Exciting Campus Research

Below is an abbreviated list of research topics explored in our courses and extracurricular research platforms.

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Testing of H.I.V. susceptibility in humans

A little known fact is that select individuals throughout the world have been repeatedly exposed to the human immunodeficiency virus (H.I.V.), yet have not tested positive for the virus. In some cases, a particular genetic mutation in the CCR5 co-receptor (one of the cell surface proteins found in humans that is required for viral entry) impairs the introduction of H.I.V. into human cells. Scientific studies have found that up to 10% of certain European groups carry this mutation, possibly due to previous exposures of their ancestors to the bubonic plague, and subsequent natural selection. If an effort to explore the prevalence of this (partially) protective mutation in local populations we have tested various University of Hawai‘i students, and indeed found a few individuals carry the mutation.

HIV gel image

Human testing of the mutant HIV co-receptor

Human DNA testing was done using buccal (cheek) samples and primers specific for the CCR5 mutation.

  1. DNA marker
  2. Normal susceptible individual
  3. Normal susceptible individual
  4. Normal susceptible individual
  5. Individual with 1 normal copy and one mutant copy of CCR5
Testing lactose intolerance in humans

Human infants produce an enzyme (lactase) that allows for the breakdown of the disaccharide lactose into glucose and galactose. In turn, these sugars are absorbed through the intestinal lining and utilized in the body for chemical building and energy.

However, in many individuals the lactase enzyme is “turned off” as infants (or even adults) grow, consequently causing lactose intolerance for the remainder of their lives. Because lactose is no longer degraded in the absence of lactase, the sugar then enters the intestines intact and is absorbed by resident bacteria there. In turn, these bacteria consume the sugar and produce gases that may cause discomfort to the human host. Interestingly though, as humans evolved numerous individuals developed mutations that allowed them to continue to breakdown lactose throughout their lives. At KapCC, our students genetically test one another to determine lactose tolerance.

lactose gel
Persistent Lactase Expressor

Confirmatory PCR and restriction fragment length polymorphism assay for a persistent lactase expressor

  1. DNA marker
  2. Buccal (cheek) DNA sample amplified via PCR
  3. The same sample subjected to restriction digestion indicating a heterozygous genotype of persistent lactase phenotype
Genotypic and phenotypic analysis of PTC paper testing

The ability to taste the compound PTC is based on a dominant trait within a person’s genome. For decades this simple test has been used in classrooms into order to examine population studies with respect to genetic traits. More recently however direct DNA tests allow for the examination the genomic sequence in particular regions of this gene.

The students that worked on this project were able to take a published experimental platform and validate it in preparation for use in various courses here at KapCC. Specifically, students took buccal (cheek) samples, extracted their DNA, performed gene amplification, cut those genes with specific enzymes, and analyzed their results using gel electrophoresis. Indeed, in every case examined the students DNA sequence matched their phenotypic ability to taste the PTC compound on a piece of paper.

PTC gel image
Confirmation of PTC Tasting via Genetic Analysis

Student took cheek samples, replicated the DNA and then cut the target PTC gene with enzymes in order to analyze the results on an agarose gel.

  1. DNA marker
  2. Taster Tt
  3. Nontaster tt
  4. Taster TT
  5. Taster TT
  6. Taster Tt
  7. Taster Tt
Detecting human papilloma virus sequences in human cancer cell lines

HeLa cervical cancer cells were extracted, isolated, and maintained over 60 years ago from a human named Henrietta Lacks (without awareness or consent from her or her family). Since that time her immortal cell line has been used in labs around the world, was important for early vaccine work, and has even been sent into space.

As a consequence of being grown in labs around the world for decades, the HeLa cells contain numerous gross genetic abnormalities and harbor additional chromosomes when compared to normal human cells. The etiology (cause) of Mrs. Lack’s cancer was an infection of human papilloma virus (H.P.V.), a common group of viruses that continue to cause cancer in humans.

In order to verify the presence of H.P.V. in HeLa cells, we perform genetic DNA tests on it, and compare those results to other human DNA sources.

HPV gel image
Detection of HPV in a Human Cell Line

The presence of human papilloma virus DNA was detected via PCR in a human cervical cancer cell line, but not in a human colorectal adenocarcinoma cell line.

  1. DNA marker
  2. HeLa cervical cancer line
  3. Caco-2 colorectal cancer line
Quantification of human gene expression

At KapCC, we have the capability to not only examine the expression of genes, but also quantify the expression levels using Real-Time Polymerase Chain Reaction (RT-PCR). This modern technology allows one to quickly and accurately assay the amount of gene expression in various cell types. It can also be applied to compare healthy and diseased tissue.

RT PCR of actin gel image
RT-PCR Detection of Beta-actin Expression in Human Cancer Cells

Human cancer RNA was reverse transcribed and amplified in the presence of a dye that allows for detection in real-time.

The arched lines represent various replicates of the human Beta-actin gene. The middle latitudinal lines are control thresholds, while the bottom latitudinal lines are assay controls.

Identifying fishes by DNA analysis

Contemporary environmental researchers commonly utilize DNA analysis to quickly and accurately determine organisms in their study. Similar methods also have a wide range of applications that include the identification of illegal contraband and endangered species.

To illustrate this ability to identify species based on their DNA we extract and query DNA from fish tissue samples retrieved from the local fish auction.

Fish gel image
Identifying Local Fish

Mystery fish samples were assessed with specific DNA primers in PCR reactions in order to determine identity

  1. DNA marker
  2. Shortbill spearfish
  3. Striped marlin
  4. Mahi mahi
  5. Ono
Confirmation of Staphylococcus aureus and M.R.S.A. in human samples

Staphylococcus aureus is a common type of bacteria found in/on humans, as well as their pets. Unfortunately though, these bacteria contain a wide array of virulence tools that promote disease, one of which may be multidrug-resistance. Unfortunately, cases of drug resistance strains increase with each year, and more Americans die of M.R.S.A. (methicillin-resistent Staphylococcus aureus) infections than H.I.V. infection. To examine the prevalence of M.R.S.A. at KapCC. students have developed a quick and simple assay that involves the detection of a drug resistant cassette found in the bacterial DNA.

MARSA agglutination
Agglutination Assay to Confirm M.R.S.A. Bacterial Strains

Four Staphylococcus aureus strains were assayed with a commercial kit to identify multidrug-resistance. The strains on the left and right tested negative, while strains 5 and 61 (in the middle) showed agglutination around the periphery, indicating that these bacteria are M.R.S.A.

MRSA gel image
DNA PCR Assay Confirms M.R.S.A. Bacterial Strains

The four Staphylococcus aureus strains used above were also assayed via P.C.R. to genetically identify M.R.S.A. strains.

  • Lane 1. DNA marker
  • Lanes 2-5 confirm the presence of Staphylococcus aureus in all strains tested.
  • Lanes 6 and 9 confirm that the non-M.R.S.A. strains are indeed negative.
  • Lanes 7 and 8 confirm the results of the agglutination assay above, that strains 5 and 61 are M.R.S.A. positive.
Detection of Toxoplasma gondii in fecal samples

Toxoplasma gondii is single-celled parasite that can infect warm-blooded animals such as humans and their pets. Local studies have shown that up to 73% of feral cats carry Toxoplasma, which may then allow for the transmission to humans via colony feeding or from pets that come in contact with them or their feces. Though normally not dangerous, toxoplasmosis may be life threatening to immune compromised individuals and developing human fetuses. Consequently, pregnant women should not come in contact with cat litter that potentially contains Toxoplasma cysts. Although infected cats only shed cysts for a short period of time, we have still explored the possibility of Toxoplasma cysts in animal feces.

Although we have previously validated primer sets to detect Toxoplasma, thus far our feline stool samples have not yielded a positive result. We believe this is due to the fact that juvenile cats only shed stool cysts for approximately two weeks. Upon further exploration, we have found that other researchers have also had inconsistent results in using a similar DNA testing method. Live and learn…

Isolation and characterization of a feline gut virus

To date, numerous studies have shown that the gut microbiome of Felidae is a complex environment containing a wide array of beneficial and pathogenic species. Genera such as Salmonella, E. coli, Campylobacter and Toxoplasma may be present within a feline, and these microbes may pose a health hazard via zoonotic transmission amongst various species. This risk includes humans who are in close or frequent contact with cats and/or their feces.

This project served as a preliminary survey to identify and characterize some of the bacterial species found within the gut of local domestic and feral felines, as well as germs derived from a tiger. To do so, fecal bacteria DNA was harvested, amplified, and cloned into stable vectors/backbones. The isolated vectors were then screened for redundancy and sequenced to identify the bacterial species present. These studies served to provide insight into the regional bacterial reservoirs found in Felidae, and may aide in the local control of pathogens transmitted from felines to humans.

Emily RFLP
Restriction Fragment Length Polymorphism Analysis of 16S rRNA Clones

Agarose gel electrophoresis of feral cat bacterial amplicons digested with HhaI shows both redundant and unique variants. Unique RFLPs were subsequently taken to DNA sequence analysis.

Emily sequence analysis
Homology Analysis

The above banner illustrates a comparison between identical and redundant sequences submitted to sequencing. DNA base-calls were aligned using the Vector NTI AlignX Analysis Tool. Yellow and blue areas indicate varying degrees of homology and conserved sequences between submitted samples.

Emily chart
Summary of DNA Analysis of Bacteria Found in Cat Stool

Mystery bacterial identification from a human skin sample

In the KapCC Microbiology 140 lab course students take skin samples in order to demonstrate the presence of a wide array of bacteria. Routinely we see a great deal of Staphylococci, as well as other bacteria. A few years ago one student sample contained no Staphylococci, but rather a strange-looking bacterium that we did not routinely observe in the laboratory. To determine the identity of the unique microbe we extracted its DNA and sequenced it.

Surprisingly, the microbe turned out to be Bacillus subtilis, a terribly common bacterium used in labs across the globe and the source of the active ingredient in Bacitracin. However, the student’s particular strain of Bacillus had a different appearance from our normal variety, and was thus not recognized in the lab until is was identified by DNA sequencing. This project also illustrates the principle that some unrecognizable bacteria may be identified by DNA sequencing.

unknown skin bacteria gel image
Amplification of Unknown Skin Bacteria

Mysterious skin bacteria was cultured, the DNA isolated, and amplified subsequent to DNA sequencing

  1. DNA marker
  2. Unknown bacterial DNA
  3. E. coli DNA
Utilizing recombinant bacteriophages to generate antibody libraries

We are currently in the process of validating a novel method to generate monoclonal antibodies via phage display. This approach will allow us to produce more unique antibodies in a shorter time frame. In addition, phage display lends itself to more advanced characterization, recombination, and improvement of selected antibodies.

Briefly, we utilize a recombinant phage library that contains a repertoire of over ten billion antibody gene fragments from naive mice. When expressed, this library generates a population of phages, each with a unique antibody on its surface. Through a series of binding, selection, and amplification in E. coli we can generate specific antibodies without the use of mice. We believe this will enhance and augment our current Monoclonal Antibody Training and Service Center found on campus.

Campylobacteri jejuni PEB1 gene cloning, expression, and purification from E. coli

Campylobacter jejuni bacteria are the most common cause of human gastroenteritis, however very little is understood on how this microbe causes illness. One of the putative virulence factors of Campylobacter is a cell surface protein called PEB1. In an effort to investigate the role this protein may play in human disease we have cloned the peb1A gene and introduced it into E. coli. We then use E. coli as a simple factory to produce this protein for us.

peb stained gel
PEB1 Protein Expression in E. coli

The peb1A gene from C. jejuni was cloned and introduced into E. coli for subsequent protein expression.

The above panel shows normal E.coli on the left and E. coli expressing PEB1 on the right. The various lines that you see are cell proteins. If you look closely, you will notice that there is one additional band found in the right lane, which indicates exogenous gene expression.

peb western image
Immunoblot of E. coli Expression of the peb1A Gene from Campylobacter jejuni

The peb1A gene was cloned behind (and in frame with) the maltose binding protein (MBP) gene in a plasmid. Subsequent expression in E. coli yielded the presence of both the MBP epitope (low band), and the PEB1-MBP protein (high band). Detection was done with an antibody specific for MBP.

  1. MBP control
  2. 24 hour expression lysate
  3. 12 hour expression lysate
  4. 6 hour expression lysate
  5. 3 hour expression lysate
  6. 1 hour expression lysate
Ongoing production of recombinant antigens/immunogens

KapCC routinely generates recombinant proteins in the Microbiology 230 course and extracurricular research.  These proteins then serve as antigens/immunogens when injected into mice.  In turn, these challenged mice produce a greater number of B cells and antibodies against these immunogens. Our students then extract these B cells and characterize the antibodies that are produced by them.

Ongoing monoclonal antibody production using mice and myeloma cancer cells

One of the ongoing research platforms that Dr. John Berestecky established at KapCC is the production of hybridoma mouse cell lines that produce antibodies. Mice are immunized with various antigens, their spleenocytes are then removed and fused with a mouse cancer cell line to create a hybridoma that produces the desired antibody, and is immortal. This work allows for the production of novel antibodies used within our labs, as well as research collaborations with a wide array of public and private labs.

Ongoing investigations into the mechanism/s responsible for Campylobacter jejuni invasion of human cells

To date, the mechanisms that allow for the invasion of human intestinal epithelial cells by Campylobacter jejuni are not determined. Therefore we are using human tissue culture methodology to investigate various Campylobacter isolates to assess the ability to adhere, and subsequently invade, human cells.

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