Laboratory Research
This section includes Class of 2022 Embark Projects within the Laboratory & Bench research disciplines. These projects include traditional bench research in a variety of basic and clinical science areas.
Relatedness Of Urinary Escherichia Coli Strains And Connection Between Strain Type And Clinical Presentation
Anet Szatkowski, B.S.1, Venkatesh Bai, Ph.D.2, Matthew D. Sims, M.D./Ph.D.2,3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Section of Infectious Diseases and International Medicine, Department of Internal Medicine, Beaumont Royal Oak, Royal Oak, Michigan
3Departments of Internal Medicine and Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, Michigan
INTRODUCTION
Urine is often not a sterile body fluid. When bacteria in the urine cause symptoms it is by definition a urinary tract infection (UTI). The specific symptoms will point toward a bladder infection (cystitis) or a kidney infection (pyelonephritis). Presence of bacteria in the urine without symptoms defines asymptomatic bacteriuria (AB). Despite requiring symptoms to diagnose a UTI, often AB is misdiagnosed as a UTI and treated with antibiotics when none are needed. Escherichia coli is the most common cause of UTIs accounting for 80-90% of community-acquired UTIs and 30-50% of nosocomial UTIs. The goals of this study are to determine whether specific strains of E. coli are associated with cystitis, pyelonephritis, or asymptomatic bacteriuria; whether specific strains are related to more serious infections; and whether specific strains can be geomapped to specific areas within Southeast Michigan using postal codes.
METHODS
E. coli isolated from adult patients (≥18 years old (yo)) either seen in the emergency department or admitted to the hospital with suspected UTIs at Beaumont Royal Oak were run on the Bruker IR Biotyper, which analyzes bacterial carbohydrates and generates a dendrogram to display strain relatedness. Each strain was tied to patients’ clinical presentations and their residential postal codes.
RESULTS
Findings suggest that no specific strain of E. coli was linked to asymptomatic bacteriuria vs. cystitis vs. pyelonephritis; nor was there a specific strain that was more likely to cause a mild vs. severe infection. Lastly, geomapping of the different clusters did not show any hot spots for certain strains within Southeast Michigan.
CONCLUSIONS
No study to date has looked at E. coli strain type as a predictor of asymptomatic bacteriuria vs. UTI. The findings suggest that the host rather than the strain type is the major driver of a patient’s presentation and disease course.
The Superior Laryngeal Nerve and Its Vulnerability in Surgeries of the Neck
Antonio Dekhou, B.S.1, Robert Morrison, M.D.2, Jickssa Gemechu, Ph.D.3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2University of Michigan-Department of Otolaryngology, Ann Arbor, Michigan
3Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, Michigan
INTRODUCTION
Anatomical considerations of the superior laryngeal nerve (SLN), a branch of the vagus,
provides information to minimize the potential for iatrogenic intraoperative injury, thereby preventing motor and sensory dysfunctions of the larynx. The present study aims to assess the variation of the SLN and its relationship to the superior thyroid artery (STA) and superior laryngeal artery (SLA).
METHODS
The study was done on 35 formalin-fixed cadavers at Oakland University in 2018–2019.
RESULTS
In our study, we found that out of 21 cadavers, 52.4% of the external laryngeal branches (ebSLN) are related posteromedial to the STA, while 47.6% are related anteromedial to it. Out of 14 cadavers, 64.3% of the internal laryngeal branches (ibSLN) are related superoposterior to the SLA, while 35.7% are inferoposterior to it. In most cases, the SLA crosses above the ebSLN while traveling to pierce the thyrohyoid membrane to reach the larynx.
CONCLUSIONS
The data demonstrate that both the ebSLN and ibSLN display variation in their relationship with the STA and the SLA, respectively. Awareness of these variable relationships is critical for identification and isolation of these structures in order to prevent consequences of nerve injury, primarily a reduction in the highest attainable frequency of the voice and aspiration pneumonia.
Kinase Inhibitor Specificity in VEGF-activated Primary Human Retinal Microvascular Endothelial Cells
Brandon Metcalf, B.S.1, Wendy Dailey, B.S.2, Kenneth Mitton, Ph.D.2,3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Oakland University, Rochester, Michigan
3Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, Michigan
INTRODUCTION
The Mitton lab elucidates the mechanisms of VEGFA165 isoform activity in primary Human Retinal Microvascular Endothelial Cells (HRMEC’s). One segment of this research is to determine the contributions of the AKT and MAPK pathways to the activation of cellular processes. Kinase inhibitors are valuable research tools but most researchers fail to confirm kinase dose-specificity. We characterized two popular inhibitors of the AKT and MEK1/2 pathway.
METHODS
In situ IR-immunofluorescence assays were used to quantify the relative amounts of AKT, MAPK (ERK1/2), and p38-MAPK, using phospho-specific antibodies. Pathways were activated with VEGFA165. The primary Human Retinal Microvascular Endothelial cells (HRMECs) were also treated with MK2206 (AKT inhibitor) and U0126 (MEK1/2 inhibitor) at various concentrations. The treatment groups were control media, VEGFA165 (5000 pM), inhibitor alone (10 um), VEGFA165 and inhibitor concentrations from 0.01 to 10 um. There were triplicates of each treatment group, and the results were averaged for each group. ED50’s were determined for the inhibitors.
RESULTS
All concentrations of the AKT inhibitor MK2206(ED50=0.01uM) decreased VEGFA165a activation of AKT. MK2206 (10 um) caused significant activation of p38-MAPK by 38%.
U0126 (inhibitor of ERK1/2 activation) significantly decreased VEGFA165a induced activation of ERK1/2 and p38-MAPK while increasing VEGFA165a mediated activation of AKT over a range of doses from 0.1-10uM (ED50=0.3uM).
CONCLUSIONS
While commonly used for inhibition of ERK1/2 activation, U0126 has strong effects on AKT activation when used at doses to completely block ERK1/2 activation. U0126 is best used at 0.5uM to significantly inhibit ERK1/2 activation with minimal effects on AKT activation. The AKT pathway inhibitor MK2206 can be used at levels below 10um without significantly affecting off-target pathways (ERK1/2 and p38-MAPK). Levels of 0.1 uM are sufficient to reduce VEGFA 165a induced AKT to basal levels.
"Support: NIH Grant: EY025089 (KPM)"
Investigating a Novel Mechanism of Soluble Splice Variants
Jamie Simpson, B.S.1, Gustavo Patino, M.D./Ph.D.2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, Michigan
INTRODUCTION
Voltage-gated sodium channels (VGSC) are multimeric complexes with a critical role in action potential generation. While most subunits of the VGSC are transmembrane (TM) proteins, the Beta-1b subunit is a secreted splice variant of the SCN1B gene. This gene codes for a V-set domain-containing protein of the immunoglobulin (Ig) superfamily, with a single Ig loop. Beta-1b is formed by retention of the intron upstream of the TM segment of the Beta-1 subunit. Because the retained intron lacks any functional domains, we hypothesize that its purpose is primarily the absence of the TM segment to create a soluble isoform. This project investigates whether this is a conserved mechanism of forming soluble splice variants in other members of this protein family.
METHODS
A list of V-set domain-containing proteins with a single Ig loop and a single TM domain was obtained from the HUGO Gene Nomenclature Committee. Each protein was searched in the NCBI database and characterized by the presence of sequenced mRNA splice variants, predicted subcellular location of the splice variants, and location of the splice sites. C-terminal domains of soluble isoforms were analyzed via BLAST for motifs and conservation among various animal species.
RESULTS
163 genes coding for V-set domain-containing proteins with a single Ig loop were identified. Of these, 44.17% were found to contain only a single V-set domain, 76.39% of which have a single TM domain, and 47.22% had characterized splicing isoforms. 50% of single-TM proteins had predicted soluble splice variants, 94.12% of which were confirmed by TM domain prediction. Of the soluble isoforms, 11.11% contained motifs within the C-terminal region, and 40.74% were conserved only in simians.
CONCLUSIONS
The formation of soluble splice variants by retention of introns lacking functional domains is a recurring mechanism for V-set domain-containing proteins with a single Ig domain.
The Prognostic Significance of Glucose-6-Phosphate Dehydrogenase as a Biomarker in Head and Neck Squamous Cell Carcinomas Treated with Conventional Chemoradiation.
Kenneth H. Barker, B.S.1, Barbara L. Pruetz BASc2, Jessica D. Arden, M.D.3, Thomas J. Quinn, M.D.3, George D. Wilson, Ph.D.3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Beaumont Health System, Beaumont BioBank, Royal Oak, Michigan
3Beaumont Health System, Department of Radiation Oncology, Royal Oak, Michigan
INTRODUCTION
The prognosis of patients with head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation can be predicted using p16 as a surrogate biomarker of Human Papilloma Virus (HPV) status, but a subset of patients continues to do poorly despite a positive or negative p16 status. This project attempted to identify another biomarker, glucose-6-phosphate dehydrogenase (G6PD) as a marker for prognosis in HNSCC patients. Radiation induces oxidative damage to destroy tumor cells, and G6PD is a key enzyme involved in protecting cells from oxidative damage. The goal of this project was to identify a prognostic biomarker that would aid in recognizing patients who would not respond well to chemoradiation and potentially respond better to other therapeutic measures.
METHODS
The paraffinized tissue blocks of 65 HNSCC samples from patients treated with conventional chemoradiation were used to create a tissue microarray (TMA) block containing two core samples from each tissue block. Thin slices from the TMA block were then immunohistochemically stained for G6PD expression. The level of expression of G6PD was then assessed under microscopy, and correlated to each patient’s overall survival time, disease free survival time, local recurrence time, and p16 status using a Kaplan-Meir (KM) survival curve analysis.
RESULTS
The clinical outcomes and level of G6PD expression in 65 HNSCC patients treated with conventional chemoradiation were assessed using KM-curves. The KM-curves for overall survival time, local recurrence time, and time to distant metastasis were found to be 0.541, 0.578, and 0.806 respectively. Additionally, association between p16 status and G6PD expression was found to be 0.475.
CONCLUSIONS
The level of G6PD expression in each HNSCC tumor core sample did not correlate with clinical outcome or with p16 status which does not support the use of G6PD as a prognostic biomarker in HNSCC patients treated with conventional chemoradiation.
Pregnancy Increases Platelet Reactivity and Induces a Thrombogenic Platelet Transcriptome in Mice
Linzi M. Hobbs, B.S.1, Marisa A. Brake, Ph.D.2, Audrey C. Cleuren, B.S.3, Dakota Redshaw, B.S.2, Kelsey A. Hobbs, B.A.4, Randal J. Westrick, Ph.D.1,2,3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Oakland University Department of Biological Sciences, Rochester, Michigan
3Life Sciences Institute, University of Michigan, Ann Arbor, Michigan
4University of Minnesota, Department of Psychology, Minneapolis, Minnesota
INTRODUCTION
Intravascular blood clotting (venous thromboembolism, VTE) is the leading cause of maternal death during pregnancy and the immediate postpartum period. Since activated platelets deliver blood-clotting proteins directly to the blood clot, molecular changes in platelets could have a major impact on risk of developing VTE. However, little is known about the transcriptional and/or functional changes occurring in platelets during the course of pregnancy. Pathophysiological states can alter platelets and their transcriptomes suggesting that physiological states like pregnancy could also affect platelets.
METHODS
To explore the role of pregnancy on platelet function, we generated pregnant C57BL/6J female mice via matings with C57BL/6J male mice. Pregnancy was confirmed by the presence of a vaginal plug, which was used to estimate embryonic day 0.5. Pregnant females were randomly assigned to one of four groups based on gestational stages corresponding to human trimesters and post-partum. Whole blood platelet aggregation studies using adenosine diphosphate (ADP) and collagen as aggregating agents were performed on right ventricular blood samples from the female mice. Platelet mRNA was isolated and comparative transcriptomic analysis was performed by RNA sequencing (RNA-seq) using the Illumina high throughput sequencing platform.
RESULTS
The late gestational group (16-20 days post coitus) had significant area under the curve differences for platelet aggregation in response to ADP (p = 0.0013, p = 0.004) and collagen (p = 0.005, p = 0.028) agonists compared to the early gestational group (7-9 days post coitus) and non-pregnant controls (n ≥ 7 per group).
CONCLUSIONS
Our results indicate that pregnancy can re-educate platelets to a prothrombotic functional state. These alterations likely play a significant role in the increased VTE risk during pregnancy and the immediate post-partum period. This knowledge opens new avenues for the development of therapeutics designed to prevent and/or treat maternal VTE.
Custom Ampliseq Targeted Sequencing Panel For Orphan Pediatric Retinal Diseases: Norrie Disease, FEVR, and Retinoschisis
Michael Sun, B.A.1, Wendy Dailey, M.S.2, Amanda Petrelli Cicerone, M.S.2, Jennifer Felisky2, Kaylee Moyer2, Naomi Haque, M.S.2, Alvaro Guzman2, Kendra Mellert2, Kimberly Drenser, M.D.2,3, Kenneth P. Mitton, Ph.D.2,6
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Oakland University, Rochester, Michigan
3Associated Retinal Consultants LLC, Royal Oak, Michigan
6Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, Michigan
INTRODUCTION
DNA-sequencing is not readily available in countries with little research resources or where health insurance does not cover the costs. This is especially true for very rare (orphan) inheritable retinal diseases. We wanted to develop a rapid targeted-sequencing protocol for eight genes involved in Familial Exudative Vitreo-Retinopathy (FEVR), Norrie Disease, and Retinoschisis, at greatly reduced cost.
METHODS
DNA is extracted from 100 uL samples of whole frozen blood. An Ampliseq targeted-panel (180 amplicons) for 8 genes was designed with illumina's DesignStudio Sequencing Assay Designer, distributed into three pools (PCR reactions) per patient sample for complete coverage of 83 exons with 25 bp adjacent intron sequence. Target Genes were: NDP (ChrX), RS1 (Chr10); CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). Ampliseq libraries were quality controlled by capillary electrophoresis (Agilent Bioanalyzer) and several sample pool sizes were tested for capacity and coverage using sequencing and variant calling on the Illumina iSeq-100 platform.
RESULTS
An average 2500-times read coverage was obtained for a pool of 16 patient libraries and 800-times for a pool of 48 patient libraries. Numerous potential disease-associated variants were detected in targeted libraries from patients diagnosed with Norrie Disease, FEVR, and Retinoschisis. Average numbers of variants per patient for the 48 patient pool were: 19.5 ± 1.6 SNVs, 0.6 ± 0.2 Inserts, and 0.6 ± 0.2 Deletions.
CONCLUSIONS
We developed a targeted exome-sequencing protocol using Illumina Ampliseq reagents and the iSeq-100 platform for three rare pediatric retinal diseases. Coverage validated the ability to pool 40-50 patients per run for eight genes and provides for excellent base call accuracy (>Q30). Over 90 patient samples were successfully sequenced during validation. Potential mono and digenic variant contributions in FEVR patients are detectable by testing multiple genes. Research analysis costs were reduced to $250 per patient.
