Development And Characterization Of Primary And Immortalized Ovine Intestinal Epithelial Cells (OIECs) Essay Sample For College

The gastrointestinal wall of an ovine intestine is comprised of four anatomically distinct layers intended for digestion, absorption, secretion and protection. The ovine intestinal epithelial cells (OIECs) are the first line of defense against pathogens and underneath, a thin layer of connective tissue- lamina propria rich in immune cells. Initiation of innate immune response is triggered by recognition of pathogen-associated molecular patterns (PAMPs) by pathogen-recognition receptors (PRRs) such as Toll-like receptors (TLR). The goal is to understand the role of ovine epithelial cells in innate immunity.

Primary OIECs from a 3-day lamb will be used to generate cell line. To sustain the OIECs for research, cell death must be prevented by immortalization using Human Telomerase Reverse Transcriptase gene. Immortalized cells will be compared to primary OIECs. Immunohistochemistry will be used to characterize specific surface markers on these cells by exploiting the principle of antibodies binding to antigens.

Lectins binding assay with flow cytometry will confirm the expression for lectin binding profile by selectively identifying antigens in cell tissue. This procedure will provide important information on how OIECs detect and interact with pathogens. Eventually, we will achieve an OIEC line, characterize these cells and examine their role in ovine innate immunity.

Development and Characterization of primary and immortalized ovine intestinal epithelial cells (OIECs); analyze the lectin binding profile on these cells and the role in innate immunity.

Background & Primary Literature Comment by Microsoft Office User: Minimum of 1 page and 3 peer reviewed journal articles.Context:Provides clear awareness of the ‘big picture’; Why is this proposed question important/interesting in this field? What are the knowns that relate directly to the question? How does this experiment address the question?Accuracy and Relevance Content knowledge is accurate, relevant, and provides appropriate background for reader including defining critical terms. Comment by Adaeze Ifesi:

While the gastrointestinal tract epithelium must be accessible for nutrient absorption, ingestion of food is a potential source of pathogenic influx (Shipra and Miwako 2011). Presence of so many bacteria triggers potential immune system response. As food is swept along the digestive tract, epithelium cells of the mucosal membrane acts as the first line of defense by producing a range of antimicrobial factors including antimicrobial peptides (Wells 2011).

Enteric pathogens pose a threat to ovine livestock and can lead to animal death and money loss (Connor and Li 2010). Knowledge of how ovine intestinal epithelial cells (OIECs) develop, differentiate and fight off pathogens can provide insight on mechanisms to enhance nutrient uptake and protect against pathogens in ruminants. OIECs interact with enteric pathogens and display a significant role in mediating mucosal immune responses. Epithelia cells of the intestine do not only act as a barrier, interactions between antigen-presenting cells and OIECs regulate B and T cell responses to pathogens (Uprety 2018).

The gastrointestinal wall of ovine is similar to that of humans. Currently, ovine intestinal epithelial cultures are not available while intestinal epithelial cultures for bovine, guinea pigs and humans are available. Because of the difficulty in preserving primary OIECs for a long period of time, these cells must be immortalized.

Toll-like receptors (TLRs) are pattern recognition receptors that detect conserved molecular structures of microorganisms and instigate immune response (Shipra and Miwako 2011). In the presence of many bacteria, intestinal myofibroblasts are activated expressing toll-like receptors. To further research on the role of OIECs in innate immunity, Lectins are proteins or glycoproteins that bind complex sugar with high affinity. Lectins play a significant role in nonself recognition and immune responses (Nilsson 2016). With the use of lectins, the immune system achieves basic functions such as cell to cell adherence, programmed cell death and inflammatory modulation (Andrews 2014). Establishing and characterizing lectin binding profile of ovine intestinal epithelial cells (OIECs) can provide significant information on infectivity and tropism of various enteric pathogens.

Hypothesis Comment by Microsoft Office User: Testable and consider alternatives: Hypothesis is clearly stated, testable, and consider plausible alternative explanationsScientific merit: Hypothesis has scientific merit Ovine intestinal epithelial cells (OIECs) express various sugar moieties kin to initiate innate immune response. Immortalized cells express the same phenotypical characteristics as primary cultured cell lines. Lectin binding profile of OIECs provide important infectivity information.

Controls and Replication Comment by Microsoft Office User: Controls and replication: Appropriate controls (including appropriate replications) are clearly explained. Primary cell line of OIEC would serve as control. Immortalized cells using immunohistochemistry and lectin binding assay will be compared for phenotypic expression, lectin binding profile in pathogenic exposure and activity of pattern of recognition reception such as toll-like receptors. Experimental Design Comment by Microsoft Office User: Experimental design is likely to produce salient and fruitful results (tests the hypotheses posed).

Primary cultured cell lines are obtained from the ileum of a 3-day lamb. Collagenase digestion of cells has already been performed. The ovine intestinal epithelial cell will be grown in a mixture of Dulbecco’s Modified Essential Medium (DMEM) and high glucose media with 10% fetal calf serum, 1% antibiotics and 1% NEAA (Non-Essential Amino Acid). This would be incubated at 37 and 5% and supplemented with growth nutrients. The cells are already immortalized. Immortalization was done using Human Telomerase Reverse Transcriptase gene (hTERT) to prevent shortening of telomere important for cell longevity. After the establishment of primary cell lines, the cells are further characterized by biochemical and immunological techniques using immunohistochemistry.

Primary OIECs are compared to immortalized cells for TLR gene expression, growth time rate and lectin binding profile in innate immunity. The role of pathogen-associated molecular patterns (PAMPs) in innate immunity would be studied by identifying some conserved nonself recognition molecules such as Lectin. Flow cytometry would be used to test for lectin ability to bind to specific sugar expressed by OIECs. We will use Inhibition assay to confirm lectin binding specificity for accuracy.

Data selection Comment by Microsoft Office User: Proposed data collection is comprehensive, accurate, and relevant. Data will include the percentage of OIECs binding to lectin in vitro and percentage that did not. ICC/IHC imagery of both primary and immortalized cell lines will be included. Data analysis Comment by Microsoft Office User: Proposed data analysis is appropriate for hypothesis tested and appears to be the correct analysis with proposed data Data will be analyzed using two-tailed t-test at 5% significant level.

By Buying A Fake, We Sponsor Child Labor

Designer names like ‘Gucci’ and ‘Louis Vuitton’ have long taken home in the ever so aspirational fancies of the Indian urban-sphere. Relegated to whimsical fancies for many the vicarious is provided by the glut of fakes and counterfeits available from the local markets to digital marketplaces like e-bay or Facebook marketplace. Even the avant garde fashionistas try to save a buck or two if they can evade policing. Recently, anti-piracy raid seized staggering total of 62 bags worth Rs 5 lacs at Oberoi Hotel’s shopping centre. So the penetration and permeation has not left the hitherto unblemished and hallowed of places.

In this particular case, the duped customers were miffed as to how a five-star hotel after many customers and complained to the LV headquarters in Paris. The oversight agency Enforcers of Intellectual Property Rights (EIPR), an anti-piracy body was contacted by LV to get to the bottom of the matter. EIPR officials then alerted the cops who conducted the raid. Two accused were apprehended to boot. So what does it mean to label a product a counterfeit and not just a cheaper knock-off. Well, saliently, it must include another party’s federally registered trademark or wittingly using the other party’s trademark without the authorization.

The inspired knockoff designs however, remain outside the remit and protection of copyright and trademark laws. In India and more so in China, early 1990s witnessed mushrooming of quality-control foibles in food, medicine, and luxury products. With the proliferation of production of such haute couture stuff in China, tracing the supply chain becomes a herculean task as the fakes are produced in the same warehouses where the original craftsmanship is exercised. The phantomous third or midnight shift is where the world of counterfeits is forged and thrived.

The gamut of counterfeited and pirated goods is infinitely pervasive. From such handbags to luxury dress to Apple smart phones faking has long been devilishly alluring. In 2016, a report published by the Organization for Economic Co-operation and Development (OECD) estimated trade figures touching $461 billion and 2.5 per cent of World’s trade– tantamount to the GDP of a small country. There are nearly3 million consumers buying fake goods each year, not for the want of the real stuff or affordability but due to uncanny resemblance and matchless replication. Today we have the age of super fakes or the fulmination of triple-A fakes.

Whether it is the logos, material or manufacture details, etc, there are few substantial distinguishing parameters. The superfakes are created with greater care and precision so much so that they remain inscrutable to an untrained eye. Precisely why we now have people learning how to spot counterfeit goods of high-end products. They sift through minutiae, infinitesimal imperfections and trace irregularities As perInternational Anti-Counterfeiting Coalition (IACC), counterfeiters are elusive to tax nets and thus aid in funneling of laundered monies across the dark world of organized crime.

Apart from the obvious follies of inadequate quality and uninspired innovation the fact that these products are manufactured in sweatshops through coerced child labor puts the nail in the coffin. Notwithstanding that there are few who argue that counterfeits can act as gateway products. Countering the perceived mal-impact, they act as free endorsements for the signature product and thus foraging market penetration and garnering popular scope. These commentators even advocate an immanent public interest in such counterfeits. The sobering reality of counterfeiting is the anchoring and breeding of organized crime syndicates immersed in terrorism, contraband and human trafficking and child labor. Concomitantly, they are causing massive damage to the brands that are at the cutting edge of fashion, design and innovation and resultantly destroying creative energies and inventive synthesis. So if one can’t muster curry and buy the high street fashion, it is best to shy away from the street corner faux pas.

Transition Metal Complexes With Non-innocent Ligands And Their Biological Applications

From the literature, I propose to design a redox-active ligand, their transition metal complexes and then to determine the molecular, electronic structure, and applications of such complexes and finally to systematically investigate the redox-driven reactivity properties at the metal sites. During this work, organic as well as inorganic synthesis, separation, purification and characterization of organic/inorganic molecules will be done.

Ligands can be broadly classified as either innocent (those that do not take part in redox reactions) or non-innocent (those that can undergo redox reactions). This redox non-innocence of the ligands has made describing the electronic structure of such complexes challenging. As these redox active metal complexes play a crucial role in catalysis and multielectron reactivity so for the next upcoming years research will be based on its application of metal complexes with non-innocent ligands. One family of bidentate non-innocent ligands includes o–phenylene diamines, catechols, and o–aminophenols. These can exist in three different oxidation states that are depicted, starting from the fully reduced o–amidophenolate dianon, in Scheme.

Non-innocent (Redox-active) ligands, o-Catecholate, o-Phenylenediamine, o-Benzene dithiol, o-Aminophenol, o-Aminothiophenol, are the best studied ligands that are capable of redox-active upon metal complexation. The coordination chemistry of the metal-coordinated ligand radicals has been intensively studied in recent years due to its occurrence in the active site of many metalloenzymes. Extensive efforts have been made to provide valuable insight into the general aspects of the structure, physicochemical properties, and functions of phenoxyl radical complexes with a series of transition metal ions.

In an attempt to understand the spectroscopic properties, redox features, stability, and chemical reactivity of phenoxyl radical a series of complexes with the metal-radical interaction have been studied by Wieghardt et al., Tolman et al., Stack et al., Whittaker et al., Pierre et al., Itoh et al., and Yamauchi et al.

The fundamental challenge associated with this idea is how to effectively interface the redox-active moiety with the ligating moiety to best affect the ligand field. Furthermore, it is necessary to probe these new complexes to determine the extent of redox-activity from the ligand to the transition metal center. We intend to use synthetic chemistry, experimental characterization and will check the biological applications.

To achieve this goal the following objectives are proposed:

  1. Synthesizing the non-innocent ligands
  2. Synthesizing and characterization of transition-metal complexes
  3. Checking the applications

Research methodology to achieve the research objectives.

Synthesis of ligands. I propose to synthesize H2L1 Ligand. H2L1: Ligand H2L1 will be synthesized by using the procedure reported in the literature.1 In Ist step, pyridine-2-carboxilic acid is refluxed with SOCl2 and pyridine-2-carbonyl chloride will obtain.

In 2nd step, A solution of 2-Amino-4-tert-butylphenol (1.0 g, 4.9 mmol) in dry THF (40 mL) was taken in a 100 mL round bottom flask. To it pyridine carbonylchloride (0.694 g, 4.9 mmol) and triethyl-amine (0.50 g, 4.9 mmol) was added and the mixture was stirred for 24 h in N2 atmosphere. It was then filtered and the residue was washed with dichloromethane. The filtrate was concentrated under reduced pressure. A solid compound thus obtained was subjected to column chromatography [ethyl acetate and n-hexane (80:20), as mobile phase] over silica gel (100–200 mesh). After solvent removal the purified compound was obtained as white crystalline solid.

Synthesis of complexes. With ligand H2L1: Synthesis of complexes from ligand H2L1 will be done using the following reported procedures. The ligand H2L (0.100 g, 0.22 mmol) was dissolved in CH3OH (10 mL) and to it was added metal salt in portions. The color of the solution changed from something to dark brown. The mixture was stirred for 2 h. After removal of the solvent a solid was obtained, which was recrystallized from Methanol/Et2O affording shining crystals.

The ligand H2L (0.100 g, 0.22 mmol) was dissolved in N, N′-dimethylformamide (DMF) (8 mL) under N2 atm., and to it was added solid NaH (0.026 g, 0.44 mmol). The combination was stirred for 15 min resulting in a light brown solution. To this solution, solid metal complex (0.11 mmol) was added portionwise. The resulting deep brown solution was stirred for 2 h and filtered. The solvent of the solution was evaporated and 1 mL CH3CN was added and 20 ml diethyl ether was added to it and kept in deep freeze. After 12 hours, the ppt. formed was filtered and washed with ether. Solid obtained was dried under vacuum.

For Characterization NMR, Mass, IR, CHN, UV-Vis Spectroscopy, Electrochemistry, Spectroelectrochemistry, EPR (Electron paramagnetic resonance), Solution Magnetic moment, Mössbauer etc. will be done and will find new properties and applications of non-innocent ligands and their transition metal complexes.

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