Targeted Proteomics for Exosome Analysis and Its Application to Develop Blood Markers of Liver Drug-Metabolizing Enzymes

Two main routes of drug elimination include renal excretion via the kidney and metabolic degradation via metabolizing enzymes (DMEs) in the liver, followed by renal excretion or biliary elimination of the now more polar molecule. Unlike renal excretion, liver metabolism tends to be highly variable between individuals due to various intrinsic and extrinsic factors such as age, genetics, smoking, and diet. The variability in liver DME activity among individuals creates a challenge in drug development and pharmacotherapy, especially for drugs that are required to be metabolized and have narrow therapeutic windows. If the liver DME activity of an individual could be predicted and each patient dosed according to their unique liver DME activity, the adverse effects or lack of effectiveness associated with these drugs may be prevented. Currently, two methods available for predicting an individual’s liver DME activity are genotyping and phenotyping. For genotyping, RT-PCR is used to determine the DNA sequence of the expressed enzyme. This reveals the specific polymorphism of the enzyme and the “typical activity” of the expressed polymorph is used to dose the patient accordingly. The issue with this however, is that it fails to account for other intrinsic or extrinsic factors that can affect the expression level of the given DME polymorph. On the other hand, phenotyping is done by administering a cocktail of drugs to a patient and monitoring the activity of the expressed enzyme(s). Because this reveals the true activity of the liver DMEs, it is more clinically relevant and used more often than genotyping. However, phenotyping is expensive and requires consistent monitoring by medical professionals in the hospital, thus is inconvenient. Accurate prediction of liver DME activity at the individual level in the clinical setting remains challenging, however if a more clinically friendly method were to be developed, it could lead to a broader range of potential drug candidates in drug development and lower risk of adverse drug responses. In this dissertation, the use of exosomes to act as biomarkers for liver DMEs is investigated. Exosomes are vesicles, typically 50 – 150 nm in diameter, secreted by cells into the extracellular space. In the human body, exosomal vesicles have been found in blood, saliva, urine, breast milk, and other bodily fluids. What makes exosomes unique from other vesicles secreted by cells, is that exosomes are formed by an endosomal route, thus contain cargo that reflecting the cell from which they are being secreted, in the extracellular space. This allows for the development of minimally invasive “liquid biopsies” to probe for markers of different diseases and cancers. While exosomes have been demonstrated as useful tools for diagnosis and monitoring patient response to treatment, they are yet to be used in the clinical setting. This is due to the lack of standardization in exosomal isolation and analysis. These challenges were also addressed in this dissertation. In summary, this dissertation describes the development of a liquid chromatography multiple-reaction-monitoring mass spectrometry (LC-MRM-MS) method for exosomal analysis and its benefits over more traditional assays. Following the method development, the presence of DMEs in exosomes were explored along with the ability of exosomal DME levels to be altered to reflect a change occurring in the secreting cell. Finally, the ability to isolate liver derived exosomes based on the expression of a liver specific marker protein, ASGR1, is explored. Further efforts of this project could lead to the development of a blood-based biopsy to evaluate the DME content of liver derived exosomes, which may correlate to liver DME activity, providing a new-found basis of personalized medicine

A nomogram to determine required seed air kerma strength in planar 131 Cesium permanent seed implant brachytherapy

Purpose: Intraoperatively implanted Cesium-131 ( 131 Cs) permanent seed brachytherapy is used to deliver highly localized re-irradiation in recurrent head and neck cancers. A single planar implant of uniform air kerma strength (AKS) seeds and 10 mm seed-to-seed spacing is used to deliver the prescribed dose to a point 5 mm or 10 mm perpendicular to the center of the implant plane. Nomogram tables to quickly determine the required AKS for rectangular and irregularly shaped implants were created and dosimetrically verified. By eliminating the need for a full treatment planning system plan, nomogram tables allow for fast dose calculation for intraoperative re-planning and for a second check method. Material and methods: TG-43U1 recommended parameters were used to create a point-source model in MATLAB. The dose delivered to the prescription point from a single 1 U seed at each possible location in the implant plane was calculated. Implant tables were verified using an independent seed model in MIM Symphony LDR™. Implant tables were used to retrospectively determine seed AKS for previous cases: three rectangular and three irregular. Results: For rectangular implants, the percent difference between required seed AKS calculated using MATLAB and MIM was at most 0.6%. For irregular implants, the percent difference between MATLAB and MIM calculations for individual seed locations was within 1.5% with outliers of less than 3.1% at two distal locations (10.6 cm and 8.8 cm), which have minimal dose contribution to the prescription point. The retrospectively determined AKS for patient implants using nomogram tables agreed with previous calculations within 5% for all six cases. Conclusions: Nomogram tables were created to determine required AKS per seed for planar uniform AKS 131 Cs implants. Comparison with the treatment planning system confirms dosimetric accuracy that is acceptable for use as a second check or for dose calculation in cases of intraoperative re-planning

Modeled Wellness: How Perceived Supervisor Wellness Explains Supervisee Personal Wellness

In this quantitative investigation of 105 counselors-in-training, we analyzed how their perceptions of supervisor wellness are related to their own levels of wellness. The supervisee’s perception of their site supervisor’s level of wellness did explain the personal wellness of the counselor-in-training and the strength of the supervisory relationship acted as a suppressor variable in the expanded regression model. Implications for counselor educators and supervisors include the importance of adopting positive wellness attitudes and behaviors. Counselors-in-training appear to notice, for good and for bad, their supervisors’ levels of wellness. Adopting positive wellness attitudes and behaviors allows supervisors to model positive approaches

Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

This work is licensed under a Creative Commons Attribution 4.0 International License.The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality

Graduate Assessment at a Predominantly Undergraduate Institution

Poster presented at the Assessment for Curricular Improvement Poster Exhibit 2023."This project focuses on improving graduate program assessment at a primarily undergraduate-serving liberal arts college. The university has 38 different graduate programs across 6 different schools. Most of the programs lack proficiency exams, a thesis, or other universal culminating experiences. Some are 4+1 programs and many cater to working professionals, and students begin their programs at different points of the year. These conditions have made developing manageable, systematic assessment challenging. In this project, assessment at the graduate level is improved with two main efforts. The first is by providing expanded resources on Educational Assessment targeting graduate programs. The second is working directly with each program to establish or modify assessment plans and, helping programs develop new approaches to assessment, including, for example, the use of portfolio methods for program-level assessment. These efforts aim to have 75% of programs develop assessment plans by the end of the 2022-2023 academic year.

Multi-Stakeholder Focus Groups on Potential for Meat Inspection Data to Inform Management of Pig Health and Welfare on Farm

peer-reviewedMeat inspection (MI) findings can act as a valuable source of information on pig health and welfare. The PIG WELFare INDicators (PIGWELFIND) project (Research Stimulus Fund 11/S/107) was developed to progress the development of ante and post mortem MI as a pig health and welfare diagnostic tool in Ireland. Three multi-stakeholder focus groups were organized to explore areas of conflict and agreement between stakeholders’ vision for including pig health and welfare indicators in MI and on how to achieve this vision. Each focus group consisted of eight stakeholders: pig producers, Teagasc pig advisors, pig processors, veterinarians involved in MI, private veterinary practitioners, and personnel with backgrounds in general animal health and welfare and food safety policy. In general, stakeholders expressed positive attitudes towards the use of MI data to inform pig health and welfare when standardization of recording and feedback is improved, and the MI system provides real-time benchmarking possibilities. Most emphasis was placed on health indicators as a first priority, while it was felt that welfare-related indicators could be included after practical barriers had been addressed (i.e., line speed/feasibility, standardization and training of meat inspectors, data ownership). Recommendations are made to further progress the development of MI as a pig health and welfare diagnostic tool and address some of these barriers

Single institution implementation of permanent 131Cs interstitial brachytherapy for previously irradiated patients with resectable recurrent head and neck carcinoma

Purpose: Permanent interstitial brachytherapy is an appealing treatment modality for patients with locoregional recurrent, resectable head and neck carcinoma (HNC), having previously received radiation. Cesium-131 (131Cs) is a permanent implant brachytherapy isotope, with a low average photon energy of 30 keV and a short half-life of 9.7 days. Exposure to medical staff and family members is low; patient isolation and patient room shielding are not required. This work presents a single institution’s implementation process of utilizing an intraoperative, permanent 131Cs implant for patients with completely resected recurrent HNC. Materials & Methods: Fifteen patients receiving 131Cs permanent seed brachytherapy were included in this analysis. The process of pre-planning, selecting the dose prescription, seed ordering, intraoperative procedures, post-implant planning, and radiation safety protocols are described. Results: Tumor volumes were contoured on the available preoperative PET/CT scans and a pre-implant treatment plan was created using uniform source strength and uniform 1 cm seed spacing. Implants were performed intraoperatively, following tumor resection. In five of the fifteen cases, intraoperative findings necessitated a change from the planned number of seeds and recalculation of the pre-implant plan. The average prescription dose was 56.1 ±6.6 Gy (range, 40-60 Gy). The average seed strength used was 2.2 ±0.2 U (3.5 ±0.3 mCi). Patients returned to a recovery room on a standard surgical floor and remained inpatients, without radiation safety restrictions, based on standard surgical recovery protocols. A post-implant treatment plan was generated based on immediate post-operative CT imaging to verify the seed distribution and confirm delivery of the prescription dose. Patients were provided educational information regarding radiation safety recommendations. Conclusions: Cesium-131 interstitial brachytherapy is feasible and does not pose major radiation safety concerns; it should be considered as a treatment option for previously irradiated patients with recurrent, resectable HNC

Teaching teachers to teach writing, critical thinking, and information literacy: A case study of a faculty learning community as a campus-wide pedagogical intervention strategy

In this session, faculty collaborative group drawn from multiple disciplines and university units reports on an initiative to improve research and writing instruction in upper-division major-specific courses. Presenters showcase the initiative’s intervention, a faculty learning community (FLC) whose members were recruited from across the university to study evidence-based best practices for writing, critical thinking, and information literacy instruction. The session will also detail the how the FLC was shaped by institutional assessment data, discuss how it builds on local faculty development traditions, and assess its impact through analysis of course documents created by faculty participants

Optimizing parametrial aperture design utilizing HDR brachytherapy isodose distribution.

Treatment of cervical cancer includes combination of external beam radiation therapy (EBRT) and brachytherapy (BRT). Traditionally, coronal images displaying dose distribution from a ring and tandem (R&T) implant aid in construction of parametrial boost fields. This research aimed to evaluate a method of shaping parametrial fields utilizing contours created from the high-dose-rate (HDR) BRT dose distribution. Eleven patients receiving HDR-BRT via R&T were identified. The BRT and EBRT CT scans were sent to FocalSim (v4.62)(®) and fused based on bony anatomy. The contour of the HDR isodose line was transferred to the EBRT scan. The EBRT scan was sent to CMS-XIO (v4.62)(®) for planning. This process provides an automated, potentially more accurate method of matching the medial parametrial border to the HDR dose distribution. This allows for a 3D-view of dose from HDR-BRT for clinical decision-making, utilizes a paperless process and saves time over the traditional technique