BIOMED Master's Thesis Defense

Title:
UV-Assisted Functionalization of 3D-Printed PEEK for Use in Spinal Fusion Cages

Speaker:
Paul M. DeSantis, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Michele Marcolongo, PhD
Department Head and Professor
Department of Materials Science and Engineering
College of Engineering
Drexel University

Details:
Treatment for intervertebral disc degeneration involves the complete removal of the disc and the fusion of two adjacent vertebrae via a biocompatible implant. The implant must be capable of both supporting the mechanical forces experienced by the spine, as well as promoting bone growth. Polyetheretherketone, or 'PEEK,' has been shown to perform well in spinal fusion surgeries, but can face challenges with osseointegration as it is highly hydrophobic. Post-processing techniques have been considered as a method to improve the osseointegration of PEEK spinal cages, with ultraviolet (UV) light assisted functionalization being one possible method. The addition of calcium phosphate to bone scaffolds has been shown to increase osseointegration due to similarities to hydroxyapatite, a major inorganic component of bone. We investigated the use of UV-assisted functionalization to add a calcium phosphate layer to PEEK samples in order to improve osseointegration.

PEEK samples with dimensions of 10x10x1 mm were obtained via fused filament fabrication/3D printing and submerged in simulated body fluid (SBF).  Samples were exposed to a 2W/cm2 UV light for six hours, and then placed in a water bath set to 37°C for a total of 72 hours, with SBF being changed after 48 hours. After functionalization, the PEEK samples were washed with DI water and dried. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed on the functionalized samples, control PEEK samples that were not treated, and control samples that were soaked in SBF for 72 hours but were not exposed to UV light. Images of the surface of the functionalized samples and controls were obtained using a scanning electron microscope (SEM) (Zeiss Supra 50VP) with EDS (Oxford) capabilities.

SEM/EDS analysis of UV functionalized PEEK samples showed morphological surface changes as well as calcium and phosphorous on the surface of the material. The functionalized PEEK samples were found to have a molar Ca:P ratio of 1.69, while natural hydroxyapatite has a molar ratio of 1.67. EDS analysis of non-functionalized control samples found no calcium or phosphorus. FTIR results found that the non-functionalized PEEK samples and the PEEK samples that were soaked in SBF but were not treated by UV were nearly identical, with no calcium phosphate. For functionalized samples, notable peaks were observed at 560 and 600 cm-1, and between 1000 and 1100 cm-1, which has shown to be a phosphate group.

Analysis of functionalized PEEK revealed that UV-assisted functionalization successfully applied a layer of calcium phosphate material to the surface of the sample. While the goal was to obtain a layer of hydroxyapatite in order to promote osteoblast adhesion, the observed ratios of calcium to phosphate obtained via EDS are more likely to indicate an amorphous calcium phosphate layer. The addition of this layer to the PEEK surface is believed to enhance the osseointegration of newly developed biomedical implants.

BIOMED PhD Thesis Defense

Title:
Cell Surface Env Transformation and Membrane Disruption by HIV-1 Entry Inhibitors
    
Speaker:
Charles G. Ang, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisors:
Irwin Chaiken, PhD
Professor of Biochemistry and Molecular Biology
College of Medicine
Drexel University

Ken Barbee, PhD
Professor, Senior Associate Dean, and Associate Dean for Research
School of Biomedical Engineering, Science and Health Systems
Drexel University

Details:
This work examined transformations of the HIV-1 fusion protein Env and its anchoring membrane that can be subverted by lytic entry inhibitors to inactivate Env on viruses and cells. Two entry inhibitors were investigated: KR13 and M*DAVEI. KR13 is a chemically synthesized peptide triazole thiol (PTT) comprised of a CD4 binding site pharmacophore linked to a disulfide-targeting thiol, while M*DAVEI is a chimeric recombinant protein containing a gp120-glycan-binding lectin linked to Trp3, a gp41-binding fragment of Env’s membrane proximal external region (MPER). Both inhibitors have previously been shown to cause virus lysis, indicating that they can trigger membrane disruption. Env-presenting cells were selected for these experiments to facilitate mechanistic studies and provide preliminary data for further cell-based studies.

To investigate KR13, we developed a flow cytometry protocol to examine the nature of its Env rearrangements by measuring changes in epitope availability for key regions of Env upon treatment with serial dilutions of KR13 or its thiol-blocked analogue KR13b. HEK293T cells were transiently transfected for surface presentation of JRFL Env and pre-loaded with fluorescent dye. Epitopes examined were: intact gp120-gp41 association (Ab 35O22), formation of the 6HB (Ab NC-1), MPER exposure (Ab 10E8), the immunodominant loop (Ab 50-69), fusion peptide (Ab VRC34.01), and membrane disruption (loss of intracellular dye). Env transformations caused by KR13 resembled several steps of native fusion, including loss of attached gp120, increase in 6HB detection, increase in MPER exposure, and membrane disruption, suggesting that the mechanism by which KR13 induces transformation may incorporate and subvert the conformational “program” Env uses to enter target cells. In turn, KR13 and PTT compounds in general may be useful not only as antagonist leads but also as probes to interrogate HIV entry and the exposure of key post-fusion epitopes from the unique approach of transforming Env and membrane without target cells. Further experiments with KR13 and KR13b showed that the membrane disruption and leakage effects of KR13 could be recapitulated by the combination of KR13b and the redox protein Thioredoxin-1 (Trx1). Virions were treated with either KR13 alone, or a combination of fixed concentration KR13b and serial dilutions of Trx1, showing comparable p24 release between the two. Along with literature showing Trx1’s presence in and necessity to native fusion, we established that the KR13’s free terminal thiol may be mimicking the reducing effects of Trx1, likely targeting Env’s C296-C331 disulfide.  

The second part of this research examined the engineered protein M*DAVEI, which causes severe membrane disruptions in virions, speculated to result from the dual engagement of Env by the gp120-binding lectin and the MPER/membrane-interacting Trp3. We investigated whether the destructive effect of lectin-DAVEI on viruses could be used to target cells presenting Env on their surfaces. HEK293T cells were transiently transfected with DNA encoding JRFL Env, pre-loaded with calcein dye, treated with serial dilutions of M*DAVEI, and stained for intact gp120-gp41 association (Ab 35O22). The observed effects of M*DAVEI on cells were proportional to individual levels of Env expression: "Low-Env" cells experienced transient poration and calcein leakage, while "High-Env" cells died. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and its associated cytotoxicity. Controls with unlinked M*DAVEI components showed no effect on calcein leakage or cell viability, confirming covalently linked DAVEI components as necessary to induce transformation.  

Cumulatively, this work has demonstrated Env metastability as an intrinsic property of the transmembrane protein complex and not limited to fusogenic or fusion-like transformations of Env alone. As such, the Env-membrane complex can be perturbed to trigger membrane disruption in virus and cell contexts, an extension of our understanding of these entry inhibitors’ effects and their mechanisms of action.

BIOMED Master's Thesis Defense

Title:
Automated Genomic Wide Variants Analysis and Reporting Pipeline

Speaker:
Nhat Duong, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Advisor:
Michael Xie, PhD
Supervisory Bioinformatics Scientist
Department of Biomedical and Health Informatics
Children's Hospital of Philadelphia (CHOP)

Details:
The field of bioinformatics contains many pipelines for different purposes. These pipelines are often for data processing, which are used to process raw files into ready to be analyzed files. Despite availability of many different pipelines, the process of going from processed files to analysis the data and making a report from the results are still largely done manually. Among different data analyses, a commonly used analysis applicable across a variety of data is the genetic/mutation load analysis on cohort data (cases vs. controls groups). It is thus desirable to produce a fully automated and flexible pipeline capable of going from the variant discovery VCF file and BED file all the way to a final interactive report file which can be presented to clinicians or principle investigators.

The pipeline was made using Snakemake as the workflow management tool. The pipeline starts with a VCF file that is derived from the user’s preferred variant discovery method. This file then gets filtered, reformatted, statistically analyzed and the results are stored and displayed in an interactive webpage. This pipeline also takes in copy number variation information in the form of a BED file and performs analyses independent of the short variants data. The MetaP Fisher method was added to the pipeline to combine the p-values from the two independent analyses (short variant and copy number variation) which can buttress any positive results. Results from these analyses would get stored in a database and displayed in an interactive webpage.

An application of the pipeline was done to study congenital heart defects (CHD) in a cohort of 22q11.2 deletion syndrome patients. 22q11.2 deletion syndrome (22q11.2DS) is the absence of a DNA segment – roughly 3 million base pairs in size – on one copy of chromosome 22. The pipeline mentioned above was used with whole exome sequencing samples of 380 22q11.2DS patients as the VCF input. After QC, there remains 147 cases (22q11.2DS patients with CHD) vs. 132 controls (22q11.2DS patients without CHD) samples. The pipeline compared the genetic loads between the case and control cohorts by performing gene-gene and functional term analyses. The goal was to identify mutations that are significantly over-represented in cases as compared to controls.

Successful run of the pipeline’s gene-gene analysis was able to identify a gene cluster on chromosome 9 – mostly 9q – that is significantly over-represented in 22q11.2DS patients with CHD. Most genes in this cluster are closely associated with early cell and embryonic development. The functional-based analysis – using GO terms and mammalian phenotypes – resulted in numerous cardiac related functions significantly over-represented in 22q11.2DS patients with CHD, as well. Some of the most significant functions include heart trabecula and cardiac ventricle morphogenesis. All of these results were reported using an HTML file. This result suggests that further interrogation of the loci on human chromosome 9 and the cardiac functions identified from the functional analysis are warranted. Most importantly, however, it shows that the pipeline was successful in performing a cohort study and generating an interactive report file.