Blog Post #2: Continuation of Research on the Methods of Extracting Bee Pollen

During the entirety of the Fall semester, I worked collaboratively with Dr. Mojica for 1-2 lab sessions per week. We met before, during, and after my time in the lab to establish the goals for the lab session, answer any relevant questions, and discuss a brief overview of the results attained. Using my lab notebook, I kept track of each step taken in this process, as well as by taking photo documentation. Dr. Mojica and I also worked to establish an abstract to submit to the American Chemical Society for the 257th National Meeting, taking place in the Spring of 2019 in Orlando, Florida. I was happily accepted, alongside some of my colleagues, to be given the opportunity to present my research at the conference with some of my colleagues. Since my study focuses on various extraction methods, it has been very beneficial to work with Dr. Mojica who, through a collaborative effort, has taught me several methods of extraction for various bee-deriving substances. With a passion for the topic of analysis, the initiative to continue my research this semester was simply to find an answer to the question at hand in hopes of using the results to help better understand how bee substances can help our society.

Throughout this semester, my UGR research has taken a turn from focusing primarily on propolis to being inclusive of pollen, as well. A recurring obstacle was noticed despite of the change in focus from propolis to pollen. It appeared that cross-contamination in the GC-MS instrument remained a prevalent issue. For this reason, we came up with a few problem-solving techniques, such as running a trial solely with the solvent before running it with the pollen. This aided in cleaning out the machine of any by-products that may have been lingering from prior research. Additionally, our addition of a new machine, described below, has helped us in the extraction process in an effort to avoid cross-contamination. Each week, we have worked to make continuous progress on this project, despite of any obstacles encountered along the way.

Fortunately, we ended the semester with a gift from NSF, the National Science Foundation. The National Science Foundation, an American government agency, provides financial aid to support research and education in the field of science. Dr. Mojica was given a grant for the Accelerated Solvent Extraction (ASE) machine, which I have had the benefit to start using. The ASE machine is used for the extraction of chemicals from a solid. I first grind the bee pollen into smaller pieces and let these pieces sit in 5 mL of the given solvent overnight. Then, I manually filter this solution using syringe filters. This is followed by an extraction in the ASE machine. Below is a photo of me with our new ASE machine and my first extraction using the machine! We are continuing to answer the question of why there are outside chemicals showing up in our results from the GC-MS and hope to have this issue solved by the end of the semester.

 

My first extract of pollen and methanol using our new ASE.

Chromatographic Characterization of Bee Propolis and Pollen from Around the World

The title of my research, as stated above, is the Chromatographic Characterization of Bee Propolis and Pollen From Around the World. I have chosen to analyze and compare the components that make up bee pollen and propolis from several species with varying origins, developing an understanding for how the type of extraction methods will affect the gathered results. The purpose of this research is to distinguish between the results gathered from using different extraction methods, while understanding the importance of the extracted components. Moving forward, we would like to understand the different health benefits that propolis can provide for humans and which benefits relate to which chromatographically discovered components.

Before stepping into the wet lab and performing physical research, Dr. Elmer Mojica and I worked collaboratively to perform literary research in order to finalize my research question and methodology being used. We studied papers from several scientific journals, such as the Food Research International and the Journal of the Science of Food and Agriculture. In today’s society, the focus of what is being put inside the foods and products consumed by humans is so vast and significant. For this reason, I expect to achieve a fully developed understanding of which extraction method is best, along with which components are found in which species of bees. I can then use this to understand which components humans are consuming when they go to health food stores to purchase propolis and pollen.

The methodology being used to answer my research question varies depending on the extraction method being used. In chemistry, extraction methods are used to separate substances that they are mixed with. I used my literary research to fully understand the Soxhlet, microwave, sonication, accelerated solvent extraction, and simple extraction. I aim to compare these methods with one another. I will first be using dichloromethane and methanol to make solutions with the propolis and pollen. I can then inject these solutions into the machine known as GC-MS, or gas chromatography-mass spectrometry. This provides us with a visual representation of the spectrum. We can then use this, alongside a library, to analyze which molecules make up the propolis and pollen. Refer to Image 1.0 for footage of the first bee pollen that we have begun analyzing.

Image 1.0 – Stingless Bee Pollen from Los Baños, Leguna, Phillippines

This semester has so far been successful with literary research and we have begun injecting samples into the GC-MS. We are looking forward to analyzing the results and successfully reaching our desired achievements to answer my research question.

The Impact of Agriculture on Water Quality in Southern Trinidad

I am spending my summer conducting field work in Southern Trinidad to find out the impact of agriculture on water quality. This area and these river systems are very important to me personally, because my family is from Trinidad and they interact with these rivers quite a bit. The rivers here are not only the recipient of a lot of agricultural runoff (which can contain animal waste and fertilizers, among other contaminants), but of great cultural and economic importance to Trinidadians. People practicing the Hindu faith use the river for religious ceremonies, and a big part of the economy here is tourism, which means that the beaches and marine wildlife need to be clean and healthy.

Due to the importance of both agriculture and water quality to the human population living there, southern Trinidad is considered an appropriate model to study human-induced water pollution and its effects on ecosystem and human health. For this reason, I am spending this summer examining how agricultural runoff contributes to microbial and nutrient (nitrogen, phosphorus) pollution.  I will also be sampling and characterizing macroinvertebrate communities in the next few weeks.  These macroinvertebrate communities consist of organisms such as worms, shellfish, and insects (e.g. dragonflies and mosquitoes) that spend their larval phase in water. The composition of these communities is an indicator for overall pollution and ecological health. I will elaborate on this more in my next blog post.

Right now, it is the “rainy season” in Trinidad, which is the time of year when most of Trinidad’s average yearly rainfall occurs. Large amounts of rainfall during the rainy season potentially will flush a hoard of pollution into nearby river systems. For the past few weeks, I have been conducting water sampling, both during rain events and between storms. I have chosen to collect water samples in two different river systems in Southern Trinidad. One is heavily developed by agriculture (the South Oropouche river) and the other has low levels of human land use (the Moruga River). By comparing levels of fecal contamination and nutrients in the two rivers, I hope to make some conclusions about the impact that agriculture is having, and how future agricultural development might influence the ecology of river systems here. Before starting my field work, I walked along each river to find sampling sites.  I found three sites on each river, determining them by accessibility.

Once samples are collected, they need to be tested for fecal contamination and for nutrients (nitrogen, ammonium, and phosphorus). Fecal contamination is found by testing for the presence of bacteria (called coliform bacteria) that are typically found only in the gut of warm-blooded animals. There are many types of coliform bacteria and not all are harmful, but they do indicate that animal waste is making its way into a river. Coliform bacteria produce acid and gas from lactose, and you can take advantage of this to perform a simple test on water to see if they are there. I add 1 mL of the river water sample into a test tube with lactose and a color solution that is sensitive to pH (i.e., acidity) – kind of like litmus paper. I then agitate the solution for about 30 seconds, then leave it for 72 hours at room temperature to let the bacteria do their thing. If the broth does not change color, fecal coliforms were not present in high enough numbers to change the acidity of the water. If it turned yellow, that means that fecal coliforms consumed the lactose, lowering the pH of the water and causing the color solution to change. Interestingly enough, fecal coliforms have been indicated in all the water samples I tested, including the samples from the Moruga River (i.e., the “pristine” river in my study). Although I am not sure of the extent of contamination (only that the samples are contaminated with some level of fecal waste), it was surprising to find that both rivers have this contamination, and that contamination was found even when it wasn’t raining.

My nutrient analysis of river samples will require the use of analytical machines, so unlike the coliform testing, I can’t perform these analyses at the house I am staying at. Nutrient concentrations can change very quickly in water if algae and bacteria are present, so I have to freeze my samples right after collecting them, and keep them stored in the freezer until they can be analyzed. Luckily, I have been in touch with a Professor at the University of Trinidad and Tobago, where I will run a portion of my samples to measure concentrations of nitrate and phosphate, to get some experience with the analysis. When I get back to NYC, I will test all samples for nutrient concentrations (nitrate, phosphate, ammonium).

 

Doing my field work has been an amazing experience, and has confirmed to me the importance of the small study I am doing. Before I came to Trinidad for the summer, I tried to find other water quality studies of the rivers here, and didn’t find any. It also seemed like most residents on the island, even though they use the rivers quite a bit, are largely unaware of the presence or repercussions of river pollution there. These findings have been reflected in my experiences in the field. While collecting samples from the South Oropouche, some of the local people noticed I was collecting water samples near their homes and asked what I was doing and where I was from. I explained to them what my research was on, and they were shocked to find out what was happening in the river system they use daily. They were glad to find out that someone was conducting water quality tests on the river system they rely on for food and water. Hearing this made me happy because I felt that my research can potentially help better people’s everyday lives.

Production Day

After a month or so of planning Linda and I were finally able to get together and work on the stop motion project. This day took almost a year to get to and was very rewarding. The production itself was pretty seamless, the only issues we ran into were minor placements of materials.

Stop motion is a very tedious process, pieces must be moved precisely and accurately to convey motion. Of course the artist can choose to embrace the chaos of choppy motion, but a lack of continuity can make the project look sloppy and not chaotic.

The main takeaway from this project was that I should have more fun with my art. It is very easy to become so obsessed in the process of making as well as the thought process of subjectivity and meaning of a piece. This project showed me that I should prioritize a solid scheduled. But that I should always take joy in what I create and embrace every curveball.

Morley’s Triangle of Some Special Triangle

The aims of this research are to study the internal and external Morley triangles whose mother triangles are (1) equilateral triangles, (2) right triangles, and (3) isosceles triangles.

So far, we have investigated the external Morley triangle of our first mother triangle, an equilateral triangle. By solving systems of linear equations with trigonometric functions, we are able to find the coordinates of the three vertices of the external Morley and how they are related to the mother triangles. (See below) Moreover, parallelism and collinearity of vertices and centroids are also studied.

Given the mother triangle ABC with A= (-a, 0), B = (a, 0), and C=(0,c), we can obtain the following figure and find the coordinates of the vertices of the external Morley triangle PQR as follows.

Phenolics in Bee Propolis

Phenolics in Bee Propolis

Research by Josephine Farshi and Professor Elmer Mojica

The primary focus of my Summer 2017 research is the analysis of phenolics in bee propolis. I aim to investigate the properties of this propolis in relation to their use by both bees and humans alike. I will achieve this by studying the antioxidant makeup and nutritional properties of propolis by means of analyzing results gathered from gas-spectrometry mass spectrometry (GC/MS). In this blog post I will describe the purpose of my research, along with how I became interested in the topic.

First, I would like to clarify that bee propolis is a product of bees, but not the commonly presumed product known as honey. The product I am focusing on comes from beehives, just like honey does (Alibino 2014). However, bees make propolis by mixing beeswax with vegetable resin, a brown substance gathered from sap (Castro et al. 2014). Propolis is commonly referred to as bee glue. During the process of GC/MS, the propolis is separated into the chemicals that it consists of during the gas chromatography and further analyzed based on these chemicals during the mass spectrometry overview (Kartal et al. 2002).

Studying the chemical properties, such as flavonoids, will provide insight on how certain chemicals can provide nutritional benefits. The nutritional and food-science aspects behind the bee propolis are what first grabbed my attention about this very topic. For instance, bee propolis is known to contain 300 active compounds, some of which have been used to fight sore throat and stomach ulcers due to the pharmaceutical activities (Rios et al. 2014). I knew that I wanted to work with something related to food-science because I am interested in all aspects of nutrition, including health benefits that certain natural products provide and the effects of socioeconomic status on global nutrient intake. From my research, I aim to see how the chemical composition of bee propolis can be used for benefitting human health.

So far this summer, I have focused on an immense amount of literary exploration. There are several reasons behind why literature is significant in my scientific research. For starters, literature provides me with an understanding of how researchers have previously analyzed the same topic of phenolics in bee propolis. Having a broad understanding of the topic at hand can therefore create a rapport between the audience and myself, as they will be able to trust the authenticity of my studies. By using the Web of Science, I have gathered over 200 abstracts from scientists, published between 2014 and 2017. I now have a broad understanding of the importance of analyzing propolis for both bees and humans alike, along with an established methodology for gathering results.

 

Quantifying the Number of GFP Expressing Trichomonas Vaginalis Adhering to the Vaginal Epithelial Cells Treated with Amyloid Dyes

Trichomonas vaginalis is a eukaryotic parasite that causes the most common non-viral sexually transmitted infection worldwide. Although it is the most common, it is also the most understudied and poorly comprehended parasite. The research that will be conducted on this organism will help to better understand host-parasite relationship as the trichomonads adhere to vaginal epithelial cells and begin to colonize. The purpose of this research is to successfully quantify the number of GFP expressing Trichomonas vaginalis adhering to the vaginal epithelial cells treated with amyloid dyes. Because there are a lot of trichomonas cells that will be seen under a microscope, the use of GFP or green fluorescent protein will help to magnify and quantify how many cells are present. The goal is to see an increase in fluorescent activity each time the cells are displayed under a microscope.

In order to carryout this research, we first must colonize GFP. To do this, we use GFP that was found in E. coli and smear it onto an agar plate with LB (Luria broth) medium and ampicillin.

(Source: http://microbeonline.com/wp-content/uploads/2016/10/Pour-Plate-and-Spread-Plate-Method.jpg)

Ampicillin is sometimes used on plates because when ampicillin breaks down, it can often lead to more colonies being formed. The plates are then incubated over night to allow for colonization to occur. Once colonization occurs, the plates are moved under a sterile hood and using a sterile pipet and tip, a single colony is extracted and placed into liquid culture made up of the same LB medium with ampicillin and relocated to incubate on a shaker plate over night. The following day, the tubes are removed from incubation and transferred to a microcentrifuge tube and centrifuged out to collect a concise pellet at the bottom of each collection tube. This pellet collected contains bacterial cells that contain GFP, which will eventually be cut out and cloned multiple times. Once the pellet is collected, the DNA is extracted from those cells using a QIA Spin Miniprep kit. The DNA collected after the end of this kit is the unclean DNA, which could possibly contain buffers and residual waste from the kit that was not completely centrifuged out. However, the unclean DNA is tested to make sure that there is indeed DNA present, and to do this, we have to set up a gel electrophoresis.

Gel electrophoresis is a technique used to separate mixtures of DNA, RNA or proteins based on molecular size. Molecules are pushed by an electric field that moves negatively charged particles through the small pores of agarose gel to the positive charge at the other end.

(Source: https://schoolworkhelper.net/gel-electrophoresis-basics-steps/)

Through this technique, we can compare the sizes of bands to a ladder, or just to simply confirm the presence or absence of a specific molecule. For this purpose, we used the gel to determine the presence of bacterial DNA from the E.coli culture. Below is a picture of the resulting gel after running this technique:

As shown by the picture above, DNA was indeed present in the unclean tubes. From those tubes, a certain amount was then collected and another kit, DNA Clean & Concentrator, was used to further clean the DNA to use for a PCR or polymerase chain reaction.

Polymerase chain reaction is used to increase a single copy or a few copies of a section of DNA. Below is a table that shows the main ingredients of a PCR and what their role is:

The main goal to achieve from the PCR is to use the primers that have been specialized to highlight the region used to code for GFP in the DNA sequence, amplify it and cut it out. Once the PCR is complete in the set conditions, another gel is run to determine if the primers worked and if the gene was amplified. However, PCR is a very sensitive technique that requires the right conditions, which have yet to be discovered. Below are the results of the gels following a PCR treatment and the conditions that were set:

1.)

 

2.)

Using these previous conditions, it will allow for us to easily manipulate the numbers to try and find the most ideal conditions. In the future, these ideal conditions will allow for us to isolate the GFP band with the correct restriction sites and insert it into the parasite.

Moving forward, the research aims to successfully obtain the GFP gene in the PCR using the specialized primers. Once this is obtained, we can perform a gel extraction that will isolate the GFP that contains the specific restriction sites that will eventually be cloned and inserted into the parasite in question. As an individual, I hope to perfect my skills in performing a PCR as well as extracting DNA in the form of a pellet. These skills will eventually aid me in my future career as a forensic scientist where the use of PCR is necessary to amplify the DNA in question to perform further tests on it.

Characterization of the Microbial Community of the Accessory Nidamental Gland of the Longfin inshore squid, Loligo pealei

The title of our experiment is Characterization of the Microbial Community of the Accessory Nidamental Gland of the Longfin inshore squid, Loligo pealei. The research that is being undertaken is focusing in on the symbiotic relationship that is present between marine organisms and the mutualistically beneficial bacteria that grows on these organisms. The foundation of this research was interest in the nidamental gland of the female reproductive organ in squids. The nidamental gland is responsible for coating the eggs with a jelly like substance that is integrated with bacteria to serve as a protective barrier against predators and fowling. If this bacteria is able to be characterized, The properties that allows it to protect the eggs will be understood and potentially be beneficial in other ways. Understanding this bacteria will be attempted through 16S rRNA gene sequencing. Results will assist in understanding of marine symbiotic organisms and how they function. This will be the first study addressing the microbial population of the Longfin inshore Squid nidamental gland as well as their egg cases.

 

So far, many efforts have been made in reference to lab training for the experimentation itself. LBS agar plates were created in order to perform 3 way streaks and isolate particular bacteria from the marine symbionts. Squid, as well as jellyfish that were collected through wading, were the subjects of bacteria isolation. The process of isolating the bacteria from the jellyfish was extensive, beginning with euthanization of the jellyfish (through freezing),  macerating it, rinsing it with 3x deionized water and then vortexing it. Through performing a three way streak of this sample, particular colonies of bacteria were able to be isolated and examined individually. Colony PCR was performed following this and the samples were then inserted into gel in order to perform gel electrophoresis. PCR clean up was then undertaken using a Qiagen kit. This is done in order to prepare the DNA samples that were amplified to be sequenced. Twelve different bacteria samples were successfully amplified through PCR and able to be sequenced in the lab so further characterization of the bacterial colonies can occur in the future.  

 

Comparative NMR Spectra of Metronidazole and Ornidazole

We successfully built structures of metronidazole and ornidazole on Gaussian, both of which we calculated the NMR spectra in chloroform. Additionally, we obtained this data using experimental spectroscopic techniques; this was then compared to the theoretical spectra from Gaussian.

Theoretical proton NMR of metronidazole indicated five different types of hydrogens; Gaussian was unable to differentiate between the hydrogens of the methylene group attached to the imidazole ring, both of which were depicted in two different peaks yet with a degeneracy of one. In comparison, our experimental proton spectra depicted these five peaks more downfield. Gaussian predicted six carbon peaks while experimental NMR resulted in five carbon peaks, three of which were shifted more downfield and two of which were very similar in chemical shifts with Gaussian. The missing peak was the carbon with the nitro group on the imidazole ring.

Gaussian provided a more complex proton NMR for ornidazole, in which the hydrogens on the methyl group and the hydrogen on the hydroxide group were reflected in similar peaks. The hydrogens on the methylene group were also reflected in two peaks. Experimental spectra provided one more hydrogen peak. In contrast to theoretical data, the methyl group was more upfield than the hydroxyl group. Seven different carbons were predicted by Gaussian; experimental spectra indicated some similarities in the chemical shifts of three carbons. The carbon with the nitro attachment on the imidazole ring was reflected in a small peak this time.

With these results we were able to adjust our objectives for the future. Gaussian provided a reasonable theoretical analysis of structural information, and in utilization with experimental spectroscopy, we can determine the reaction mechanism of metronidazole and ornidazole. We want to investigate what the activated complex in the interaction between the drug and the microbe looks like, as well as how it forms. This is particularly an area of interest for ornidazole, which has been verified as more potent than metronidazole because of the smaller amount of drug required to kill a larger number of microbes.