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Microbial Community Structure of Canine GI Tract Analysis

Summary:
For this study, metagenomics was utilized to assess the genetic material of a group of canine fecal specimen.
To begin the metagenomics project, fecal material from 184 unique species of Labrador retrievers was obtained.
The total fecal microbial complement was determined by isolation, amplification of the 16S rDNA, and sequencing.
The entire project lasted 8 weeks. Weeks 5-7 were practice for isolation and amplification of soil samples. Week 7 involved the extraction of gDNA from the canine fecal samples. For week 8, PCR reactions were set up to amplify the 16S rDNA gene sequence from the isolated fecal gDNA. Finally, the amplified genomic DNA samples were run on agarose gel electrophoresis. Samples were sent to Anschutz Medical Campus to be sequenced.
The documentation of the data was provided on an online based platform known as QIIME.
Analysis on the findings was based on the success of the sequenced fecal samples, phylogenetic diversity, abundance of species, and correlations between gut microbiota and existing health conditions.
Abstract:
Gut microbiota is diverse as well as complex and plays a substantial role in the body of its host. This study was designed to test the microbial community structure of the GI tract of those canines that were treated with filtered and unfiltered well water and its effects on health. Bacterial species of the gastrointestinal tract was analyzed by using a combination of molecular techniques. Microbial community composition of the GI tract was assessed by extracting genomic DNA from canine fecal matter. The extracted gDNA was then run onto a gel made of a substance called agarose and the 16S rDNA was later amplified. The DNA obtained provided results in bacterial composition, diversity of taxa, and the types of microbial species. Microbiome profiles revealed no reported links between the composition of gut microbiota and the influence of water quality in canine fecal samples. Further research is needed to understand how water type impacts dispersal of microbiota in the gastrointestinal tract of canines and the effects on health.
Introduction:
Complete genome sequencing provides a comprehensive and detailed overview relative to an organism’s composition such as structure and function (Bello et al. 2012). Much research has been conducted relative to the types of microorganisms present in the gastrointestinal tract and the overall effects on the host’s health and behavior. Microorganisms inhabiting the gastrointestinal tract that of a dog plays an integral role that facilitates in nutriment, immune defense, and susceptibility to local and systemic disease. The ever-diverse microbial community with its high population density, form a myriad of networks inside a closely integrated ecologic unit in organ systems.
Organisms across all spectrums are exposed to various sorts of bacteria that can influence the genetic and metabolic diversity situated in the gut (Caporaso et al. 2014). Research suggests that ecological factors and host activities are important indicators of gut health immunity (Kirchoff et al. 2019). In this case, water content plays an important part in the overall concentration and dispersal of microbiota. Fecal pollutants vary depending on water filtration measures. Ingestion of water contaminated with several impurities may lead to a distressed system and cause various waterborne linked diseases. Pathogenic strains can challenge the immune system by colonizing the space and disrupt cell functions. Enzymes produced by microbes can influence the rate of reactions required for the breakdown of complex substrates (Bird and Conlon 2015). However, distinctions between health associated microbiota and harmful pathogenic microbiota are not always clear. Depending on the bacterial species, some may be induced with certain strains which promote health or cause disease (Browne et al. 2017).
Given these observations, I thereby hypothesized that water type and quality influences composition of the canine gut microbiome between different species’s communities. So far, no studies have explored a potential link between the gut microbiome and the effects of water quality that dogs are exposed to. Similar works point out a potential interaction between the dispersal of fecal bacteria in sea creatures and water type. For example, harmful bacteria found in aquatic environments such as metals, or toxins can disrupt several of the host’s health systems and cause proliferation of certain microbial communities (Hintz et al. 2005).
The objective of this exploratory analysis is to examine variability in the composition of bacterial communities between canines treated with unfiltered and filtered well water along the gastrointestinal tract and to measure the effects on health.
Materials and Methods:
Fecal Sampling
A total of 206 fecal samples were obtained from 184 pure bred Labrador retrievers along with complete medical and behavioral histories from the Morris Animal Foundation. Out of the 206 samples, 11 were repeated 3 times.
DNA Extraction
Total genomic DNA was extracted using the DNeasy Powersoil Kit made by a company called Qiagen per manufacturer’s instructions. To release bacterial DNA effectively from the fecal matter, chemical lysis was performed. Fecal samples were placed in PowerBead tubes already containing C1 solution and incubated at 60?C for 10 min, followed by a vigorous bead beating process with a vortex adapter, until thoroughly homogenized. The DNA in the tubes were then pelleted and transferred into a centrifuge and spun for a minute at 10,000 x g. The remaining solution (supernatant) was collected and pipetted into a clean 2 ml collection tube, whereby the pellet containing non soluble debris was discarded. The rest of the protocol was followed as recommended by the manufacturer. All samples were eluted in 50 ul of buffer and stored at -20 C until analyzed. The integrity of the nucleic acids was determined visually using the Lonza FlashGel ® System stained with agarose. The DNA concentration was measured using ultraviolet light illumination.
PCR and 16S rDNA Sequencing
PCR was performed from each sample to produce a fragment of the 16S rDNA gene and obtain profiles of the type and abundance of bacterial communities present in the canine fecal genomic DNA. Different pairs of barcoded primers were used to distinguish individual student samples. To analyze the PCR products, the amplified samples were run though gel electrophoresis stained with agarose using the Lonza system with modifications to input volume to prevent overloading the column. Those reactions that worked were combined into one pool of DNA. Subsequently, DNA was rid of its reagents and purified using the DNA Clean

Stem Cells and its Use in Therapy

Introduction
Stem cells are unspecialized cells found in the human body with the potential to become specialized. Stem cells have the ability to become and replace any cell in the body. This characteristic ability of stem cells has “great potential for future therapeutic uses in tissue regeneration and repair” (Biehl and Russell, 2009). In addition, stem cells can be directed to differentiate into different cells and used to treat diseases including macular degeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis (“Stem Cell Basics IV.”, 2016). These diseases are hard to cure and potentially life-threatening with a bleak outlook. Thankfully, after decades of research, doctors, and scientists can finally improve their patient’s conditions with stem cell therapy. Stem cell therapy is an umbrella term and refers to a wide variety of different treatments. However, stem cell therapy is relatively unexplored and still needs an enormous amount of additional research before the true potential and safe application of stem cells is discovered. The three most viable stem cell therapies are autogenic and allogeneic transplant, mesenchymal stem cells therapy, and therapeutic cell cloning.
Stem Cells Types
The term “stem cells” collectively refer to all the different types of stem cells found in the human body. The two main categories of stem cells are adult stem cells and embryonic stem cells. As the name suggests, embryonic stem cells are found in embryos and adult stem cells are found in adults. Embryonic stem cells are significantly more powerful than adult stem cells, being able to form nearly any cell in the body. Stem cells can also be categorized by the ability they have. Stem cells with the ability to replace any cell in the body are referred to as totipotent. These stem cells can develop into a fully functioning body. These cells are briefly present in the early stages of the zygote. Stem cells with the ability to replace most, but not all, cells in the body is referred to as pluripotent. Pluripotent cells are found in the inner mass cells of the blastocyst. Stem cells with the ability to replace a limited number of cells are referred to as multipotent. An example of multipotent stem cells is mesenchymal stem cells (MSCs). MSCs can differentiate into many different cells such as osteoblasts, chondrocytes, and myocytes.
Autogenic and Allogenic Transplant
Autogenic and allogeneic transplant has been around for nearly three decades and is an approved stem cell therapy. Allogeneic stem cell transplantation involves transferring the stem cells to a patient after high-intensity chemotherapy or radiation. Here lies the difference in an allogenic and autogenic transplant. In allogeneic stem cell transplant, the stem cells, specifically bone marrow stem cells, come from a donor but holds the risk of transplant rejection. In allogeneic stem cell transplant, the stem cells are harvested from the patient’s body and therefore free of any risk of transplant rejection. This treatment is often used for patients suffering from leukemia and other related diseases. When doctors use chemotherapy with or without radiation to kill cancerous cells, the blood-forming stem cells in your bone marrow also dies. To alleviate this, doctors usually add the collected stem cells into the patient’s bloodstream a day or two after the chemotherapy. The stem cells are added in the same way as a blood transfusion. As the days go by, the transplanted stem cells move to the marrow space in the bones where they gradually start to produce new blood cells. After a few weeks, newly formed blood cells such as red blood cells, white blood cells, and platelets should appear in the patient’s bloodstream.
Mesenchymal Stem Cells Therapy
MSCs are the major stem cells for stem cell therapy and is particularly effective in the treatment of tissue injury and degenerative diseases. Mesenchymal stem cells (MSCs) “exist in almost all tissues” (Wei, X., Yang, X., Han, Z., Qu, F., Shao, L., and Shi, Y., 2013). They can be easily isolated from the bone marrow, umbilical cord, fetal liver, muscle, and lung and can be successfully divided in laboratory settings. Despite its lack of cell variety when compared to ESC and iPS, MSCs hold one clear advantage over them. One key characteristic of ESC cells and iPS cells is their high potential for teratoma formation. A teratoma is an uncontrolled tumour made out of multiple tissues. MSCs, on the other hand, have nearly no chance of a teratoma formation. In nearly all of the clinical studies, “the engraftment of MSCs into damaged tissues via migration to enhance tissue repair/regeneration is a crucial process for clinical efficacy”, regardless of the type of organ or specific disease (Wei, X., Yang, X., Han, Z., Qu, F., Shao, L., and Shi, Y., 2013). Another key characteristic of MSC is that they have a tendency to travel towards to damaged tissue and inflammation. This allows for the continual repair of damaged tissue throughout the body. Although the treatment model is in place, product quality standards, and safety controls are still not available in most countries.
Therapeutic Cell Cloning and Drug Testing
Therapeutic cloning produces stem cells with the same DNA as the patient. To achieve this, the nucleus of the donor stem cells are removed and the patient’s nucleus is injected into them. With the transfer of the patient’s own cell nucleus, the issue of immunocompatibility and transplant rejection of human ESC cells would thereby be solved. Furthermore, stem cells are also expected to significantly shorten the time drug companies take to test new drugs for side effects. Modern drug testing involves extensive animal trials and even after animal trials, there is no guarantee that the drug will be safe for humans. However, using human tissue grown with stem cells to test drugs would be much more accurate. The genetic makeup of the grown tissue would be the exact same as the patient. Since the most prominent drug side effects affect the heart, liver, and kidney, doctors will grow large amounts of these tissues to test with. This would significantly lower the testing costs and decrease the time it takes to develop a new drug. Another benefit of therapeutic cloning is the introduction of personalized medicine. Therapeutic cloning will allow drug companies to develop and tailor drugs that are safe and effective for the patient’s needs and specifications. However, Article 18 of The Convention on Human Rights and Biomedicine or the Oviedo Convention declare that “the creation of human embryos for research is prohibited.” There is also a blanket ban on human cloning. These bans severely limiting the research capabilities of scientists around the world.
Current and Future Research
Naturally “much more research is needed to understand the full nature and potential of stem cells as future medical therapies” (Stem Cell Basics, 2015). Scientists still do not know how many kinds of adult stem cells exist, how they interact, and how they evolve. Current research is focused on the application of induced pluripotent stem cells (iPSCs) in an attempt to treat age-related macular degeneration, a common cause of blindness in older people. This small scale research is designed to analyze the safety of iPSCs transplantation into patients’ eyes. In addition, scientists are also focused on finding new methods of acquiring new stem cells. Future research is focused on the negation of iPSCs ability to promote tumour growth and metastasis. iPSCs have a tendency to form tumours in vivo because using viruses to genetically alter the cells can trigger the expression of cancer-causing genes or oncogenes. These are just a few of the many formidable hurdles that researchers still face. Researching the characteristics of stem cell is difficult and elusive and researching the safe application of stem cells are even more so. “Those are things we have to continually learn about and try to address. It will take time to understand them better,” Dr. Owens says. With all the scientific, regulatory, and financial challenges that lie ahead, “It’s unlikely that one entity could do it all alone. Collaboration is essential” (Stem Cell Basics, 2015).
Conclusion
All in all, there is a range of stem cell treatments ranging from allogeneic stem cell transplant and autologous stem cell transplant to mesenchymal stem cells therapy to therapeutic cell cloning. Despite the over exaggerated and often times completely false promises about stem cell therapy, there truly is great undiscovered potential for stem cell research and stem cell therapy to cure hard-to-cure diseases such as macular degeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis (“Stem Cell Basics IV.” , 2016). In order to ensure further progress in the field of stem cells, more funding must be dedicated to its research. Additionally, the public must be informed and familiarized with stem cells in order to alleviate the ongoing ethical debate over stem cells. Hopefully, in the future, stem cell research and therapies will be widely accepted by all, ensuring the advancement of research and enabling doctors to cure difficult diseases. Knowing that stem cells exist is different from knowing how to use them safely.

Biehl, J. K.,

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