was observed. CA-MRSA, community acquired MRSA, is the most common antimicrobial drug resistant pathogen found in US hospitals and became part of Endemic infections at that time. Along with MRSA there are other bacterial strains that are found to have resistance including Enterococcus faecium (penicillin resistant), in 1983, (vancomycin resistant) Enterococcus in 1987 and linezolid resistant Enterococcus in the late 1990’s. However group A Streptococcus pyogenes remained sensitive to penicillin. Later penicillin resistant Staphylococcus pneumonia emerged worldwide. The main gene responsible for beta lactamase and penicillin resistance is mec-A which encodes PBP2a proteins.

There are several problems caused by increasing antibiotic resistance that have become a major threat in various sectors including health, food, security, clinical sectors, agricultural sectors, It is most commonly prevalent among frontline workers like military officers, hospital staff and patients. It can affect anyone of any age, gender and any country. It occurs naturally, but misuse of antibiotics in humans and animals accelerate this process. Due to antibiotic resistance, the effectiveness of antibiotics is decreasing. As a result, it is becoming difficult to treat various diseases including pneumonia, tuberculosis, gonorrhea and salmonellosis.It leads to higher medical costs, longer hospital stays and increased mortality. Plasmids are one of the major important vectors which are responsible for spreading resistance among the strains in soil in agroecosystems. PBR3222 is one such resistant plasmid which carries genes for tetracycline and ampicillin resistance. HGT is one of the most common mechanisms by which bacteria either acquire or exchange resistance from other bacteria. Mobile genetic elements (MGE) like integrons (which are gene cassettes) usually carry antibiotic resistant genes to bacterial plasmids and transposons. Most of this resistance is due to the presence of certain proteins and genes, mainly the mecA gene which was originated from coagulase negative Staphylococci and is associated with a mobile genetic element called Staphylococcal chromosomal cassette mec (secmec) and integrates in the Staphylococcal chromosome. mecA encodes for the PBP2a protein, which is a transpeptidase and has a low affinity for beta lactam antibiotics like methicillin and tetracycline. PBP2a being a transpeptidase is involved in the continuous synthesis of the cell wall during antibiotic attack. PBP2d protein along with beta lactamases are mainly responsible for the broad spectrum resistance in MRSA.

Discussion

The genes mec-A for the PBP2a protein and bla gene for beta lactamases are mainly responsible for resistance in bacteria like MRSA. To know the mechanism behind the resistance and to study the location and properties of the active site, the study of crystalline structures of PBP2a is very important (Lim and colleagues, 2002). For the crystallization process, cells were grown on m9 medium to which 50 g of protein solution containing thr, leu, met, selenomethionine were added per liter of culture. After 15 minutes, protein expression was induced and the colonies were subjected to further purification by hydrophobic affinity chromatography and gel filtration exchange chromatography by adding 0.1% thiodiglycol. Cells were lysed and native Staphylococcus aureus (Sau) PBP2a was extracted as a soluble protein by a purification process using a hydroxyapatite column.

Further purification was done by gel filtration chromatography on a sepharyle 1000 column apparatus, which is equilibrated with 5 mM NaHCO3. Extraction of PBP2a was carried out using a hanging drop vapour diffusion method. Then, exposure of PBP2a with chemical compounds like NaCl, PEG and HEPES at pH 7 led to the formation of crystals and the crystals were extracted by a cryopreservation technique in which the N terminal anchor of PBP2a was replaced with a methionine and was observed under electrospray mass spectrometry. When beta lactams are introduced, they inhibit beta lactam sensitive PBP’s. Strains containing PBP2a have low affinity for beta lactams, conferring antibiotic resistance by the continuous synthesis of the cell wall, which prevents the cell from lysis. Beta lactam antibiotics like penicillin and methicillin are substrate analogs of PBPs and catalyze the process of cell wall lysis and eventually lead to death. However,acquisition of mec-A genes by MRSA confers resistance. The mec-A gene is highly conserved among MRSA isolates with > 90% sequence identity and encodes the PBP2a protein. This PBP2a is resistant to beta lactam and does not have any sensitive analogs or homologs. It has a large molecular mass of 78 kD and belongs to class B PBPs which usually have broad spectrum for methicillin and other beta lactam antibiotics. This study on the crystal structure of PBP2a reveals the structural features responsible for its beta lactam resistance and provides important insights for the design of novel antibiotics against MRSA. Sau PBP2a has a transmembrane anchor which can be removed for the studying of beta lactam binding kinetics without affecting the beta lactam activity. The soluble derivative residues of PBP2a were determined by MAD method (Mean Absolute Deviation) using selenomethionine substituted protein. The extraction of this molecule has revealed structural conformations of the PBP2a protein and the location of active sites present within the non penicillin binding domain in the PBP2a. The active site along with the serine 403 at the N terminal helix of the alpha2 fold of Sau PBP2a along with a conserved oxidation hole containing serine 403 and Thr 600 as a nitrogenous backbone.During the acylation of beta lactam, the serine active site is mostly responsible for the process of inducing resistance towards the antibiotic. However, continuous acylation of serine molecules leads to dehydration. And may result in decreasing the activity of the active site, resulting in cell lysis and death. In order to make this process continuous, the serine 403 associated with the N terminal end of lys 406, present in beta lactam carboxylate enzyme. This forms hydrogen bonds, thus helping the serine present in the active site undergo deacylation, to further continue the process of cell wall protection by nucleophilic attack during the resistance mechanism.

This serine-lysine bond promotes the PBP2a conformational change, thus providing unfavourable conditions for the antibiotic to attack the complex. The low affinity binding towards the beta lactam induces slow acylation, leading to a rate limiting step responsible for the beta lactam resistance in MRSA. In normal methicillin sensitive PBPs, higher acylation rates are one of the major reasons for the antibiotic sensitivity and lead to cell death.

Location and function of the mec-A gene

The mec-A gene is located on a mobile chromosomal cassette called secmec. In this paper (Katayama, et al. 2003), use of naive cells (strains without mec-A genes) and experienced hosts (methicillin susceptible strains in which mec-A was excised) was mainly done to demonstrate the expression of mec-A genes and PBP2a protein activity towards the beta lactam resistance. A plasmid called pYK-20 was used as a carrier for the mec-A gene and was introduced into experienced and naive strains. The excision of this mec-A gene from the methicillin resistant strain was done by CC5-1, CC5-2 restriction enzymes and were introduced into the different vector plasmids like pYK-644, pYK-20, Hind 3, Coln plasmid, pNR-5542 and pCN-2278 and some of the plasmids were introduced with both mec-A and bla genes. These plasmids were amplified using PCR and these plasmids were introduced into naive hosts and experienced hosts through the process of micropropagation and growth curves were measured to screen for the transformants containing the plasmid vectors. These cells were grown on nafcillin containing media to test for antibiotic resistance. Naive cells containing the plasmids that carried only mec genes showed weak growth of 2.5% and the naive cells containing plasmids which are introduced with both mec-A and bla genes showed resistance to nafcillin. Transformed experienced strains showed normal resistance as that of parents containing mec-A genes. Taken together, these experiments suggest resistance is driven by transformants containing the plasmids with both mec-A genes and bla genes, indicating that combined activity of mec-A and bla are required for resistance. As, the experienced strains showed resistance and the naive cells did not express due to the host barrier. Tranformants were then tested to analyse the expression of the PBP2a protein by western blot. After the electrophoresis separation the separated proteins are loaded onto the nitrocellulose membrane containing monoclonal anti PBP2a antibodies. The desired PBP2a proteins bind with the antibodies. After the washing the unwanted proteins are removed. The secondary labelled antibodies are introduced and bind to the desired protein-antibody complex and develop colour and the PBP2a are visualised as thick bands depending upon the concentration under autoradiography. It was observed that Colnex with pYK 20 showed the maximum PBP2a production indicating high resistance, containing a plasmid carrying both mec and bla genes.

Conclusion

Both PBP2a and beta Lactamases are important for antibiotic resistance, and the structural confirmation of Ser403 present in the active site of the PBP2a protein is most important to confer proper and effective resistance towards the antibiotics. As in the studies we observed that the cells with plasmids carrying both mec and bla genes showed more resistance than cells with plasmids containing a single gene. What this suggests is although PBP2a is required for antibiotic resistance, it is not sufficient to induce resistance. In fact, additional genes such as bla are also required to induce resistance. More studies and research on new antibiotics targeting Ser403 and the active site of PBP2a, possibly designing new antibiotics towards PBP2a and designing antibiotics that target bla gene products would be effective. Combinatorial antibiotic treatment targeting both PBP2a and bla may help in effective treatment towards these antibiotic resistant diseases.

References

Julin Davis and Dorothy Davis, September 2010. Origins and Evolution of Antibiotic Resistance.Microbiol Mol Biol Rev. V 74(3): 417–433.

Alfredo Tello, Brain Austin and Trevor C Telfer, Aug 2012. Selective pressure of antibiotic pollution on bacteria of importance to public health. PubMed.

Charlie Ye Mo, January 2016. Make Antibiotics Great Again: Combating Drug Resistance By Targeting Lexa, A Regulator Of Bacterial Evolution. Researchgate.

Susana Gardete and Alexander Tomasz, Jul 1 2014. Mechanisms of vancomycin resistance in Staphylococcus aureus. The journal of clinical investigation, V 124(7): 2836–2840.

Daniel Lim and Natalie C. J. Strynadka, 21 October 2002. Structural basis for the -lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nature structural biology, Volume 9: 870-876.

Yuki Katayama, Hong-Zhong Zhang, Dong Hong, and Henry F. Chambers, 23 June 2003. Jumping the Barrier to -Lactam Resistance in Staphylococcus aureus. JOURNAL OF BACTERIOLOGY, p. 5465–5472.

About the author

Harshini Balaga

The post Antibiotic resistance in Staphylococcus aureus and its adverse effects on global health appeared first on Exploratio Journal.

Abstract

Heart attacks, or Myocardial Infarctions (MI), lead to death of tissue due to lack of blood supply to the portion of the organ. Resulting scar tissue does not contract or function as well as healthy muscle tissue. On the other hand, stem cells have shown propensity to be guided into becoming specific cells that can be used to regenerate and repair diseased or damaged tissues in people. This paper will explore the latest research that supports using induced pluripotent stem cells for tissue reparation in cardiovascular disease.

Introduction

Over the past few decades, stem cell therapies have evolved considerably and one of their many potential applications could be to repair the scarring caused by myocardial infarctions. Myocardial Infarction (MI), which is a reduction or blockage of blood flow in the coronary arteries, commonly referred to as heart attack, is one of the leading causes of death in the United States with 805,000 people experiencing one every year (CDC). Unfortunately, out of those 805,000, 12% will die (CDC). Following MI, the inadequate blood flow to the infarcted tissue causes a severe reduction of oxygen and nutrients, leading to cardiomyocyte necrosis (reduced contractility), and therefore compromised heart function. MI does not traditionally have any treatment since once the tissue has necrotized, it can not regain its function. MI’s can only be managed with preventative measures taken to inhibit another incident. Medicines like aspirin and other anti-clotting drugs are used to keep clots from forming and causing another MI (CDC). ACE inhibitors reduce the strain on the heart by lowering blood pressure and this helps to not weaken the damaged tissues any further (NIH). Similarly, Betablockers also reduce the strain on the organ by blocking the release of stress hormones like noradrenaline and adrenaline to keep heart rate constant (NIH). All of these, however, only reduce the risk of a recurrence and do not regenerate the dead tissue, whereas a different form of therapy for the damaged tissue could bring about a brighter prospect. Stem cells can regenerate tissues suitable to one’s own body without having to use a transplant. This makes it less risky when it comes to a patient’s body rejecting the cells. Some varieties are also easily accessible, usable, and effective in their respective needs.

Out of the many varieties of stem cells, induced pluripotent stem cells, are some of the most promising to study. Induced pluripotent stem cells (iPS cells) are derived from somatic cells that are reprogrammed into iPS cells. These cells can then be made to differentiate into whatever tissue cell is needed (Shi et al, 2016). They are also important to observe because of their accessibility and high turnover rate (Krzysztof et al, 2018). In this review, we will focus on two types of stem cells: induced pluripotent stem cells, and their abilities in tissue regeneration in regards to therapies to treat the infarcted myocardium (Yoshida et al, 2017).

Induced Pluripotent Stem cells

Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells that are reprogrammed into a pluripotent state. These cells are adults and unipotent, meaning they are capable of regenerating only their own specific tissue type (Tweedell, 2017). For example, an adult somatic cell in the skin could only generate skin cells. When these cells are reprogrammed into iPSCs, they become pluripotent, and are able to differentiate into any type of tissue with appropriate differentiation factors (Tweedell, 2017).

The use of iPSCs for regenerative medicine bears significant advantages. In fact, the somatic cells generally used for reprogramming are highly accessible and they are already part of the body of the person who needs them. Therefore, there is no risk of rejection when they are implanted for regenerating damaged tissues or organs (Arjmand et al, 2017). One further advantage is that they are not controversial like embryonic stem cells that are isolated from embryos while having similar properties. Generating the iPSCs is completed by taking any healthy adult somatic cells from the body and reverse engineering them into a pluripotent state where they can then differentiate into whatever cell type is needed. How this occurs is that first, the cells organize spatially and then divide into three areas. The middle section, differentiates into the middle portion of the three’s lineage and this activates certain genes.

Cardiovascular Regeneration

As mentioned above, one of the capabilities of iPSCs is tissue regeneration, which is paramount for cardiovascular tissue regeneration. The basic process for cardiovascular tissue engineering consists in isolating somatic cells of the patient or from healthy donors, which are then reprogrammed to iPSCs. Next, the obtained iPSCs are differentiated into the specific cell type that is needed (such as cardiomyocytes, cardiac fibroblasts, or endothelial cells). The differentiated cells must be cultured in the lab to grow, and during this process they can be stimulated with chemical or physical cues to mimic the mechanical properties of the beating heart. The last step is to inject or implant the cells into the patient.

Cardiovascular tissue engineering has shown promising results in vitro and in preclinical in vivo studies. Several groups have used small animal models, including mice and rats model of myocardial infarction to assess the ability of repairing the damaged heart tissue with iPSCs-derived cardiomyocytes. An example of implantation of cardiac engineered tissues in a small animal model is provided by (Tompkins et. al. 2018), that used 3D bioprinted iPSC-derived cardiomyocytes, fibroblasts and endothelial cells to produce 3D patches that were implanted in n=6 infarcted rats.

Additionally, vivo models further demonstrate that this path of study is incredibly promising. The work of Tompkins et. al. describes small animal models where iPSCs are implanted. This article demonstrates how this work is viable in live models as they tested various species of small animals to prove efficacy. Moreover, the same study considered large animal studies and deduced that they too have promising results. More specifically, in swine models, which are known to have extremely similar cardiac structure to that of humans, these studies further the thought that using iPSCs to repair tissues is a viable solution. Kawamura et al. placed a sheet of dermal fibroblast-derived hiPSC-CMs over the infarcted area in an ischemic swine model, which produced improved cardiac performance, angiogenesis (increased number of blood vessels in the infarct), and an attenuated LV remodeling 8-weeks post implantation.

While in the lab, stimuli of stretching and current are used to help the cells mature faster and grow more resilient. This is one place of research that is continuing to challenge researchers, since they do not have years to culture mature cells and there is risk with implanting immature cells regarding their ability to adapt to the heart’s environment. However, it can and has been done, as explained above, which has drawn tremendous attention to this field of pursuit. Moreover, cardiovascular regeneration is one of the newest technologies in repairing damaged tissues in the heart. This breakthrough has made it possible to just regrow healthy and functional tissue instead of needing a transplant since it is already known that once tissue is dead from a myocardial infarction, there is no way to salvage it. As the MI damages the tissue, it makes it impossible for the original tissue to be functional, so inputting fresh, cultivated tissues open up new possibilities in life for the patient after their MI episode.

Drug Screening

During the process of drug screening, various drugs are tested on the cardiac engineered tissues to gauge safety and efficacy of the tested molecules and drugs. One of the commonly tested side effects of newly developed drugs are for drug induced arrhythmias. By testing in-vitro with iPSCs outside of a patient’s body, it is not only more convenient to do so but also safer so as to not involve a live subject (Smith et. al. 2017). Various types of cell models are used, ranging from flat, 2D monolayers to more complex 3D tissues, organ-on-a-chip models show a wide range of functionality. Each of these model types show a range from the least to most complex levels of organization in order to understand how drugs can affect the cardiomyocytes on a basic to fully vascularized level (Smith et. al. 2017 Fig. 1). This is one place of development in the field of iPSCs that would be of great benefit to the scientific community and to the general population as well. If drugs can be screened and tested within a lab without having to use in vivo models until much later in the process, it can be much more ethical and more varieties of medicines that may or may not be viewed as viable could potentially be trialed in this way due to the reduced ethics concerns.

One example of a clinical trial is in the research of Blinova et. al. which shows a personalized drug screen model that highlights how iPSCs derived from 22 healthy subjects can be grown and tested within a dish. Safety and efficacy of two drugs, dofetilide and moxifloxacin (hERG‐blocking and QT prolonging), were tested on iPSCs isolated from the peripheral blood mononuclear cells and differentiated in cardiomyocytes. There were no drug induced arrhythmia-like events observed at the studied drug concentration rate. In vivo model of testing that highlights how tissue can be grown and tested within a dish. In this trial, the researchers tested and analyzed for arrhythmias in the iPSCs. This demonstrates how various environments of the heart can be simulated in the lab and that it is necessary to do so (Blinova et. al. 2019)

Various different types of trials can be used to screen for arrhythmias in a drug screening. One method researchers historically and commonly use is the analysis of hERG channel response which is the standard procedure for in vitro preclinical trials of drug screening. While this is a method commonly used, it is not as accurate as could be desired since false positive results are frequent occurrences (Smith et. al. 2017). This is why iPSC-CMs are making headway in the field of drug screening since they offer a more accurate option. There are various tests researchers can run with in vitro models of iPSCs to represent the function of the heart more fully and effectively. Out of the multitude of options researchers now have with iPSC-CMs as an option, an example presented in the above research is that researchers measure cell contraction to observe the cardiomyocytes’ contractile function (Smith et. al. 2017).

Conclusion

After an episode of myocardial infarction, heart tissue is damaged irreversibly and the prognosis only entails either drug therapeutics or organ transplant. Cardiovascular regeneration is one of the newest technologies in regards to repairing damaged tissues in the heart. With reprogrammed iPSCs , the patient is able to have their healthy cells cultured in a lab and remediate the scarred tissue resulting from an MI episode. Furthermore, progress has also been made in labs to accommodate the new research and to screen drugs to ensure their safety with the cultured tissues when implanted in a person. All together, these breakthroughs have made it possible to regrow healthy and functioning tissue and using iPSCs could make this possibility a reality.

References

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Blinova K, Schocken D, Patel D, Daluwatte C, Vicente J, Wu JC, Strauss DG. Clinical Trial in a Dish: Personalized Stem Cell-Derived Cardiomyocyte Assay Compared With Clinical Trial Results for Two QT-Prolonging Drugs. Clin Transl Sci. 2019 Nov;12(6):687-697.

CDC, Heart Disease in the United States, 2020 Sept.

Csöbönyeiová M, Polák Š, Danišovič L. Perspectives of induced pluripotent stem cells for cardiovascular system regeneration. Exp Biol Med (Maywood). 2015 May;240(5):549-56.

Hoang P, Wang J, Conklin BR, Healy KE, Ma Z. Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells. Nat Protoc. 2018 Apr;13(4):723-737.

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Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi T, Kawamura T, Kuratani T, Daimon T, Shimizu T, Okano T, Sawa Y. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation. 2012 Sep 11;126(11 Suppl 1):S29-37.

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About the author

Rishya Gutti

Rishya is a junior at Neuqua Valley High School. She is interested in biological sciences and is an aspiring medical student. Research programs like RISE (Research, Inquiry Skills & Experimentation) have equipped Rishya with necessary skills to conduct independent research. She is a third degree black belt in Taekwondo and has won several national titles in her age group. Rishya enjoys volunteering her time to teach mathematics to younger students and to promote mental health awareness through a non-profit organization. In her free time, you will find her reading, working out, or watching her favorite tv shows.

The post Using induced pluripotent stem cells for tissue regeneration in cardiovascular diseases appeared first on Exploratio Journal.

Abstract

While the bone can naturally regenerate itself, in larger bone defects, oftentimes a biomaterial must serve as a scaffold to hold the structure together while the bone regenerates. Calcium phosphates, calcium sulfates, and bioactive glass are common examples of biomaterials that can be used to repair or regenerate the lost or damaged bone. Depending on the situation, the biomaterial may need to be modified to better suit its purpose. This modification can occur solely on the surface (surface modifications) or change the entire material itself (bulk modifications). This paper introduces a few different approaches that have been investigated to improve the biomaterials properties for making them more amenable for bone tissue regeneration.

Introduction

More than 20 million patients worldwide are affected by the loss of bone tissue caused by trauma or disease annually (Iaquinta et al, 2019). The human body can naturally regenerate bone tissue (Dimitriou et al, 2011); however, when such regeneration is needed at a larger scale, surgeons can use a multitude of strategies to address the issue: growth factors such as bone morphogenetic proteins (BMPs), mesenchymal stem cells (MSCs), gene therapy, and biomaterials (Dimitriou et al, 2011). Biomaterials as scaffolds provide a substrate for cell proliferation and adhesion while regulating the cell activity and function–all important factors to induce bone regeneration (Gao et al, 2017). Cell proliferation is the division of cells, a mechanism to induce rapid cell and thus tissue growth. Biomaterials induce cell proliferation through promoting growth factors, proteins that stimulate cell division (Gao et al, 2017). Cell adhesion is organically between cells and cells or cells and the extracellular matrix (ECM), allowing communication and mechanical force to be transferred between the two bodies without separation; in the context of biomaterials, this is important for communication and the clinical performance of the bone, and a cell’s adhesion abilities are usually referred as its biocompatibility (Gao et al, 2017).

Currently, there is no possible way to synthetically produce an entire bone, so biomaterials must be utilized to modify, augment or treat current bone structures (Dimitriou et al, 2011). The biomaterials most commonly used for promoting bone repair include calcium-phosphate ceramics, biopolymers, bioactive glass, calcium phosphates (such as hydroxyapatite),calcium sulphates (such as -tricalcium phosphate), and composite biomaterials (Trimeche, 2017). In developing new biomaterials and strategies to effectively promote bone regeneration, scientists have to meet specific requirements. Function-wise, biomaterials must be osteoinductive (recruitment and proliferation/differentiation of progenitor cells), osteoconductive (bone growth on the biomaterials), and able to perform osseointegration (integration into surrounding bone) (Stevens, 2008). In addition, biomaterials must meet biocompatibility, bioactivity, and controllable biodegradability standards (Gao et al, 2017). 

The porous structure of the biomaterial can affect the type, structure, and function of the regenerated tissue (Gao et al, 2017). As the pore allows for cell movement and oxygen flow, depending on the porosity, cells will be restricted or allowed to penetrate, proliferate, and differentiate. Additionally, the rate at which the degradation occurs will vary depending on the size and dispersion of porosity For example, more porous materials favor greater cell growth and transport of nutrients and waste while less porous layers favor external mechanical loading.

There are a few approaches to address the issue of creating optimal biomaterials, including surface and bulk chemical modifications. Surface modifications adjust the physicochemical behavior, interaction attributes, and structural properties of the biomaterial by increasing its compatibility at its surface level (Raval et al, 2019). Conversely, bulk modifications adjust the compatibility of the biomaterial by modifying the entire biomaterial itself, which affects its innate attributes and functions (Raval et al, 2019). Through new developments in technology and the neverending search to modify current biomaterials, scientists aim at developing more types of and more unique uses of biomaterials in the field of bone regeneration. 

1. Surface Modifications

The surface of biomaterials are uniquely reactive, often influencing the environment around the biomaterial (Ratner et al, 1996). There are a multitude of ways to measure the distinct characteristics of biomaterial surfaces, of which include contact angle methods, electron spectroscopy, secondary ion mass spectrometry, scanning electron microscopy, and others (Ratner et al, 1996). Surface modifications refers to changes to the outer layer of the biomaterial, involving controlling the surface to modulate its influence and optimize the biomaterial’s compatibility and use (Raval et al, 2019). Surface modifications can be categorized by physical, also called mechanical, and chemical changes (Raval et al, 2019). This section will describe how surface modifications impact on different properties of biomaterials to improve adhesion and biocompatibility to the physiological  environment. 

1.1 Hydrophilicity and Hydrophobicity

Hydrophilicity refers to when biomaterial surfaces attract water because the surface is polar while hydrophobicity refers to when biomaterial surfaces repel water because the surface is non-polar, and it is measured with the contact angle (hydrophillicity is considered below 90°) (Paterlini et al, 2017). Hydrophilic materials have a much higher wettability, which makes hydrophilic materials a favorable choice through its interaction with the body and improved cell viability and proliferation (Paterlini et al, 2017).

The components of the biomaterial and its hydrophilicity are ultimately dependent on its environment. In a more hydrophobic environment, such as environments with more air, more hydrophobic components may migrate to the surface of the cell; in more hydrophilic environments, such as an aqueous solution, more hydrophilic components would surface (Ratner et al, 1996). 

The hydrophilicity and hydrophobicity of a material affect cell behavior. Hydrophobicity increases the stiffness of the biomaterial, and hydrophobic materials are less adhesive and hinders the growth of cells, while hydrophilicity does the opposite (Ferrari et al, 2019). Also, hydrophobic surfaces favor protein absorption while hydrophilic surfaces doesn’t (Paterlini et al, 2017). However, hydrophobic surfaces may induce irreversible absorption of those proteins and even possible denaturation. 

Hydrogels are hydrophilic when placed in water, their cross-linked structures begin to swell (Ratner et al, 1996). Its ability to swell, and thus effectively support protein absorption and cell adhesion, makes hydrogel a very common scaffold in bone tissue regeneration (Yue et al, 2020). On the other hand, polytetrafluoroethylene (PTFE) is hydrophobic and thus very lubricant (Ratner et al, 1996). Unlike hydrogels, PTFE does not rely on water to serve its purpose; on the contrary, its repulsion to water actually makes the material more effective. 

By changing the surface in such a way to significantly change the contact angle of polymer biomaterials such as polyethylene (PE) or Teflon (FEP), the surfaces of these materials become “super hydrophobic” (Paterlini et al, 2017). Oftentimes, plasma cleaning is used to change the surface of the biomaterial, decreasing in water contact angle and increasing in wettability, thereby making the material more hydrophilic (Ratner et al, 1996). This is specifically done through providing the surface with more “adhesion power and cell proliferation”, making the surface more hydrophilic (Paterlini et al, 2017). 

Biodegradable synthetic polymers such as polylactide (PLA) and poly(lactide-co-glycotide) (PLGA) are common orthopedic biomaterials, but are not bioactive enough nor induce enough cell regeneration. Thus, researchers have modified the surfaces of many biodegradable synthetic polymers to improve their biological response, including active gases (radiation or vapor), plasma treatment, layer-by-layer deposition, or self-assembled monolayers (Paterlini et al, 2017). By making the surface more porous or more hydrophobic, biodegradable synthetic polymers, when used, have increased cell adhesion and proliferation (Paterlini et al, 2017).

Depending on the situation, making a surface more hydrophobic or more hydrophilic can help serve its purpose better. However, either feature has its advantages and disadvantages, and many other modifications are used to combat the effects of a biomaterial’s hydrophilicity/hydrophobicity. 

1.2 Peptide Binding

An alternative to artificial biomaterials such as ceramics and synthetic polymers, natural biomaterials, namely natural polymers, are known to be more biocompatible, more biodegradable, and have no toxicity whilst still efficiently engineering a bone graft (Guo et al, 2021). They are most commonly categorized as either protein-based biomaterials (such as collagen or gelatin) or polysaccharide-based biomaterials (such as hyaluronan or cellulose) (Chen et al, 2015). However, peptide-based biomaterials are less frequently used for bone regeneration (Collier et al, 2011).

Between protein-based and peptide-based biomaterial, peptide-based biomaterials can in fact be a better alternative for it is more chemically defined, meaning that it is easier to modify precisely (Collier et al, 2011). When using protein-based biomaterials, contaminants like lipopolysaccharide often affect the effectiveness of the biomaterial. The chemical definition and precision of peptides makes it easier to take away those contaminants (Collier et al, 2011). Furthermore, using peptide sequences in enzyme substrate sequences help precisely control the degradation of the matrices itself, suggesting that modifying a biomaterial using peptides is an efficient way to control the biodegradability of the material (Collier et al, 2011). Peptides may be conjugated to a biomaterial through specific chemistries  and use of varying crosslinkers (Collier et al, 2011).

Furthermore, peptides are highly capable of binding to growth factors, which are proteins vital to the regeneration of bone (Collier et al, 2021).

The most studied peptides that are used with biomaterials for biomedical applications are RGD, dominant in fibronectin, and YIGSR, dominant in laminin, two of the main components of the extracellular matrix (ECM).Though many studies have demonstrated improved regeneration and cell adhesion when coupling growth factors with biomaterials (Collier et al, 2011), it is important to note that the RGD peptide in modification to a common natural polymer hydroxyapatite (which is actually found in bone itself) actually resulted in little benefits and was worse off for the regeneration process by inhibiting bone formation (Hennessy et al, 2009). 

However, RGD as a modification to polycaprolactone (PCL) two-dimensional film has improved bone marrow stromal cell (BMSC) adhesion to the films because of its induced cell adhesion and thus cell-substrate interaction. PCL is a biomaterial very suited for the needs of tissue engineering and bone regeneration primarily because of its mechanical properties, affordability, and fabrication with proper porosity (Zhang et al, 2009). PCL lacks proper cell adhesion due to its hydrophobicity, which is a modification that RGD helps enhance. For three-dimensional PCL scaffolds, the RGD was crosslinked to modify the scaffold and, despite resulting in inconsistent cell distribution, still improved BMSC adhesion (Zhang et al, 2009).

Thus, in consideration of RGD as a modification in theory and in practice for two different biomaterials, RGD, while flawed, is a modification with much potential and could perhaps even be re-introduced for future biomaterials. 

1.3 Calcium Phosphate Coatings

A common strategy used primarily in orthopedics and dentistry, calcium phosphate coatings are a surface modification evolved from the traditional calcium phosphate ceramic materials (CPC). Calcium phosphates are primarily coated onto metal biomaterials, such as titanium and magnesium (Jeong et al, 2019). Calcium phosphate appeals as an orthopedic biomaterial modification primarily because of its quick and strong bond and integration to bone tissue (Yoshinari et al, 2002). Furthermore, the coatings are often applied for increased bioactivity and bone regeneration (Jeong et al, 2019). Calcium phosphate is very porous, which helps contact with physiologic fluids and protein absorption. Moreover, depending on the pore size, calcium phosphates have been shown to induce bone ingrowth and angiogenesis (Jeong et al, 2019).

Sol-gel and electrodeposition methods are often used to coat surfaces with calcium phosphates (Jeong et al, 2019). Physical vapor deposition (PVD), ion sputtering, and ion plating are also techniques to adhere a thin even coat of the calcium phosphate. The ion beam dynamic mixing (IBDM) method combines PVD and ion implantation not only provides a thin even coat of the calcium phosphate, but also has a higher deposition rate and better adhesion qualities (Yoshinari et al, 2002). Calcium phosphates have tendencies to be amorphous (Yoshinari et al, 2002). Referred to as ACP (amorphous calcium phosphate), ACP is a hydrated, loosely structured, and often unstable biomaterial (Habraken et al, 2016). However, because of the effectiveness of ACP as a biomaterial for biological pathways and bone regrowth, namely being a potential precursor for bone growth as having similar chemical composition of bone mineral, a solution has been found (Habraken et al, 2016). Specifically, the amorphousness of ACP is mitigated by rapid infrared heat-treatment using hydroxyapatite. (Yoshinari et al, 2002).

2. Bulk Modifications

Bulk modifications most commonly refer to the chemical modifications to a material, affecting both the material core  in addition to the surface. Unlike surface modifications, where the improvement is uniquely achieved on a small superficial layer, bulk modifications often change the properties of the material through tweaking its chemical composition. This section focuses on different properties that can be achieved with bulk chemical modifications.

2.1 Degradability

The degradability of a biomaterial, or biodegradability, refers to a material’s ability to break down chemically into non-toxic components. And, depending on the application of the material, the implanted biomaterial could stay stable in the system for long periods of time or be rapidly dissolved in its environment. A too fast degrading biomaterial may result in a collapsed porous structure, no mass transfer, and thus necrosis (cell death); on the contrary, a too slow degrading biomaterial may cause a fibrotic encapsulation, which would hinder tissue regeneration (Park et al, 2010). Degradable biomaterials can help scaffold the defected bone area while new bone tissue forms and eventually seamlessly replaces the biomaterial as the biomaterial degrades without producing toxic  (Davison et al, 2014). 

A well-known biodegradable and biocompatible biomaterial is chitosan, a natural polymer that, because of its porosity, helps in the bone implantation process (Zhang et al, 2019). Its biodegradability is dependent on its degree of deacetylation, or the amount of glucosamine in the chitosan; highly deacetylated chitosan degrades much slower than less deacetylated chitosan (Zhang et al, 2019). In this sense, the content of glucosamine can be manipulated to control the biodegradability of the chitosan (Zhang et al, 2019). 

Alginate is a natural polymer that is biodegradable, non-toxic, can become gel, biocompatible, and easy to process (Sahoo et al, 2021). However, despite being widely researched and used for its use as a drug delivery and being cell carriers, it rapidly degrades (Birdi et al, 2011). This makes alginate harder to use as an orthopedic biomaterial since bone regeneration often takes a long time to occur. Through the addition of orthosilicic acid (OSA), an alginate hydrogel was able to slow down degradation in tests using a potent calcium chelator (EDTA) (Birdi et al, 2011). Through these tests, the OSA integrated alginate hydrogel resulted in no detect of calcium release than alginate hydrogel alone (which resulted in significant calcium release), suggesting that OSA inhibits the release of calcium through forming calcium cross-links, thus resulting in a slower degradation speed (Birdi et al, 2011). 

When developing new composite biomaterials, biomaterials, particularly polymers, are chosen based on biodegradability, biocompatibility, and mechanical strength. For this reason, the polymers are commonly PLA or PGA polymers. PCL is also becoming more common for long-term bone regeneration due to its low degradation rate, being biocompatible, and mechanical strength. Together with zein through solvent casing particle leaching (SCPL), the zein/PCL composite scaffold’s degradability is controlled by zein (Zhu et al, 2020).

2.2 Drug Release

While most biomaterials in bone regeneration serve as a scaffold for the bone to regenerate by itself, some biomaterials have been modified to serve as a drug carrier to quicken the regeneration process. However, drug delivery has been historically hard to implement; drug are commonly delivered through a noninvasive systematic delivery, but this may result in a poor distribution of the drug throughout the bone (Newman et al, 2016). By using carriers such as peptides, aptamers, and phosphate-rich compounds—all materials that are biocompatible with the environment—specific to proteins and other cells unique to the environment, these carriers can deliver the drugs with great distribution while maintaining its systematic function (Newman et al, 2016). Also, these carriers further support on-demand drug delivery and tissue engineering (Vashist et al, 2017).

An example of an excellent biopolymer with drug release is elastin-like polypeptides (ELP), which is based off of human elastic (MacEwan et al, 2014). Specifically for bones, coacervate ELP nanoparticles physically encapsulate BMP-2 and BMP-14 cytokines, growth factors that induce bone growth (MacEwan et al, 2014).

Using biomaterials as drug carriers requires the biomaterial chemical composition to be adjusted to hold onto the drug. Hydrogels are commonly used for scaffold due to their properties being similar to that of the extracellular matrix of organs or tissue. It fulfills the role as a scaffold because of its mechanical strength, porosity, and diffusive properties. In the field of nanomedicine, hydrogels are now being developed into injectable hydrogels, in which hydrogels serve as a drug carrier that can be made into a desired shape depending on the function of the biomaterial. This was made through a series of polymers and live cells, which formed a hydrogel depot which was processed using the sol-gel method. By making the hydrogel injection, it not only served as a drug carrier, but also a sealing agent, tissue-engineering product, and surgical glue (Vashist et al, 2017).  

Electrospun nanofibers are seen as great drug carriers due to their high surface area and mechanical properties, depending on what polymer the nanofibers are derived from. Silk fibroin protein has also become a common drug carrier partly due to its purification, sterilization, lack of cross-linkers, and great biocompatibility amongst other qualifications (Farokhi et al, 2020). 

Amphiphilic block copolymers (ABC) is commonly applied in the pharmaceutical field and extensively used as a drug carrier (Adams et al, 2005), and is also being researched as a modification to scaffolds to induce their drug delivery abilities (Kutikov, 2015). Polyesters, while great as scaffold, contain many limitations due to their hydrophobicity, with drug delivery being one of them (Kutikov, 2015). By integrating hydrophobic poly(ethylene glycol) (PEG) blocks with hydrophobic polyesters to become an ABC, the characters of the copolymer completed shifted from its physical properties to biological performance, with positive but not conclusive results in drug delivery for the repair of bone tissue (Kutikov, 2015).

While drug delivery as a bulk modification is still being researched, drug delivery systems in biomaterials that naturally contain them are essential to inducing bone growth. 

Conclusion

In this review paper, different properties of both surface and bulk modifications in biomaterials primarily used for bone regeneration were assessed. While these are the most common properties, there are far more to be looked at and each property is far more complex than the overview and insight that this paper provides. However, this paper does provide a holistic view on the innovative aspect of new techniques and primary goals in biomaterials for bone regeneration.

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About the author

Cathleen Chow

Cathleen is currently a junior at the Cate School in Carpinteria, California. She loves playing volleyball and waterpolo and is on the school varsity team. In addition, Cathleen plays the violin and you’ll frequently find her in the studio practicing for an upcoming chamber piece. Despite her numerous interests, she always makes time to research and learn more about the sciences that she finds fascinating and the sciences that affect our daily lives.

The post Surface and Bulk Modifications of Biomaterials Used in Bone Regeneration appeared first on Exploratio Journal.

Abstract

Migraine is the sixth most disabling illness in the world and the third most common (Saper, 2021). Many studies have found that migraines have numerous causes, which creates a wide variability in the effectiveness of treatments. In recent years, there has been exploration of alternative methods of treatment, such as music therapy. There have been many studies regarding music therapy and pain treatment, but very few specifically for headaches, and results have been inconclusive. The proposed research seeks to examine the extent to which predisposed beliefs about music therapy impact its efficacy when treating chronic migraines. Over the course of a six-week study, patients will be randomly assigned to one of two experimental treatment conditions whereby they will receive a combination or standard pharmaceutical treatment plus music therapy or standard pharmaceutical treatment plus therapeutic rhythmic class. Patients will also report the degree to which they perceive music therapy and other alternative treatments as effective pain-relieving measures. Patients will also report their pain symptoms weekly. It is anticipated that patients with stronger belief in the efficacy of music therapy, in particular, will experience greater reduction in pain symptoms when in the music therapy condition. The implications of an interaction between treatment efficacy beliefs and actual treatment effectiveness are discussed.

The Impact of Belief in Treatment Methodology on the Efficacy of Music Therapy in Those with Chronic Migraines

While many people have friends or family members who suffer from migraines, individuals who do not experience migraines themselves may be unaware of how chronic, unpredictable, and often mysterious migraine symptoms are. Approximately 1 billion people around the world have migraines (Pool, 2021), making migraines the third most prevalent illness in the world, along with being the 6th most disabling (Saper, 2021). Lipton et al. (2007) reported that 53.7% of those with migraines were severely impaired.  Munakata et al. (2009) found that those with episodic migraine had direct and indirect costs of $1757 per year, and those with transformed migraine had costs of $7750 per year.  Those who experience migraine–as well as their closest family and friends–are more likely to pursue knowledge regarding migraine causes and treatment (Saper, 2021), but they often find that migraines have many different causes and thus there is wide variability in types of treatment and their efficacy (Dodick & Gargus, 2008). Recently, there has been increasing interest in alternative treatments for migraine sufferers such as yoga (John et. al, 2007), risk factor modification (Schwedt, 2014), and music therapy (Diamante & Roxas, 2020). However, the degree to which such alternative treatments alleviate symptoms may be influenced by patients’ beliefs in their efficacy (e.g., Horne, 1999). Accordingly, the current research proposal seeks to examine how much predisposed beliefs about music therapy impact its efficacy, and if music therapy is actually effective in treating chronic migraines.

Music Therapy

Music therapy has been examined as a potential treatment for dementia (Vink et. al, 2003), generalized anxiety disorder (Gutiérrez & Camarena, 2015), as well as chronic migraine symptoms (Diamante & Roxas, 2020). Research on the use of music therapy as a treatment for migraines has demonstrated that it shows a significant improvement from no treatment (Oelkers-Ax, 2008). Music therapy has been shown to be effective for those seeking treatment for chronic migraine and tinnitus (Nickel et al., 2005). Additionally, Langenburg and colleagues (1995) did a case study on music therapy as treatment for someone with chronic migraines which demonstrated that it does lessen migraine symptoms. While additional studies have demonstrated similar efficacy for MT as a treatment for migraines (e.g., Diamante & Roxas, 2020), other studies have found that MT, while effective, is no more effective than other alternative treatments (Koenig, 2014). Moreover, music therapy can better improve executive functioning when it is able to provide functional support (Thaut & Hoemberg, 2017). Accordingly, we should expect music therapy to reduce migraine symptoms in patients when used in conjunction with standard treatment, but perhaps not more so than other distracting treatments. However, there are moderating factors that could influence the degree to which individual patients respond to alternative treatments such as music therapy–one of which may be their belief in the efficacy of music therapy.

Patient’s Beliefs in the Efficacy of Treatment

The ability of a patient’s belief in the efficacy of certain treatments to affect treatment outcomes has been examined in a variety of contexts. Patients’ beliefs about the type of treatment they’re receiving has been shown to impact the efficacy of that treatment (Horne, 1999). The symptoms of patients suffering from various illnesses were shown to respond more strongly to medical treatment among patients who believe more strongly in the effectiveness of traditional medicine as a treatment (Foulks et. al, 1986): the expectation of a drug’s impact on symptoms strongly influences the therapeutic efficacy of that drug (Bingel et al, 2011). Similarly, Clatworthy et. al found that patients better adhered to their treatment plan when they believed that it would work, and thus they found more benefits from their treatment (2007). In addition, a study regarding the effects of methotrexate on rheumatoid arthritis symptoms found similar results (de Thurah et. al, 2009). Studies have also demonstrated that beliefs regarding severity of the patient’s illness impact the efficacy of the treatment (Marks et al., 1986). However, to the best of our knowledge, no research has yet investigated this moderating influence regarding the efficacy of music therapy in migraine treatment. Accordingly, the current research will assign patients to two conditions of music therapy, one who has strong beliefs in the efficacy of music therapy, and one who does not. These two groups will demonstrate the relationship between belief in the efficacy of treatment and pain reduction.

Research Hypothesis: We expect that the efficacy of music therapy, in comparison to a different alternative treatment, in reducing migraine symptoms will be moderated by patients’ beliefs in music therapy’s effectiveness. Specifically, we expect that the relative efficacy of music therapy, in comparison to yoga therapy, in relieving chronic pain symptoms will be greater among individuals who already perceive music therapy as an effective treatment.

Method

Participants

Participants will be recruited from a hospital circuit, through compliant doctors asking their patients with chronic migraines. They will be given a small monetary sum, and the study will be conducted in an academic center with music facilities.  

Measures

Belief in Treatment Efficacy. Prior to being randomly assigned to a treatment condition, patients will fill out a brief survey asking them to report how effective they believe each of the following therapies would be for reducing their migraine symptoms: music therapy, rhythmic class, yoga, mindfulness meditation, traditional western medicine, and pet therapy (Appendix A). Brief descriptions of each therapy type will accompany the items. Participants will indicate their belief in the efficacy of each therapy on a 1 (“Not at All Effective”) to 7 (“Highly Effective”) Likert-type scale. The variable of interest is patients’ beliefs in the efficacy of music therapy, and patients will be told that their beliefs will not affect the treatment condition to which they are assigned. 

Treatment Condition. Patients will be randomly assigned to receive one of two treatments in addition to traditional pharmaceutical treatment: music therapy or rhythmic class This treatment manipulation is similar to one used in previous research (Gutgsell et. al 2013).  In each condition, patients will undergo their additional treatment 3 times per week. Patients in the music therapy condition will go into a dark room without distractions for 20 minutes and have a music therapist play harp pieces for them. Patients in the rhythmic class (Bozorg-Nejad et. al 2018) condition will go into a dark room without distractions for 20 minutes and will be taught how to control the various paces of the body with breathing. These conditions will last for 6 weeks.

Improvement of Symptoms. The dependent variable that will be measured is the difference in pain before treatment and after treatment using a standardized scale. Patients will rate their overall severity of their symptoms at the end of each week on a scale from 0 (“No Symptoms”) to 10 (“Emergency Treatment Necessary”) similar to measures of migraine pain used in previous research (Jensen et. al 1999). Average scores from the final three weeks will be subtracted from average scores from the first three weeks to determine the change in symptoms over the treatment period.

Procedure

 When first agreeing to participate in the study, the participants will complete an informed consent document. They will be given their monetary reimbursement of $50 for their time, and then will be separated into two groups, after being sent and completing the survey about their beliefs in the efficacy of music therapy remotely. From those two groups they will be randomly assigned to either the music therapy condition or the rhythmic class, depending on what time of day they fill out the survey. They will then go to these therapies at least three times per week for 20 minutes for six weeks. Over the course of these six weeks their pain symptoms will be monitored and recorded through their respective nurses on the standardized scale in Appendix B. 

Results

Correlational Analyses

A zero-order Pearson correlational analysis will be conducted to assess the relationship between belief in the efficacy of music therapy and improvement of pain symptoms for patients in the MT condition only. We hypothesize that, among patients in the MT condition, higher belief in MT will predict more improvement of symptoms. We expect to see a similar correlation between belief in the effectiveness of rhythmic class in the comparison condition.

T-Test

A t-test will be conducted to compare symptom improvement between the two experimental groups. We expect that without taking into account the patients’ beliefs in MT’s effectiveness that patients in the MT conditional will not show significantly more or less improvement than patients in the rhythmic class condition. 

 Moderation Analysis

To test our primary research hypothesis, a moderation analysis will be conducted using the PROCESS macro (Hayes, 2013) Model 1. Treatment condition will be added to the model as the independent variable (x), belief in the efficacy of music therapy as the moderator (w), and symptom improvement as the dependent variable (y). We predict that belief in the efficacy of MT will moderate the difference between treatment groups such that MT will be more effective than rhythmic class among participants with higher beliefs in MT’s efficacy. The conceptual model is illustrated in Figure 1.

Discussion

The proposed study seeks to explore the relationship between patients’ beliefs in the efficacy of alternative forms of treatment for chronic pain and the actual effectiveness of those treatments. In particular, this study intends to focus on music therapy (MT) as a potential treatment. Patients’ beliefs about MT, and other alternative treatments, will be measured prior to random assignment to a treatment condition that includes typical pharmaceutical treatment plus either music therapy or a comparison treatments condition. It is hypothesized that beliefs in the efficacy of MT will interact with assignment to the MT condition such that patients who perceive MT as more effective and are assigned to the MT condition will experience more pain symptom relief over the course of the study. 

Implications

Approximately 5-12% percent of people worldwide suffer from migraines (MacGregor et. al, 2003), which is millions of people. Therefore, the majority of people at least know someone who is a victim of this kind of pain. Additionally, making strides within the treatment of these migraines could vastly improve quality of life. Music therapy specifically is an instance of alternative methods, based on other facts. If the experimental hypothesis was supported, then pain treatment would be reimagined to adjust to the beliefs of the patient. To ensure the best outcomes, physicians could prescribe whichever alternative treatment an individual patient has the most positive views of in addition to pharmaceutical treatment. Additionally, results that support our hypotheses would suggest that physicians should emphasize scientific support for alternative methods to their patients. Doing so may make beliefs about such treatments more positive, which would in turn make the treatments more effective.

Limitations

This study would be done on adults, so it probably would not be as conclusive for the same study done on children or adolescents. The proposed study also does not account for potential disparities among those who have chronic migraines. This study also would not discuss the musical aspect involved in rhythm. The proposed study also does not account for the influence of those providing the treatment on the patient’s belief in efficacy. This study also only compares two types of alternative therapies, when there are many others which could have different results.

Future Directions

In future studies, there should be much deliberation regarding the control therapy and how it relates to music therapy. There could be focus on how different types of music impact the efficacy, or how it relates to what is being treated. . For instance, it has been shown that music can help those with dementia express themselves with language (Brotons & Kroger, 2000). Moreover, connecting these two things is something worth looking into, based on how music has a unique impact on the brain and has helped lessen the impacts of other neurological disorders. As far as pain management goes, it is important to consider the different ways that people can feel pain and how each experience it unique, along with the wide variety of treatment methods- beyond the ones discussed in this proposal.

References

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Bozorg-Nejad, M., Azizkhani, H., Ardebili, F. M., Mousavi, S. K., Manafi, F., & Hosseini, A. F. (2018). The effect of rhythmic breathing on pain of dressing change in patients with burns referred to ayatollah mousavi hospital. World journal of plastic surgery, 7(1), 51.

Brotons, M., PhD, MT-BC, Koger, S. PhD, The Impact of Music Therapy on Language Functioning in Dementia, Journal of Music Therapy, Volume 37, Issue 3, Fall 2000, Pages 183–195, https://doi.org/10.1093/jmt/37.3.183

Clatworthy, J., Bowskill, R., Rank, T., Parham, R., & Horne, R. (2007). Adherence to medication in bipolar disorder: a qualitative study exploring the role of patients’ beliefs about the condition and its treatment. Bipolar disorders, 9(6), 656-664.

de Thurah, A., Nørgaard, M., Harder, I. et al. Compliance with methotrexate treatment in patients with rheumatoid arthritis: influence of patients’ beliefs about the medicine. A prospective cohort study. Rheumatol Int 30, 1441–1448 (2010). https://doi.org/10.1007/s00296-009-1160-8

Foulks, E. F., Persons, J. B., & Merkel, R. L. (1986). The effect of patients’ beliefs about their illnesses on compliance in psychotherapy. The American Journal of Psychiatry, 143(3), 340–344. https://doi.org/10.1176/ajp.143.3.340

Gutiérrez, E. O. F., & Camarena, V. A. T. (2015). Music therapy in generalized anxiety disorder. The Arts in Psychotherapy, 44, 19-24. 

Gutgsell, K. J., Schluchter, M., Margevicius, S., DeGolia, P. A., McLaughlin, B., Harris, M., … & Wiencek, C. (2013). Music therapy reduces pain in palliative care patients: a randomized controlled trial. Journal of pain and symptom management, 45(5), 822-831.

Horne, R. (1999). Patients’ beliefs about treatment: The hidden determinant of treatment outcome?[Editorial]. Journal of Psychosomatic Research, 47(6), 491–495. https://doi.org/10.1016/S0022-3999(99)00058-6

Jensen, M. P., Turner, J. A., Romano, J. M., & Fisher, L. D. (1999). Comparative reliability and validity of chronic pain intensity measures. Pain, 83(2), 157-162.

John, P., Sharma, N., Sharma, C.M. and Kankane, A. (2007), Effectiveness of Yoga Therapy in the Treatment of Migraine Without Aura: A Randomized Controlled Trial. Headache: The Journal of Head and Face Pain, 47: 654-661

Koenig J. Music therapy in the treatment of primary headache disorders. OA Alternative Medicine 2014 Jan 18;2(1):1

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MacGregor, E. A., Brandes, J., & Eikermann, A. (2003). Migraine prevalence and treatment patterns: the global Migraine and Zolmitriptan Evaluation survey. Headache: The Journal of Head and Face Pain, 43(1), 19-26.

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Appendix A

Questionnaire for measuring belief in efficacy of music therapy.

How confident are you in the ability of pet therapy to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

How confident are you in the ability of rhythmic classes to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

How confident are you in the ability of yoga to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

How confident are you in the ability of music therapy to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

How confident are you in the ability of traditional western medicine to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

How confident are you in the ability of mindfulness meditation to treat your pain?

  1                   2                 3                   4                  5                 6                 7

Not at all                                                                                                  Extremely 

confident                                                                                                  confident

Appendix B

Measurement of pain

How would you rate your pain symptoms this week??

  1               2              3                 4              5              6              7                8               9            10

No symptoms                                                                                                                                                                                                                                                                                                    

About the author

Karishma Kulshrestha

Karishma is currently a Senior at the Thomas Worthington High School in Ohio. She has a strong passion for people, and her extraverted nature is where this seed was planted. As someone with chronic migraines herself, she has seen a lot of the medical systems from the patient perspective which has allowed her to think about things others might not normally. Karishma is in marching band and was a drum major this year. She also plays volleyball, and started a ski club at her school.

The post The Impact of Belief in Treatment Methodology on the Efficacy of Music Therapy in Those with Chronic Migraines appeared first on Exploratio Journal.

Abstract

The ability of stem cells to self-renew and form different mature cells expands the possibilities of application in cell therapy, such as tissue reorganization in regenerative medicine, drug detection, and treatment of neurodegenerative diseases. In addition to stem cells found in embryos, several adult organs and tissues also have stem cell niches in an undifferentiated state. In the central nervous system of adult mammals, neurogenesis occurs in two areas: the subventricular zone and the dentate gyrus of the hippocampus. The different nervous systems originate from adult neural stem cells, which can self-renew or differentiate into astrocytes, oligodendrocytes, or neurons that respond to specific stimuli. The regulation of the fate of neural stem cells is a finely controlled process that relies on a complex regulatory network that extends from the epigenetics to the translational level and involves components of the extracellular matrix. Therefore, a better understanding of the mechanisms by which neurogenesis is induced, regulated, and maintained will provide clues for the development of new strategies for neurodegenerative treatment. In this review, we focus on the regulatory mechanisms of transcription factors, microRNAs, and components of the extracellular matrix in neuronal differentiation.

1. Introduction 

Neocortical circuits consist of profoundly interconnected excitatory glutamatergic and inhibitory GABAergic neurons, which are produced from unmistakable pools of RGCs. During embryo development, the excitatory neurons are formed from RGCs confined in the ventricular zone of the dorsal telencephalon and relocate radially toward the pial surface in a back to the front way (spiral movement). Then again, inhibitory neurons fundamentally begin from the ventral telencephalon and relocate digressively into the neocortex (distracting movement). Regardless of such unique formative starting points, both excitatory and inhibitory neurons go through the multipolar stage with a few minor cycles in the neocortex before axon augmentation. Then, at that point, they go through dynamic morphological changes to start axon development, in particular, neuronal polarization. Sakakibara and Hatanaka () assessed the consecutive occasions in polarization cycles of both excitatory and inhibitory neurons, and they talked about the hidden atomic instruments. At the multipolar stage, the excitatory neurons fleetingly utilize a multipolar relocation mode, to be specific movement with no fixed heading, in the subventricular and middle of the road zones. Then, at that point, they receive a bipolar shape during neuronal polarization and relocate rapidly toward the pial surface along with RGC measures, which is called headway mode.

Many types of molecules are involved in these powerful changes in neuron morphology and migration. The small GTP binding protein plays an important role in regulating the development of the cortex and the formation of neurons. The Rnd protein, “abnormal” relatives of Rho, was displayed in neuronal movement, and their ascending and descending pathways were discussed. Many cytoplasmic protein elements, including parts of the cytoskeleton, are managed through phosphorylation and dephosphorylation measures. Ohshima focused on protein kinases, including CDK5 and JNK, and examined their paperwork on cytoskeletal associations during multipolar bipolar progression and extended movement. OhtakaMaruyama and Okado comprehensively summarized the atomic pathways involved in these formation cycles and emphasized the importance of subplate neurons in steering events and the development of the six-layer neocortex design. 

 Pluripotent stem cells are usually derived from embryonic tissue. At least three different types of mammalian pluripotent stem cells have been identified: embryos or cancer cells (CE), embryonic stem cells (ES) derived from the inner cell mass of blastocysts, and embryonic germ cells (EG) derived from post-implantation embryos. In the early 1990s, several groups reported the existence of a subset of stem cells found in the central nervous system (CNS). These cells form the brains of fetal mice or mice that grow in culture and show an almost unlimited lifespan. However, compared to embryonic stem cells, their differentiation potential is more limited and they mainly produce three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes, hence the name NSC. These cells have also been isolated from the adult central nervous system, although it is not clear whether these dividing cells are truly pluripotent or whether their fate becomes more restricted during development. For the purposes of this review, NSCs are defined as nerve cells that have the potential to self-renew and generate all the different types of cells in the nervous system after differentiation. 

2. Method

Neuronal differentiation is an early event in mammalian embryogenesis, occurring shortly after germ layer differentiation. The organization of the central nervous system is derived from a well-defined neuroectoderm, the neural plate, which is located in the dorsal midline of the embryo. It appears that neural plaque is produced by signals that locally inhibit or avoid inducing non-neural differentiation. Examples of such signals come from bone morphogenetic protein (BMP) and other molecules of the transforming growth factor-beta (TGF beta) superfamily, which direct epidermal differentiation when gastrulation

Neuronal fate is inhibited by BMP. In the body, several molecules that promote neuronal differentiation, such as noggin, follistatin, and chordates, are antagonists of BMP. Although noggin antagonizes BMP signaling, it is not necessary for the induction of early neurogenesis, because knockout mice are normal at embryonic day 8.5 (although they die at birth). These data indicate that other BMP antagonists can compensate without Noggin expression and illustrate the concept of redundant signaling pathways during embryonic development. The role of BMP is further regulated by the presence of two ubiquitously expressed BMP receptors, BMPR1A and BMPR1B, which do not appear until the 9th day of the embryo. The expression of BMPR1A induces the expression of BMPR1B, and this process is inhibited by the sonic hedgehog. The further development of the neural lamina into mature cells of the central nervous system is clearly and precisely regulated by spatial and temporal differentiation patterns. The growth and proliferation of cells in the early neural plate eventually lead to the closure of the developing nerve sulcus and the formation of a hollow neural tube. The neural tube cavity produces the ventricular system and the epithelial layer contains stem cells that will produce neurons and glial cells of the central nervous system. One of the central problems in developmental neurobiology is the mechanism by which a simple neuroepithelium (only one cell thick) can produce the various cell types that make up the mammalian central nervous system. Currently, there is a large number of studies that have determined the internal factors and external soluble signals that affect this regional pattern and specific neural differentiation. An example of this invertebrate neurogenesis is the transmission of dorsal and ventral patterns of opposing soluble signals: sonic hedgehog (Shh) and BMP antagonists, chordin and noggin, are secreted from the bottom plate, while other signals are emitted from the top plate. Form a gradient. Signal concentration. The precise concentration and ratio of each signal in the neural tube is critical to the development of specific neuronal phenotypes at different points along the gradient. For example, there is a concentration-dependent induction of model genes in progenitor cells encoding homeodomain transcription factors. These differences in expression patterns produce neuronal clusters with different division and differentiation patterns (and ultimately different phenotypes) along the dorsal-ventral axis of the plaque. In humans, the neural tube is formed during the third and fourth weeks of pregnancy. Initially, the neuroepithelial lining consisted of a single layer of neural stem cells with similar morphology. These cells then divide symmetrically to enrich the pool of NSCs or divide asymmetrically to produce a more differentiated progeny from which neurons and mature cells of the glial mass line develop. Although the BMP family of molecules may be involved again, the signals that determine symmetric or asymmetric division are not yet fully understood. 

Retroviral marker studies have been used to identify dividing cells in the ventricular region. Approximately 48% of the labeled cells remained in the colonies in the ventricular zone, indicating self-renewal at this site. In humans and other mammals, the active proliferation of such progenitor cells is likely to be balanced by apoptosis to maintain a stable population, but the exact mechanism has not been determined. As development continues, neurons migrate, partly guided by radially oriented glial processes, and the size of the ventricular area decreases. Neural stem cells are still attached to the basal layer. It has been shown that these cells can divide asymmetrically, and the more differentiated offspring migrate from the ventricular area to the overlying cortex. By obtaining specific phenotypic markers, these cells can divide further and distinguish them from neural stem cells. During CNS development, the temporal pattern causes oligodendrocytes to generate neuronal cell types earlier. In the spinal cord, these two cell populations appear to be produced from a common precursor, and the final decision of fate depends on external signals and specific patterns of transcriptional activation. There are two main types of transcription factors that can determine the fate of neurons or the glia. These are homeodomain factors, an example is NKx2.2, and the basic helix loop helix family of transcription factors, including Olig1 and Olig2. The expression of Olig transcription factors is regulated by external signals (such as Shh), and their expression has been shown to clearly define cells as oligodendrocytes. However, in mice, Olig1 and Olig2 are expressed from E9, long before the presence of oligodendrocyte precursors. At this early stage, Olig2 is known to be the core of neuronal development in specific areas of the ventral spinal cord and is found in cells that can track the fate of motor neurons. The importance of Olig2 in neurodevelopment has been demonstrated in functional gain experiments. As development proceeded, the expression of Olig1 and Olig2 persisted and began to overlap with the homeodomain transcription factor NKx2.2. These doubly positive progenitor cells migrate from the ventral midline and mature into oligodendrocytes. Olig1 and Olig2 have been developed in double-mutant mice. These animals lack oligodendrocytes and also have a considerable loss of motor neurons. In the Olig1 / Olig2 double mutant, the offspring of stem cells in the pMN region of the developing spinal cord generally develop into motor neurons, then oligodendrocytes, but form V2 interneurons and then astrocytes. These results indicate that the expression of the combination of transcription factors determines the fate of the stem cell bank in the developing embryo. However, this does not rule out the existence of more restrictive dividing cell populations. The expression of proglia transcription factors can be regulated by cell surface receptors such as gaps. The jagged1 (notched ligand) signal of neurons has been shown to inhibit the phenotype of oligodendrocytes. Presumably, when the number of neurons is sufficient, jagged1 is down-regulated, and a signal from the original oligodendrocytes (one of which may increase electrical activity) triggers myelination. Once the neural precursor determines the fate of the oligodendrocyte lineage, the last step of honey-forming cell formation needs to exist.

3. Conclusion

All in all, corticogenesis from mouse ESCs shows a setup collection of parent-of-beginning articulation and DNA methylation of engraved loci. This model could be utilized dependably to unwind the atomic components engaged with choosing the communicated parental allele with regard to engraving during the cortical turn of events. Our discoveries likewise give support to utilize ESCs to demonstrate cortical turn of events and for drug screening. The in vitro corticogenesis framework could be an integral asset to pinpoint sedates that de-curb the quieted parental allele in specific mind sicknesses related to irritated IG articulation or to gauge the effect of ecotoxic compounds on the epigenetic marks and the advancement of cortical cells. The cerebral cortex creates through the organized age of many neuronal subtypes, yet the systems included stay hazy. Here we show that mouse undeveloped undifferentiated organisms, refined with no morphogen except for within the sight of a sonic hedgehog inhibitor, summarize in vitro the significant achievements of cortical turn of events, prompting the successive age of a different collection of neurons that show the most notable elements of veritable cortical pyramidal neurons. When joined into the cerebral cortex, these neurons foster examples of axonal projections relating to a wide scope of cortical layers, yet in addition to exceptionally explicit cortical regions, specifically visual and limbic regions, subsequently showing that the character of a cortical region can be determined with no impact from the mind. The disclosure of natural corticogenesis reveals new insight into the instruments of neuronal particular and opens new roads for the demonstrating and treatment of neural disorder.

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About the author

Harsheel Dhruva

Harsheel is currently a Senior at Irvington High School in Fremont, California. Growing up in the Bay Area, he is very interested in the natural sciences and the potential of discovery from advancing technology. He is pursuing studies in Neuroscience and Biotechnology in hope of pioneering his own research in the future and create technology to improve the human condition. Outside of his study, Harsheel loves completing challenging physical activity or listening to music whenever he gets the chance, and is always happy to read something new.

The post Exploring Corticogenesis: Pluripotent Stem Cells into Cerebral Operations appeared first on Exploratio Journal.

Abstract

Chinese herbal medicine (CHM) has been used in ancient China for over 2500 years, with its prevalence continuing to grow ever since. In recent years, traditional Chinese medicine (TCM) methods such as acupuncture, tai chi, and herbal medications have gained a global presence. However, its basis has not been proven scientifically, and anecdotes or observations of successful treatments have led to the foundations of CHM. Thus, believers of CHM and TCM practitioners have not encouraged the publication of many clinical studies on the efficacy and safety of herbal medications. Some experimental studies have led to promising results, but more research needs to take place to confirm such effects and any adverse effects medications may bring. The purpose of this paper is to examine the basis and research behind CHM as well as the risks of ingesting CHM.

Introduction

Rooted in Chinese culture, TCM has become a staple for the holistic treatment of illnesses. Although China’s healthcare system has embraced western medicine (WM) for the treatment of some illnesses in recent years, the prevalence of TCM is nonetheless prevalent across the nation’s citizens. According to the China Health Statistics Yearbook 2018, there were a total of 2,641 million visits to TCM practitioners, making up 32.3% of all clinical visits in 2017 (Liu et al.).

Many patients with chronic illnesses look to natural treatments such as TCM after WM has been unsuccessful. Moreover, when illnesses can only be treated with symptom relievers such as the common cold, TCM has been able to fill a gap by claiming to address the root cause (Marshall, 2020). With a growing number of patients utilizing TCM and success stories beginning to rise, it has begun to gain worldwide acceptance, with the World Health Organization adding a chapter regarding TCM in the International Classification of Diseases (Nature, 2019).
Essential components of TCM such as acupuncture and Tai Chi are deemed by the National Institutes of Health to be relatively safe due to their non-invasiveness (Hopp & Shurtleff, 2019).

Although these practices are often scrutinized due to their lack of scientific basis, there are generally fewer risks in participating in acupuncture or Tai Chi than ingesting CHM. However, the effects of acupuncture or Tai Chi are often credited to placebo effects due to their lack of scientific explanations. CHM seems to be the main concern, due to its lack of research into its adverse effects and long-term use by patients of all ages (Liang et al., 2018). The concern behind CHM is attributed to its extensive usage, especially in children and older adults, as well as the lack of randomized clinical trials required to validate claims of efficacy or safety.

Basis Behind Chinese Medicine

TCM is a medical system based on the Chinese belief of balance, where the equilibrium between Yin and Yang can ensure the health of a person (National Cancer Institute). Yin and Yang represent opposite elements that coexist to create a steady cycle between all entities. If an entity has an imbalance, it is deemed as unhealthy and may require a method of TCM to resolve the imbalance. Furthermore, the Chinese believe that among the balance exists ‘Qi,’ a vital source of energy that circulates the body. Based on this belief, traditional Chinese practices focus on the circulation of Qi and restoring the flow of Qi within the body by working to clear the path of the Meridians, channels that Qi flows through. This is a practice that has been passed down in Chinese society for thousands of years. The creation of Chinese herbal medicine is often attributed to Emperor Shen Nong, who sampled more than 100 herbs where he later introduced them to his subjects (Ergil et al., 2002). Illnesses and diseases are caused by disturbances in the balance of Yin and Yang and the flow of Qi within one’s body. Thus, TCM practitioners aim to reinstate the natural balance and flow in order to heal a patient. CHM supporters claim herbal remedies utilize a slow healing method that can eliminate the root of a problem and achieve a balance between one’s emotional, physical, and spiritual health.

In contrast to the western approach on fast responding medication that can suppress symptoms as well as a standard methodology to treat diseases and symptoms, CHM judges a patient as a whole and utilizes the syndrome differentiation theory. This theory analyzes the harmonic pattern inside a patient’s body based on the core beliefs of Qi to make a diagnosis of the illness (Jiang et al., 2012). As such, most CHM has not been clinically tested due to their belief that isolating a symptom and targeting it is an incorrect way of treating a patient which would only temporarily fix the problem.

Although the beliefs surrounding the Chinese medical system have been utilized for over 2500 years, it is important to note that all aforementioned beliefs are not defended by clinical research or trials and cannot be deemed reliable.

Dosage and Usage of TCM

In China, the Chinese Pharmacopoeia outlines stipulated dosages of CHM as a guideline for Chinese practitioners. However, a 2014 study found that practitioners often overlook the guidelines and prescribed quantities far greater than stipulated dosages. The study concluded that guidelines in the Chinese Pharmacopoeia are not comprehensive enough for clinical usage to match with stipulated dosages (Ni et al., 2014).

The majority of CHM is often primarily based on the experience of the TCM practitioner due to unspecified guidelines in the pharmacopoeia. Furthermore, there is no dosage guideline for pediatric patients, nor patients who are overweight (Marshall, 2020). This may lead to overdosing or underdosing of the medication. In contrast to WM which is set on a predetermined dosage, including medications used by children, there are no dosage guidelines for the pediatric use of CHM. Practitioners may use either their own clinical experience or the estimated body weight and age of the patient.

Among TCM practitioner visits, respiratory illnesses and symptoms were the most common reason for treatment. Respiratory symptoms are especially common in children. In a study based in Taiwan, 63.2% of children were found to have been treated with CHM at least once (Liang et al., 2018). However, due to the lack of dosage guidelines for pediatric patients, the usage of CHM may pose dangers.

Development and Research behind Medications

Chinese herbal medicine has been in clinical application for thousands of years and has also been under continuous and extensive research in hope of developing new formulas. As previously stated, the discovery of CHM is attributed to the Chinese belief in Qi as the vital life force, as well as the balance between Yin and Yang; however, while that may be true, Chinese herbal medicine is a collection of theories that have been altered based on years of experiments, observations, and anecdotes. According to Doctor Chen-Pien Li, herbal medicine was slowly developed by ancient scholars and governmental institutions, creating remedies and formulas that have since been passed down (Li, 1974). However, questions regarding its scientific basis and reliability have continued to grow with the development of modern science. In fact, many claim that certain CHM can be harmful to the human body.

However, it should be noted that some CHM has been the foundation of some WM. For instance, the calming effects of chamomile tea can be attributed to discoveries by Chinese herbal medicine in the Tang Dynasty. It was said to act as a healing coolant against the fire and heat within one’s body in the Ming Dynasty (Yang et al., 2014). Researchers are now conducting studies with regards to the concepts of TCM to see if tea plants can act as preventive medication. Furthermore, many current researchers aim to further develop CHM. Professor Si-Yuan Pan of the University of Beijing stated that researchers have been exploring ancient remedies and modernizing them. Studies now aim to modernize the method of consumption as well as some formulas (Pan et al., 2011). Researchers have been attempting to alter the method of consumption partly by extracting key ingredients from herbs and increasing the concentration, which then enables the possibility of forming it into granule pills.

Nevertheless, the modernization of formulas is essential. Some risks regarding the ingredients used in CHM have been identified by Chinese scholars, which are now under research and analysis. According to Dr. Gao RanRan of the Institute of Medicinal Plant Development, many scholars have been undergoing research involving the careful inspection of the quality and safety of the products (Gao et al., 2019). Therefore it is important to acknowledge that although TCM is one of the oldest forms of medical practice that still exists today, the medicine was slowly acquired through a collection of experiences. Since not all herbal medicines have been carefully and thoroughly inspected, patients should be cautious and always refer to a physician before ingesting CHM.

Adverse Effects of Chinese Herbal Medication

The lack of clinical trials and sufficient research into adverse effects in TCM has called its use in question. Natural remedies such as TCM often advertise as side-effect free and fail to record complications such medication can result in. Users of TCM may be unaware of adverse effects and may be unaware of complications from TCM. There are currently few regulations regarding herbal medications and the lack of strict oversight of TCM can lead to overlooked ill-effects (Chua et al., 2015). Thus, complications and contributions to illness or death from TCM are unable to be determined. Although few clinical studies have been conducted in China, other countries have begun to research adverse effects some herbs and traditional medication can result. For example, an Australian journal noted that because the contents and preparation methodology of herbal medications is not tightly controlled, products may contain illegal or harmful ingredients (Byard et al., 2017). Concentrations and ingredients may also be labeled incorrectly, which could lead to users ingesting the incorrect dosage.

According to the National Center for Complementary and Integrative Health, there have been documented cases of TCM containing traces of unapproved animal or plant products. Furthermore, some herbal medications contain drugs such as arsenic, lead, and pesticides. Combined, such ingredients can result in serious injury or death, especially in younger children (Hopp & Shurtleff, 2019).

Another possible complication is the possibility of drug interactions when treatment is utilized by both WM and TCM. When WM is used as a primary drug and TCM is added as a supplement, herbal medications may potentiate or antagonize the properties of WM, resulting in under absorption and possible ineffectiveness of WM. For example, one known interaction is between Danshen and Warfarin (used for blood-thinning properties). A case report found that the interaction between Danshen and Warfarin resulted in significant anticoagulation in patients (Chua et al., 2015). Danshen has also been reported to increase the concentration of warfarin by inhibiting hydroxylation. However, sufficient clinical research has not been conducted between the two medications, and patients are encouraged to avoid Danshen while taking Warfarin.

Conclusion

Although a handful of research studies have already been conducted on CHM and TCM, more clinical studies are needed in order to confirm the safety and efficacy of many herbs and treatments. While some research has shown that TCM does correlate to modern science, other studies have shown adverse effects instead. Therefore, it would be critical for the public to be educated on the risks and to be cautious of the consumption of CHM, especially for younger children and older adults.

Furthermore, the increased consumption of TCM worldwide should encourage further extensive research on treatments that address the complications and possible interferences of taking both TCM and WC. Steps should also be taken by both western and traditional Chinese physicians to inform the patients of the risks of a combination of WM and TCM.

Works Cited

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https://www.mja.com.au/journal/2017/206/2/what-risks-do-herbal-products-pose-australian-community.

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Chua, Y. T., Ang, X. L., Zhong, X. M., & Khoo, K. S. (2015, January). Interaction between warfarin and Chinese herbal medicines. Singapore medical journal. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325561/.

Ergil, K. V., Kramer, E. J., & Ng, A. T. (2002, September). Chinese herbal medicines. The Western journal of medicine. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1071750/.

Gao, R.-ran, Hu, Y.-ting, Dan, Y., Hao, L.-jun, Liu, X., & Song, J.-yuan. (2019, December 20). Chinese herbal medicine resources: Where we stand. Chinese Herbal Medicines. https://www.sciencedirect.com/science/article/pii/S1674638419301029#.

Hopp, C., & Shurtleff, D. (n.d.). Traditional chinese medicine: What you need to know. National Center for Complementary and Integrative Health. https://www.nccih.nih.gov/health/traditional-chinese-medicine-what-you-need-to-know.

Jiang, M., Lu, C., Zhang, C., Yang, J., Tan, Y., Lu, A., & Chan, K. (2012, January 31). Syndrome differentiation in modern research of traditional Chinese medicine. Journal of Ethnopharmacology. https://www.sciencedirect.com/science/article/pii/S0378874112000463.

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Liang, H.-F., Yang, Y.-H., Chen, P.-C., Kuo, H.-C., Chang, C.-H., Wang, Y.-H., & Wu, K.-M. (2018, June 22). Prescription patterns of traditional Chinese Medicine amongst Taiwanese children: A POPULATION-BASED cohort study. BMC complementary and alternative medicine. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013984/.

Marshall, A. C. (2020, March 2). Traditional Chinese medicine and CLINICAL Pharmacology. Drug Discovery and Evaluation: Methods in Clinical Pharmacology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356495/.

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Ni, S. L., Chen, C. R., Fu, Y. L., Zhang, L., & Song, J. (2015). Chinese herbal medicines – comparison of DOSES prescribed in clinical practice and those in CHINA PHARMACOPEIA. Tropical Journal of Pharmaceutical Research, 14(1), 171. https://doi.org/10.4314/tjpr.v14i1.24

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About the author

Nicole Zhou, Tsu-Yi Zoe Lee

Nicole and Zoe are aspiring physicians with interests in subjects regarding Biology, Chemistry, and Health Studies.

The post An evaluation of the methods and research surrounding the usage of Chinese Herbal Medicine appeared first on Exploratio Journal.

1. Introduction

Curious about what affects the death rates of respiratory diseases, especially for young children, I use statistics to discover the factors related to the death of respiratory diseases, such as region, income group, and the trend in recent 20 years. Wondering whether there may be a link between air pollution and respiratory diseases death rate, I also explore some data on PM2.5, the suspended particles in the air with a diameter less than 2.5 microns. RStudio is used to process, visualize and analyze data.

2. Explanation of Data Sources

Generally, three data sources are used, one about the deaths of lower respiratory diseases, one on populations, and the other on PM2.5 – the links are attached in the end, in the reference list. 

The first source is on the deaths of lower respiratory diseases from World Health Organization. This source contains the data on the number of children’s death from different diseases in different countries from 2000-2017, with the purpose of finding the causes of children deaths to increase children survival. According to World Health Organization (n.d.), while it has data from “civil registration with complete coverage (80% or over) and medical certification of cause of death, or nationally representative epidemiological studies of causes of child death”, it also has some kinds of estimations from WHO and the Maternal Child Epidemiology Estimation group (MCEE), using the original data and computing it with regard to the proportion of population of young children. Also, for low-mortality countries without adequate data, a multinomial model is applied; for high-mortality countries without adequate data, “a multinomial model applied to (largely) verbal autopsy (VA) data from research studies” is used. Useful data can be extracted from this, as the number of children deaths on lower respiratory diseases can be attained through filtering, and the age group is filtered to be 0-4 years. In this case, the data of the deaths of lower respiratory diseases can be attained. 

The second source is on population, from The World Bank. As it’s reasonable for countries with greater population to have more deaths from lower respiratory diseases, it’s necessary to attain the data on population. In this data set, we have population from different countries in 1960-2019. The population here is about all residents, either citizens or legal status, and it’s a midyear estimate. Also, what’s important here is the metadata of this data set, which categorizes countries by region and income group (Worldbank, n.d.). The categorization of income group is based on its GNI per capita (Serajuddin and Hamadeh 2020). 

Last but not least, data on PM2.5 can also be attained from The World Bank. It’s about the exposure of PM2.5 air pollution in micrograms per cubic meter. The exposure is calculated from mean annual concentrations of PM2.5 and the population in the region (Brauer et al, 2017).

3. Data Analysis

3.1 Preparation Work

To begin with, some preparation work was necessary. The tidyverse package was loaded, and also a RData file, named “AllData”, for the prepared data that has been cleaned and processed orderly was loaded. Also, the income group was reordered from high income group to low income group, instead of mere alphabetical order. Meanwhile, a list of years has been created for later usage.

3.2 What Affects Death Rate in a Given Year

3.21 Death VS Population Plot

For this first plot, this is about Deaths VS Population. It appears that the direct plot of 2017 is hard to analyze since many dots are extremely near each other (and same for other years).

Therefore, both deaths and population have been taken log in order to make the plots more wide-spread for better analysis, which is gg1. The plot appears to be related to region. In the plot of 2017, it can be discovered that most dots at the top of every vertical line are in pink, which indicates that with the same population, regions of Sub-Saharan Africa tend to have more deaths of lower respiratory diseases. For those at the bottom, they are likely in yellow, which shows that they are in Central Asia or Europe. Also, there are some correlations with income groups as well. Dots at the top are likely to be in either circle or triangle, indicating that they are in either lower or lower-middle income. Also, for almost all the dots that are at the bottom, they are in plus sign, which shows that countries in high income usually have less deaths. This pattern appears the same throughout the years, and its correlation with income group is even stronger in earlier years. Therefore, its relationship with regions and income groups can really be further explored.

 Regression lines for different regions in 2017 are drawn here. Since some death values are 0 and can’t be logged, all death values are added by 0.01, so that those values can be shown on the graph while the graph aren’t being affected too much. It appears that the linear regression here works well, as most points fall near the line, so the lines fit the regions well. The regression lines of most regions follow the pattern of the same slope, meaning they have the similar death rates, especially the red line, about East Asia & Pacific. Also, some distinctions between regions can be observed, such as Sub-Saharan Africa is often at the top, indicating a higher death rate while North America, Europe & Central Asia are always at the bottom, with a relatively lower death rate. Also, the variation can be observed, as pink dots of Sub-Saharan Africa are closer to the line, indicating higher death rates are consistent across those countries, while the yellow dots of Europe & Central Asia are more wide-spread, so that variation occurs – there are even several dots at the very bottom of the graph.

 Next, the regression lines for different income groups in 2017 are drawn using the similar method, and the difference is even more distinct – there are no crosses and intercepts of any two lines. At any population level, the deaths from lower respiratory diseases are always higher at lower income countries and decreases as the income group goes from low to high. Also, it appears that each income group has a fixed death rate and there is smaller spread or outliers, besides the five higher-income countries with an extremely low death rate.

 After that, in order to see how important the factor of income group is, the death per population in a specific region is plotted by income group. In general, the distinctions between lines in each region are clear, with lower income at top and higher income at the very bottom. However, we do have some lines tangling together, and one possible reason is that there are few data available here. For example, there are only two red plots (lower income) in “Middle East & North Africa”, so its regression line is definitely highly inaccurate, affected by one outlier. However, in a more general perspective, the death per population is still highly related to the income level, which shows that economic factors still play an important role in this process.

 Similarly, the death rate in a specific income group by region can be plotted. In all the four graphs below, there are lots of tangles between the lines, and the differences between the lines aren’t very significant, while the differences between income groups are rather more significant, as the slope become steeper from higher income groups to lower income groups (besides the several outliers in high income groups in Europe & Central Asia). However, there are some subtle distinctions here that the pink line, about Sub-Saharan Africa, is usually slightly higher than other regions, even in the same income group; meanwhile, the yellow line, about Europe & Central Asia is slightly lower than others in the same income group. Perhaps these interesting facts are due to something besides economic factors, and related to some environmental and geographical factors. Besides, there may be some inaccuracy here due to lack of data, as there are only two pink dots (Sub-Saharan Africa) in high income countries, one light green dot (Latin America & Caribbean) in low-income group. 

3.22 Display Death Rate Directly

Then in order to evaluate the ratio of deaths and population directly, a new variable of “Death / Population” (dividing deaths by population) is created, which is literally the death rate.

Firstly, the histograms of the logs of death rates by different income groups are plotted. It’s necessary to mention here that since some countries have 0 deaths, the death rate value can be 0 – so all values are added by 0.0000000001 ( ) to make those value appear on the graphs (same process is done for all plots below related to the new variable of “Death / Population”, which won’t be repeated later). As shown in the plot of 2017, the difference between different income groups is vast. Lower income countries have a significantly higher death rate than higher income groups. The low income has almost all countries greater than the median death rate (except one outlier, which is Syrian Arab Republic). Lower-middle income has majority of countries’ death rate higher than median value while higher-middle income has majority to be lower than median value. For high income countries, they are almost all below median death rate (except one outlier, Nauru). This shows the direct link between income groups and death rates. Also, for low-income groups, the data appears to be more concentrated, mostly between -9 to -7 on the histogram. However, for upper middle income countries and high income countries, their data are pretty wide-spread. Therefore, this reveal that the variations of death rates between different richer countries are greater than in poorer countries. Perhaps this is due to some environmental factors, or people’s attitudes towards the lower respiratory diseases in developed countries, since economic factor is no longer significant when the comparison occurs inside one income group and all countries are rich. 

Similar situations can be observed throughout the years from 2000 to 2017 – the general pattern is rather static. However, such distinction between income groups does become clearer throughout the years. In earlier years, the difference of the pattern between lower middle income and low income isn’t as significant as it is today while for upper middle income, the death rate is half greater than median, half smaller than median. At that time, the advantage of high income countries is massive. Perhaps this is a good thing that through the recent decades, middle income countries become better and better at health care so that they have a relatively lower death rate in those years. However, problem still remains for lower income countries.

3.3 Trend of Death Rates Throughout the Years

3.31 In a Given Country

To begin with, the trend of death rates in several countries are explored – US, China, India and Haiti are selected, representing different income groups and different regions. It’s exciting to discover that all four countries experience a significant decrease in the death rate in 2000-2017. Also, it appears that countries in relatively lower income groups (such as India and Haiti) have a much sharper decreasing trend, almost linear. For China, as upper middle income, its death rate decreases intensively and the curve is growing flatter over time, just as a parabola. For US, the high income, the decreasing trend is much flatter and there are more twists and turns in the 18 years.

3.32 By Income Group

After that, a boxplot of different income groups’ death rate trend by year has been made. Obviously, throughout the years, the death rate of lower income countries is always higher than higher income countries and all of them are decreasing over time. The death rate of lower-middle income and upper-middle countries are decreasing steadily and most intensively among all income groups. 

 For low-income countries, they do decrease constantly in the 18 years, but the scale is relatively small, so their death rates still stay high over the years. However, we have an outlier here among low-income countries, always lower than usual low income boxplots, which is Syrian Arab Republic (Syria).

For high income countries, the decrease over the 18 years isn’t significant though, and there are a lot of twists and turns throughout the decreasing process. However, what’s impressive is that there are several countries that have death rate to be 0 for years (revealing here:  , which is the set of dots at the very bottom). For the dot that decreases intensively from 2009-2011, it’s Andorra – the country decreases intensively and steadily in those years and the death rate becomes 0 since 2012. Besides, countries such as Iceland, Cyprus also have multiple years of death rate being 0. 

 Besides the boxplot, geom_smooth is also used to regress the lines for different income groups. The first graph is what I plot at first, without log. While all income groups are drawn in one graph, the low income countries appear to decrease dramatically, and the slope is flatter for middle incomes and almost totally flat for high incomes. Also, the gray confidence interval for mean death rate appears wide-spread for low income countries while tiny for high income countries.

  This is confusing, since from all my exploration above, high income countries’ death rates are often widespread and differ from each other a lot. Perhaps it’s because we didn’t take log at this graph. So the second graph is plotted by taking the log of the death rate. This time, as predicted, the gray confidence interval is extremely big for high income countries; this gray confidence interval is relatively small for low income countries. Also, the decreasing trend is most intensive in middle income countries, which is consistent with what we’ve observed in the boxplots. The reason behind is possibly related to the percentage of the decrease: for middle income countries, they experience a dramatic decrease in their percentage of death rates in log scale, while lower income countries, although decreasing a lot in number, don’t have a significant decrease in the percentage. Besides, it appears that in any cases, the gray confidence interval is always constant throughout the years, revealing that the changing rate of the death rate is often constant throughout the years.

3.33 By Region

  Using the same method, the boxplot for different regions throughout the years can be drawn as well. For all regions, the decreasing trend isn’t as significant as income groups, but still exists. Europe & Central Asia and North America typically have low death rates, and Europe & Central Asia contains some countries with death rate of 0. In contrast, South Asia and Sub-Saharan Africa countries usually have higher death rates. However, they often have several outliers with death rate lower than usual. Probably this result is also somehow related to the economic situation, as Europe & Central Asia or North America has countries typically of high income or upper middle income while South Asia or Sub-Saharan Africa are typically of lower income groups.

Also, the smooth plot for different regions is here. Still, without log scale, regions with higher death rates (Sub-Saharan Africa & South Asia) decreases more intensively. 

Things become uncertain after it’s logged. South Asia appears to decrease the most greatly this time and Sub-Saharan Africa is flat this time. East Asia & Pacific decreases slightly but steadily over time. For other regions, it’s no longer linear, as the line goes up and down and even the decreasing trend is unclear, showing that on a general level, the change in percentage doesn’t vary much by regions.

 3.4 About PM2.5

As respiratory diseases are related to respiratory system, and the health of respiratory system is easily affected by air pollution, an assumption is made initially that there may be some links between the death rate of lower respiratory diseases and PM2.5 exposure. This is explored with the PM2.5 data.

3.41 By Income Group

 Firstly, the relationship between PM2.5 exposure and income groups is analyzed, as shown in the histogram below. For lower income groups, they are more likely to have a higher PM2.5 exposure than the median value. This is consistent with the initial assumption and observation of death rate, as lower income groups have a higher death rate. However, when it comes to higher income groups, for countries in upper middle income and high incomes, their PM2.5 exposure spreads almost evenly on the two sides of the median value. Meanwhile, for death rate of respiratory diseases, both two income groups are always on the smaller side compared to the median value. So this makes people question whether there is really a link between PM2.5 exposure and death rates.

 3.42 By Region

For the histogram of PM2.5 by regions, similar situation occurs. For Sub-Saharan Africa and South Asia, countries are likely to have a higher PM2.5 exposure than median and they are the regions with higher death rates. However, for Europe & Central Asia, it’s evenly spread on both sides of the median value. For North America, it’s even greater than the median value. At the same time, the two regions have the least death rates among all, which is out of the original expectation.

3.43 Trend of PM2.5 through Time

The following two graphs are about PM2.5 and income groups, not logged and logged. In either case, the lower income group increases greatly while all other income groups mostly remain constant and may increase a little bit. This reveals that the original assumption may not be correct, since PM2.5 generally increases while the lower respiratory diseases death rates decrease dramatically over time, among all income groups.

The same process has been done for different regions. As shown, the gray confidence interval is extremely wide separated, revealing how different countries vary even though they are in the same region. Meanwhile, the general trend is still increasing for almost all regions, inconsistent with the decrease within death rates. Also, what’s worth noticing here is that for North America, its increase is massive throughout the years, but it usually has relatively low death rates constantly. Throughout such observations, it can be concluded that perhaps there isn’t much correlation between PM2.5 and death rates of lower respiratory diseases.

4. Conclusion

After this data analysis, it finally comes to some conclusions about what influences the death rate of lower respiratory diseases. 

Firstly, the death rate is strongly related to the economical situations of countries. In general, higher income countries usually have a lower death rate, while lower income countries have a higher death rate. Perhaps this is related to the level of health care in the countries. Obviously, for richer countries, they are able to provide better health care service to children, so that for children who have lower respiratory diseases, they can attain enough caring and get cured as soon as possible. For poorer countries, they don’t have enough money to provide such health care. In this pattern, the death rates can also represent how health care grows in 2000-2017. Throughout the years, even though all income groups have their death rate decreasing, lower income countries experience a more intensive decrease in death rate while higher income countries remain stagnant. Also, the decrease in middle income countries is significant under log scale. All of these reflect the link between income groups and death rates. 

There are also some factors besides mere economical factors. As can be seen, under same category of income groups, still regions such as Sub-Saharan Africa or South Asia still experience a higher death rate. Therefore, perhaps there are some other factors besides income groups, such as cultural factors, or environmental factors. This still needs further exploration to decide what makes some regions have a higher or lower death rate under the same economical level. 

Meanwhile, regarding to the assumption of the link between death rate and PM2.5 exposure, it appears that perhaps there aren’t much correlation between the two. While the PM2.5 exposure increases generally in 2000-2018, the death rates decrease intensively in any cases in those years. For the only consistence that low income countries have high PM2.5 exposure and high death rates, possibly it’s just a coincidence since the two matters can be explained in the perspective of income group separately. PM2.5 exposure is high because the country doesn’t have money to spend on clearing the air pollution. Death rates of lower respiratory diseases are high because the country doesn’t have enough money to provide children with good health care. Thus, PM2.5 and lower respiratory diseases death rates aren’t correlated. 

But anyway, children health is often an essential topic in our society. As more countries become richer and our levels of health care improve, there will definitely be less children dying from lower respiratory diseases. The trend is pretty good in 2000-2017, and hopes the death rate will continue to decrease in the future, so more children can live more happily throughout their childhood without the pain of diseases and death.

5. Reference List

Mentor: Dr. Peter Kempthorne, Massachusetts Institute of Technology

• Brauer, M., et al. “PM2.5 Air Pollution, Mean Annual Exposure (Micrograms per Cubic Meter).” Data. The World Bank, 2017. https://data.worldbank.org/indicator/EN.ATM.PM25.MC.M3.
• “Number of Deaths in Children Aged <5, by Cause.” World Health Organization. World Health Organization. Accessed April 5, 2021. https://www.who.int/data/gho/data/indicators/indicator-details/GHO/number-of-deaths.
• “Population, Total.” Data. The World Bank. Accessed April 5, 2021. https://data.worldbank.org/indicator/SP.POP.TOTL.
• Serajuddin, Umar, and Nada Hamadeh. “New World Bank Country Classifications by Income Level: 2020-2021.” World Bank Blogs, July 1, 2020. https://blogs.worldbank.org/opendata/new-world-bank-country-classifications-income-level-2020-2021#:~:text=The%20World%20Bank%20assigns%20the,i.e.%202019%20in%20this%20case.

About the author

Yi Zou

Yi is a rising junior at the Starriver Bilingual School in Shanghai, China.

The post Factors behind the Death Rate of Lower Respiratory Diseases appeared first on Exploratio Journal.

Abstract

 Stem cells have been a prevalent research field for their versatile use as a potential treatment of several different diseases that have remained untreatable, such as Alzheimer’s disease. These cells are unspecialized cells that can replicate themselves indefinitely (self-renewal) and give rise to more specialized cells (differentiation). Due to these unique properties, stem cells can be described as the foundation for all tissues and organs in the human body. Stem cells can be mainly classified into 3 categories: embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and adult stem cells. The different distinctive characteristics of each category of stem cell as well as their applications in the potential treatment development of Alzheimer’s Disease are discussed in this manuscript.

1. Introduction

In the last decade, stem cells have raised great interest in the scientific community due to their promising use in the fields of regenerative medicine, drug testing and discovery and modeling of healthy and diseased tissues. Stem cells are undifferentiated cells that can be isolated from embryos (embryonic stem cells, ESCs), from adult tissues (adult stem cells) or can be generated by reprogramming somatic cells, such as dermal fibroblasts (induced pluripotent stem cells, iPSCs) (Zakrzewski et al, 2019, Shi et al, 2017).  The three different types of stem cells each have potential advantages and disadvantages that may encourage or limit their use for medical applications. Embryonic stem cells are pluripotent, meaning that they can differentiate or specialize in any cells of the body, with the exception of the extraembryonic cells, which constitutes the placenta and the umbilical cord (Zakrzewski et al, 2019).  While pluripotency and plasticity makes ESCs the most desirable cell type for regenerating diseased tissues, ESCs raise the ethical issue of using them since their isolation from the inner cell mass destroys the embryo (Abdulrazzak et al, 2010). Additional to their ethical concerns, because of their pluripotency and proliferative ability, they can form tumors after transplantation (Hess et al, 2019). Because of their limited proliferative capacity, adult stem cells do not raise this issue, however they are much harder to culture in vitro and are considered multipotent or unipotent, in that they can only differentiate into fewer or just one specific cell type. Induced pluripotent stem cells are a rather new discovery so not much is known about them on their use for potential treatments, however they are the most promising because of their versatility and easy access (Shi et al, 2017). Similar to ESCs, iPSCs have the potential issue of tumor formation due to their proliferative ability. 

2.1. Embryonic stem cells (ESCs) 

Embryonic stem cells are stem cells that are found in the inner cell mass of a blastocyst. A blastocyst is a bundle of cells formed 5-6 days after fertilization of the oocyte by the sperm cell that undergoes cell division by meiosis (Yu and Thompson, 2006). ESCs have been differentiated in multiple  cells types such as cardiac cells (Liu et al, 2018) , vascular smooth muscle cells (Cheung et al, 2011) , nerve cells (Magown et al, 2017, Jones et al, 2018) and retina cells (Lakowski J, 2015,Mehat et al, 2018) to form healthy tissues that can be transplanted in injured or diseased areas of the body. ESCs can be easily identified and isolated from the inner cells of the blastocyst (Sills et al, 2005) and can be cultured and grown indefinitely in a lab setting to be used for research in medicine and science. These extracted stem cells are extremely valuable and have shown great potential for regenerative treatments as well as for drug development and testing (Yu and Thompson, 2006).

2.2. Induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cells are derived from somatic cells such as skin or blood cells and are reprogrammed into an embryonic-like state (Shi et al, 2016). Researchers associated pluripotency, unique to ESCs, with genes or factors that are only expressed by ESCs. In 2006, Shinya Yamanaka, identified four genes (Myc, Oct3/4, Sox2 and Klf4) with encoded transcription factors that could convert somatic cells into pluripotent cells (Zhao et al., 2013). The reprogramming of somatic cells with the introduction of these four genes led to the discovery and use of iPSCs which have paved a way to more efficiently identify and model disease cells that were not very successful in animal models. After their discovery, iPSCs have provided a strong headway into regenerative medicine, to repair damaged cells, tissues or organs. When conducting a normal tissue or organ transplant, it is imperative that the cell, tissue or organs donor’s physiological profile matches that of the patient. Not being able to meet these specific conditions is one of the common reasons patients die in urgent situations of accidents or patients who have been suffering degenerative diseases. The use of iPSCs, that can be reprogrammed from the healthy cells of the same patient, can greatly reduce the risks that come with transplants. Since iPSCs can be directly generated from skin and blood cells of patients, the cells which will be transplanted are from the patient’s own body. In addition, patient-specific iPSCs allow researchers to look more closely at the disease relevant cells in the patient’s body.

2.3. Adult stem cells 

In contrast to embryonic stem cells, adult stem cells or somatic stem cells are stem cells found in the adult body, especially in the bone marrow, blood vessels and in the adipose tissue (Stem cells, 2001). They are found in the adult tissues and are more difficult to expand in a lab setting since they do not duplicate as easily as ESCs and iPSCs (Stem cells, 2001), therefore they can be cultured for a lower number of passages in vitro, yielding smaller numbers of cells. These cells are more specialized compared to ESCs and iSCs and usually only give rise to limited types of cells, dependent upon the tissue type they have been isolated from (Stem cells, 2001). For example, stem cells found in the bone marrow differentiate into red blood cells, white blood cells, and platelets, however, they will not differentiate into cells of other tissues, such as liver, or brain cells. In a few cases, it has been demonstrated that adult stem cells of various tissues could be reprogrammed into IPSCs (Labusca et al, 2019) 

3. Use of stem cells for treating Alzheimer’s disease (AD)

Alzheimer’s, a disease characterized by memory loss and cognitive impairment has troubled society due to its unknown causes as well as lack of treatment for the disease. With the transplantation of stem cells, researchers can investigate how these regenerative cells could potentially slow down the development of AD in the brain as well as using them as new drug screening platforms by differentiating them into multiple brain cell subtypes.

3.1 In vitro models of AD 

The use of stem cells for the development of in vitro platforms for drug screening and discovery allow to create patient-specific models of diseases, including AD. With these models, it is possible to examine the effects of promising drugs on the cell types most relevant to AD and to screen through a variety of compounds that directly target parts of the brain in relation to the disease itself.

In this context, stem cells are used as a source to differentiate healthy cells of the brain for studying molecular pathways and physiologic/pathologic cell phenotypes in vitro. In particular, ESCs and iPSCs have been differentiated into different cells of the brain, including distinct neuronal and glial cell subtypes, as well as astrocytes (Little et al, 2019).

Furthermore, cells from patients carrying specific mutations associated to AD have been isolated and reprogrammed to iPSCs and further differentiated into neurons, to determine the optimal, patient-specific treatment to attenuate the effects of the pathology (Yagi et al, 2011, Israel et al, 2012, Muratore et al, 2014).

3.2 Stem cell therapy for AD

In addition to drug screening applications, stem cells can be directly used as therapeutic agents to slow down the progression of the disease. 

Arnhold et al. demonstrated for the first time the possibility of differentiating ESC-derived neural precursor cells into neurons and astrocytes directly after transplantation in adult rat brain (Arnhold et al., 2000). The authors also demonstrated that the precursor cells were mature and fully functional after transplantation. Several studies showed that after transplantation, ESCs are able to integrate with other cell types in the brain (Nasonkin et al., 2009) and secrete reparative molecules to regenerate injured regions (Zhang et al., 2006). Furthermore, it has been demonstrated that the ESC-derived neuron precursors are able to integrate with the host brain tissue. Aubry et al (year) suggested that the optimal number of cells to be implanted has been found to be 15×103 cells per animal.

iPSCs therapy is relatively new for Alzheimer’s disease. There are several studies in literature focused on the use of iPSCs for developing 2D and 3D models of brain diseases, including Alzheimer’s disease. Since iPSCs can be derived from AD patients, researchers have the ability to look at the specific genes and molecular pathways  associated with the disease in the neural cells (Shi et al., 2017). Furthermore, since these cells self-renew themselves, researchers can recapitulate how the disease grows and progresses and behaves in simple in vitro disease models (Shi et al., 2017).

Furthermore, it has been recently demonstrated that neurons can be successfully differentiated from fibroblasts (skin cells) reprogrammed into iPSCs (Liu et al, 2012). These cells have demonstrated similar morphology and function as these of the neural cells in the brain. Transplanted iPSCs-derived neurons are able to survive, maintain their function and even mature into the brain of mice models (Eckert et al 2015, Fujiwara et al, 2013). Promising therapeutic effects have been shown by Eckert et al, that demonstrated attenuated post-stroke effects and augmented neurological function in a mice model of stroke starting only 24 hours after the injection of iPSC-derived neurons (Eckert et al, 2015).

Fewer research studies are available that use adult stem cells for brain regeneration and AD. Stem cell therapy with adult stem cells often involves the use of mesenchymal stem cells (MSCs) and their expansion and  differentiation into neural cells (Liu et al., 2020) however these cells exhibited a low yield of differentiation and decreased stability compared to ESCs- and iPSCs-derived neurons when transplanted in vivo.

4. Conclusion

This paper describes just a few out of many examples of how stem cells can be used for the cure and research of AD. There are countless diseases in the world today with no known cure, but extensive research of stem cells could bring about countless possibilities and opportunities in which they can be used to cure untreatable diseases through applications in regenerative disease as well as disease modeling. Stem cell ability to differentiate into any cell type  and to recapitulate patient-specific diseases will allow for more ground-breaking methods and treatments to be discovered by studying more thoroughly at how the disease behaves. With more research on the use of stem cells for Alzheimer’s Disease, researchers will be able to look at the development of AD in neural cells and hopefully show promising results for a treatment that no one will fail to remember.

Stem cells which could be described as the most basic form of life were discovered about 30 years ago. These small cells which are in their simplest forms not only give rise to the most complex structures in the body but also show a hopeful future and solution for devastating diseases in the world today. It’s a gift in the simplest of essences yet in the most intricate of ways.

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About the author

Dana Chung

Dana is a student at the Coventry Christian School in Pennsylvania.

The post Stem Cells Therapy for Alzheimer’s Disease appeared first on Exploratio Journal.