CHARACTERIZATION OF VOLATILE ORGANIC COMPOUND (VOC) RELEASE AS A POTENTIAL BIOMARKER OF BOVINE HERPES VIRUS 1 (BOHV-1) INFECTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1021922
• Grant No.: 2020-38821-31119
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2019-03291
• Project Start Date: May 1, 2020
• Project End Date: Apr 30, 2025
• Grant Year: 2020
• Program Code: [EQ]- Research Project

Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853

Performing Department
Pharmaceutical Sciences

Non Technical Summary
Description of the current problem:Bovine herpesvirus type 1 (BHV-1) infection of cattle caused a number of serious complications such as upper respiratory tract disorders, conjunctivitis, and genital diseases. Furthermore, the virus may induce an immune suppression called bovine respiratory disease complex (BRDC) in which the U.S. cattle industry suffers approximately 3 billion dollars loss every year. BHV-1 belongs to the superfamilyof herpesviruses such as Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). They share a variety of similarity in terms of the genomes, structure, and infection modes.Early detection of BHV-1 is very important for agriculture and commercial purposes but not readily available.Approaches:Emission of volatile organic compounds (VOCs) upon viral infection is a recently investigated topic that holds great promise for early disease detection. Our recent report showed that gamma-butyrolactone (GBL), a VOC, was released upon HSV-1 acute infection, and that it could restrict viral replication, making the first reported discovery that a herpesvirus could trigger the release of VOC from infected cells. Given the fact of similarity between HSV-1 and BHV-1 and that GBL serves as a stress signal for bacteria, it is important to understand the role of GBL during the viral assault of cattle cells as a potential molecular biomarker.In this proposal, we will study the mechanism of GBL induction upon acute infection using BHV-1. Our Central Hypothesis is that GBL is part of the cellular self-defense mechanism against the released virus attacking neighboring cells. Our goal is to use a combination of biological/chemical tools to identify how the virus modulates the cells to generate GBL during and after infection.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1000	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

biomarker

bovine herpes virus

cattle

infection

volatile organic compound

Goals / Objectives
Description of the problem: Bovine herpesvirus type 1 (BHV-1) infection of cattle can result in a number of serious complications such as upper respiratory tract disorders, conjunctivitis, and genital disorders. Furthermore, the virus may induce an immune suppression called bovine respiratory disease complex (BRDC) in which the U.S. cattle industry suffers approximately 3 billion dollars every year. BHV-1 belongs to the familyof alphaherpesvirinae including some human herpesviruses such as Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV). They share a variety of similarity in terms of the genomes, structure, and infection modes.Early detection of BHV-1 is very important for agriculture and commercial purposes but not readily available.Goal, aims, and specific objectivesEmission of volatile organic compounds (VOCs) upon viral infection is a recently investigated topic that holds promise for early disease detection. Our recent report showed that gamma-butyrolactone (GBL), a VOC, was released upon HSV-1 acute infection, and that it could restrict viral replication, making the first reported discovery that a herpesvirus could trigger the release of VOC from infected cells. Given the fact of similarity between HSV-1 and BHV-1 and that GBL serves as a stress signal for bacteria, it is important to understand the role of GBL during the viral assault of cattle cells as a potential molecular biomarker. In this proposal, we will study the mechanism of GBL induction upon acute infection using BHV-1. Our Central Hypothesis is that GBL is part of the cellular self-defense mechanism against the released virus attacking neighboring cells. Our goal is to use a combination of biological/chemical tools to identify how the virus modulates the cells to generate GBL during and after infection. Based on our Central Hypothesis, we suggest three specific hypotheses and propose three specific objectives to test them.Hypotheses H1: Viral protein synthesis is required to modulate the infected cells for GBL induction (Objective 1).H2: Infected cells produce gamma-hydroxybutyrate (GHB) as a key cellular pathway intermediate for the subsequent production of GBL (Objective 2).H3: GBL generated from the 1st infection decreases further replication of the virus (Objective 3).Objective 1: Determine how the virus modulates the cells to release GBLWe will first test H1 to determine whether viral replication and de novo viral protein synthesis are required for this induction, using specific inhibitors such as acyclovir and cycloheximide, respectively.Next we will investigate if GBL can be induced by other cells or by other α-herpesviruses. Several epithelial and cornea cells will be tested with Vero as a control.Objective 2: Elucidate how the infected cells produce GBLFirst, we will systemically confirm that GBL was not produced through experimental procedures nor via components of media or cells not related to infection.We will next test H2 to define if GHB is a pathway intermediate for GBL production upon viral infection via a succinate pathway. A Radio-labelled feeding experiment using (13C)-isotope labeled GHB 9 will be used to feed the culture for validation.Objective 3: Characterize how GBL restricts virus spreadingWe will first test H3 to verify if GBL affects viral productivity. RNA isolated from infected cells with or without GBL will be added to a plate coated with primers against all known HSV-1 genes and subjected to qRT-PCR. Putative GBL regulatory effects will be monitored.Next we will determine if GBL has effects on host cell susceptibility to the virus. Infection at 4°C or by a UV-inactivated virus will be used to investigate the induction effects of virus attachment and entry.Evaluation PlanWe have developed a rigorous evaluation plan to monitor the progress of this project.Lab meeting will be held every Monday for faculty and students to discuss their progress and challenges.Faculty researchers will have faculty-only lunch meetings every Friday to recapitulate the project development and discuss alternative approach if necessary.The statistics will be performed by respective investigator and verified by a biostatistician.An appropriate Timeline is developed for initial monitoring.

Project Methods
In this proposal, we will study the mechanism of GBL induction upon acute infection using BHV-1. Our Central Hypothesis is that GBL is part of the cellular self-defense mechanism against the released virus attacking neighboring cells. Our goal is to use a combination of biological/chemical tools to identify how the virus modulates the cells to generate GBL during and after infection. Based on our Central Hypothesis, we suggest three specific hypotheses and propose three specific objectives to test them.Hypotheses H1: Viral protein synthesis is required to modulate the infected cells for GBL induction (Objective 1).H2: Infected cells produce gamma-hydroxybutyrate (GHB) as a key cellular pathway intermediate for the subsequent production of GBL (Objective 2).H3: GBL generated from the 1st infection decreases further replication of the virus (Objective 3).Objective 1: Determine how the virus modulates the cells to release GBLWe will first test H1 to determine whether viral replication and de novo viral protein synthesis are required for this induction, using specific inhibitors such as acyclovir and cycloheximide, respectively.Next we will investigate if GBL can be induced by other cells or by other α-herpesviruses. Several epithelial and cornea cells will be tested with Vero as a control.Objective 2: Elucidate how the infected cells produce GBLFirst, we will systemically confirm that GBL was not produced through experimental procedures nor via components of media or cells not related to infection.We will next test H2 to define if GHB is a pathway intermediate for GBL production upon viral infection via a succinate pathway. A Radio-labelled feeding experiment using (13C)-isotope labeled GHB 9 will be used to feed the culture for validation.Objective 3: Characterize how GBL restricts virus spreadingWe will first test H3 to verify if GBL affects viral productivity. RNA isolated from infected cells with or without GBL will be added to a plate coated with primers against all known HSV-1 genes and subjected to qRT-PCR. Putative GBL regulatory effects will be monitored.Next we will determine if GBL has effects on host cell susceptibility to the virus. Infection at 4°C or by a UV-inactivated virus will be used to investigate the induction effects of virus attachment and entry.Evaluation PlanWe have developed a rigorous evaluation plan to monitor the progress of this project.Lab meeting will be held every Monday for faculty and students to discuss their progress and challenges.Faculty researchers will have faculty-only lunch meetings every Friday to recapitulate the project development and discuss alternative approach if necessary.The statistics will be performed by respective investigator and verified by a biostatistician.An expert Dr. Clinton Jones from Oklahoma State University will be invited to evaluate the progress.An appropriate Timeline is developed for initial monitoring.Project plan/methods/approach and logic modelPlan:We will first characterize the release kinetics of BHV-1 in comparison to other herpesvirus using model cells. Next we will analyze how the virus influence the infected cells to release GBL. Finally we will elucidate how the infected cells produce GBL and determine how GBL restrict virus spreading.Methods:All cell lines used in the study will be obtained from American Type Culture Collection (ATCC) in Manassas, VA. Viruses for proposed studies were described previoulsly6. The BHV-1 will be a gift from Dr. Clinton Jones from Oklahoma State University with appropriate approval and documentation. All the methods used in the proposal such as GC/MS measurement analyses, fluorescent microscopy, electrophysiology, quantitative PCR/RT-PCR, plaque assays were previously described6.Summary of novel finding:GBL was detected from HSV-1 infected Vero cells using the GC/MS.The VOCs from the infected cells were collected via headspace sampling using a 50/30 μm Divinylbenzene/Carboxen Solid Phase MicroExtraction (SPME) device followed by analysis using 2D GC/MS. The compound identification was performed by comparing the measured mass spectrum of a VOC against spectra in the NIST mass spectral library to find the best match. The identified compounds were further confirmed using a standard. Within the 5-10 minute heartcut periods, the GBL concentration from the cell pellet peaked at 17.1 minutes.Approach:Objective 1: Determine how the virus modulates the infected cells to release GBLElucidate the GBL release kinetics/patterns upon BHV-1 infection. We will perform infection using BHV-1 at different multiplicity. The infections will be completed at 1, 5, 10, and 24 h followed by analyses via GC/MS, HPLC, and LC/MS.Determine whether viral replication or viral protein synthesis is required for GBL induction. Replication inhibitor acyclovir at 100 µM will be added to the infections and the headspace samples will be collected at 10 and 24 hpi followed by GC/MS and HPLC detection. To test if de novo protein synthesis is necessary for GBL induction, cycloheximide at 100 µg/ml will be used during infection and the headspace samples will be collected at 10 and 24 hpi. The samples will be subjected to GC/MS for GBL validation.Objective 2: Characterize how the infected cells produce GBLDefine if gamma hydroxybutyrate (GHB) is a pathway intermediate for GBL production upon viral infection. We propose that the conversion of succinate semialdehyde into g-GHB via a one-step reduction reaction catalyzed by succinate semialdehyde reductase is responsible for GBL production. We will test it via a radio-labelled feeding experiment using (13C)-isotope labelled GHB.9 The conversion of labelled GHB to GBL upon feeding in the culture will offer a reliable validation of whether the former molecule serves as a biosynthetic pathway intermediate for GBL. In parallel, we will employ a phenacyl bromide based derivatization method to overcome the analytical challenge posed by the high polarity and poor UV activity of GHB. This derivatization will enable us to reliably measure the concentration of GHB by both HPLC and GC-MS. 10, 11Validate the production of GHB and GBL by LC-MS/MS. Control samples will be prepared by spiking GHB or GBL to yield a series of calibrators at the following concentrations: 0, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L. In addition, an internal standard solution at a concentration of 2 mg/L will be added to the sample. LC-MS/MS measurement will be conducted using an Agilent 6545 Accurate Mass Q-TOF System. The column of the LC is an Agilent ZORBAX Eclipse Plus C18 RRHD, 3.0 × 50 mm, 1.8 μm (p/n 959757-302).Objective 3: Analyze how GBL restricts virus spreadingVerify if GBL affects viral productivity. We will further examine the regulatory effects of GBL on viral gene expression profile using qRTPCR assays. PCR primers against 75 HSV-1 genes 12 will be coated on plates and the RNA of infected cells in the presence or absence of GBL, and will be followed by qRTPCR analyses. We plan to perform infections with different moi and time points. Using an moi of 1, the RNA will be isolated at 2 hpi (α), 8 hpi (β and ?1), and 12 hpi (?2). If moi of 0.1 is used, RNA isolation will be performed at 6 hpi (α), 12 hpi (β and ?1), and 24 hpi (?2).Determine if GBL has effects on host cell susceptibility to the virus. We will continue to test H3 to determine if GBL has effects on virus exposure to the cells. Infections at 4 °C followed by GC/MS or HPLC will be used to investigate the induction effects of virus attachment. Next, we will perform infection using a UV-inactivated virus to verify if both attachment and entry are required for GBL Induction.

Progress 05/01/20 to 04/30/25

Outputs
Target Audience:Previously we participated inthe 2022 American Chemistry Society (ACS) annual meeting in San Diego, CA. We also attended theInternational virtual conference on "DRUG DISCOVERY, DEVELOPMENT AND LEAD OPTIMIZATION" on DECEMBER 16-17, 2022. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My former PhDstudent, Faith Osinaga, brought the NIFA project with her totheMerck companyand she completed a6 month on-site internship, which was highly successful, leading to apeer-reviewed publicationand ajob offer as a senior scientistat the same company. After returning, shefinished her dissertation, defended it successfully, and earned her doctoral degree within 5 years. Another graduate student, partially supported by the NIFA grant, Dr. Chen, also completed his PhD study, and he is now a research scientist in a NIH-sponsored project, BRAIN-STORRM (https://www.brainstorrm.org/). How have the results been disseminated to communities of interest?My student, Faith Osinaga, supported by the grant, delivered a platform presentation (Paper ID: 3641319) at the 2022 American Chemical Society (ACS) Annual Meetingin San Diego, CA. Her research waswell-received, garnering significant recognition from the audience. During the conference, sheestablished valuable connectionsand was subsequently offeredtwo 6-month internships--one atMerckand another atPfizer. Both companies expressed interest in expanding their research efforts in her field of study. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Previous research found that the herpes virus infection triggers the release of gamma-hydroxybutyrate (GHB), but the mechanisms behind its production were unclear. That study was successfully published in a peer-reviewed journal. Our recentstudy investigated the biochemical pathway responsible for GHB release during infection. Using methods like UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) to block different stages of viral replication, researchers found that UV-inactivated virus reduced GHB, while ACV and CHX had no effect. Additionally, inhibiting the glycolytic enzyme enolase with sodium fluoride decreased GHB production, suggesting glycolysis plays a role. The study also observed reduced levels of succinic semialdehyde dehydrogenase (involved in the TCA cycle) during infection, possibly leading to succinic semialdehyde buildup and subsequent GHB production. Themanuscript was published ina peer-reviewed journalPharmaceuticalsin 2023.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2021 Citation: Method validation of gamma-Hydroxybutyric acid detection upon Herpes Simplex Virus-Type 1 infection using LC-MRM-MS with 3- nitrophenylhydrazine derivatization

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:My PhD students Faith Osinaga gave a platform presentation at the University Research Symposium. The title is"Gamma-Hydroxybutyrate (GHB) Production Depends on Early Post-Entry Events in Herpes Simplex Virus-Type 1 Infection" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My former PhD student, Faith Osinaga, successfully collaborated/extentedto Merck during her highly productive 6-month internship. This collaboration yielded a peer-reviewed publication and resulted in her being offered a Senior Scientist position at Merck. Upon completing her internship, Dr. Osinaga successfully defended her dissertation and earned her PhD within an impressive 5-year timeframe. Additionally, another NIFA-supported graduate student, Dr. Chen, completed his doctoral studies and is now contributing to neuroscience research as a scientist on the NIH-funded BRAIN-STORRM project (https://www.brainstorrm.org/). How have the results been disseminated to communities of interest?Dr. Faith Osinaga, supported by our research grant, presented her work as a platform presentation (Paper ID: 3641319) at the American Chemical Society (ACS) Annual Meeting in San Diego. Her research attracted considerable attention from conference attendees and led to valuable professional connections. Notably, her presentation resulted in competitive internship offers from both Merck and Pfizer, with each company offering a 6-month research position. These opportunities reflect the industry's growing interest in her research area. What do you plan to do during the next reporting period to accomplish the goals?We have preliminary data suggesting the technique we developed can be used for more disease models. We are working together to streamline the process and hope to implement to other applications.

Impacts
What was accomplished under these goals? Prior studiesdemonstrated that herpes simplex virus (HSV) infection induces gamma-hydroxybutyrate (GHB) release, though the underlying mechanisms remained unclear. These findings were published in a peer-reviewed journal. Our subsequent study identified the biochemical pathway driving GHB production during HSV infection. By employing UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) to inhibit distinct stages of viral replication, we observed that UV-inactivated virus significantly reduced GHB release, whereas ACV and CHX had no effect. Furthermore, pharmacological inhibition of the glycolytic enzyme enolase (using sodium fluoride) diminished GHB production, implicating glycolysis in this process. We also found that HSV infection downregulates succinic semialdehyde dehydrogenase--a key TCA cycle enzyme--potentially leading to succinic semialdehyde accumulation and subsequent GHB synthesis. These results were published inPharmaceuticalsin 2023 (manuscript details).

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection

Progress 05/01/22 to 04/30/23

Outputs
Target Audience:2022 International conference on "DRUG DISCOVERY, DEVELOPMENT AND LEAD OPTIMIZATION" 16-17 DECEMBER 2022 Changes/Problems:I requested a 12-month no cost extension to complete the project objectives based on three justifications. 1) COVID-19 lab closure: The award started on May 1, 2020 while the labs and campus were closed due to quarantine. The lab was reopened approximately a year later. 2) Manuscript revision: We have a manuscript which is being revised. We need extra time to finish the required experiments. 3) Lab moving to the new SOPH Building: Our research slowed down in late January due to the moving of equipment. We expect to resume in April, 2023. The request was approved on April 10, 2023 and we are grateful for the assistance and support from the UMES administrator, staff, and funding agency. What opportunities for training and professional development has the project provided?The PhD student Faith Osinaga made a decision to pursue a 6-month on-site internship at Merck. This training appeared to be successful since she was offered a job at the same company as a senior scientist. She came back finished her dissertation, gave a successful dissertation defense, and earned her doctoral degree in 5 years. How have the results been disseminated to communities of interest?My student Faith Osinaga supported by the grant gave a platform presentation (PAPER ID: 3641319) at the American Chemical Society (ACS) annual meeting technical program last year in San Diego. Her research received great recognition from the audience. Druign the meeting, she established many good coonedctions and she was offered two 6-month internships from Merck and Pfizer. Both companies want to expand their research interests in that direction. What do you plan to do during the next reporting period to accomplish the goals?We will finalize this manuscript to be published at this peer-reviewed journal. Another trainee Teddy Chen will continue and finish the experiments. We plan to expand our research based on the foundation of these two articles to a comprehensive metablome study. We will contact the university public relation to compose an article for press release.

Impacts
What was accomplished under these goals? Previously we published that gamma-hydroxybutyrate (GHB) is released upon herpes virus acute infection but the mechanisms involved in the production of GHB in infected cells are unclear. This present study is focusing on the biochemical pathway responsible for the GHB release in infected cells. We used a variety of methods such as UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) treatments to control herpes virus replication/life cycle at various stages. It appears that UV-inactivated herpes virus significantly decreased GHB production but treatments with ACV or CHX did not affect. We also showed that inhibition of enolase by sodium fluoride significantly reduces GHB production upon infection. This finding suggests that suppression of glycolytic activity negatively affects cellular GHB production. Our data further indicated that succinic semialdehyde dehydrogenase, an enzyme involved in the shunt of the tricarboxylic acid (TCA) cycle to generate succinic acid, was decreased upon infection, suggesting that infection may trigger the accumulation of succinic semialdehyde, causing the production of GHB. We submitted a manuscript in 2023 entitled "Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection" to the journal "Pharmaceuticals". It is accepted but undergoing language/grammar editing.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection. Faith O. Osinaga, Yu-Chih Chen, Madan K. Kharel, Yan Waguespack, Sichu Li, and Shaochung Victor Hsia, Pharmaceuticals 2023

Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The participants of the 2022 American Chemistry Society (ACS) annual meeting in San Diego Changes/Problems:We do not have major change or problems! What opportunities for training and professional development has the project provided?My PhD student Faith Osinaga received further training at the Waters Coorporation. We offered training opportunity to a cohort of high school students under NIH-supported SEPA (Science Education Partnership Award) Program. How have the results been disseminated to communities of interest?My student Faith Osinaga gave a platform oral presentation (PAPER ID: 3641319) in the technical program of American Chemical Society (ACS) Spring 2022 in San Diego. Her research was supported by NIFA and she received great recognition from the audience. Our published abstract can been seen from the NIH Pubmedhttps://pubmed.ncbi.nlm.nih.gov/35042145/ and the full manuscript was available from the website, free of charge. https://www.sciencedirect.com/science/article/pii/S0731708521006580?via%3Dihub What do you plan to do during the next reporting period to accomplish the goals?We will finish and submit another manuscript for peer-reviewed publication in September, 2022. The trainee Ms. Faith Osinaga is preparing her thesis for her PhD dissertation defense. We will contact the university public relation to compose an article for press release.

Impacts
What was accomplished under these goals? We published an original research article in the Journal of pharmaceutical and biomedical analysis (J Pharm Biomed Anal) in Feb. 2022(doi: 10.1016/j.jpba.2021.114547).The title is "Method validation of gamma-Hydroxybutyric acid detection upon Herpes Simplex Virus-Type 1 infection using LC-MRM-MS with 3-nitrophenylhydrazine derivatization". NIFA support was acknowledged. In this article, we hypothesized that infected cells produce gamma-Hydroxybutyric acid (GHB) as a key pathway intermediate for the subsequent production of GBL. This is based on our previous report that gamma-butyrolactone (GBL), a VOC, was released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. There is no stable analytical technique for the rapid detection of GHB, and it is crucial for further understanding its role in the cellular response to HSV-1 infection. To address this issue, we have developed a sensitive, reliable, and specific methodology for the detection and quantification of GHB in mammalian cell culture using a pre-column derivatization approach. Our results indicated that the carboxylic acid functional group of GHB could be derivatized with 3-nitrophenylhydrazine hydrochloride (3-NPH) to produce its hydrazineyl derivative. Unlike GHB, the derivative could be detected seamlessly in HPLC-MS. We also demonstrate quantitative conversion of GHB into the derivative with over 95% yield at a range of 1 μg/mL- 6 μg/mL GHB concentration. This protocol provides a rapid quantification of GHB in aqueous mixtures, especially in cultured extracts.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Method validation of gamma-Hydroxybutyric acid detection upon Herpes Simplex Virus-Type 1 infection using LC-MRM-MS with 3-Nitrophenylhydrazine derivatization
• Type: Journal Articles Status: Published Year Published: 2022 Citation: J Pharm Biomed Anal. 2022 Feb 20;210:114547. doi:10.1016/j.jpba.2021.114547. Epub 2021 Dec 29.

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The participants of the 2021 University of Maryland Easterrn Shore Regional Research Symposium Changes/Problems:We do not have major change or problems except the COVID-19 pandemic. The campus closed for a while and we resumed the regular lab schedule as soon as it reopens. What opportunities for training and professional development has the project provided?The PI holds weekly meeting with scientists and students every Thursday. An expert was also invited to provide suggestions. The results were submitted and the student gave a platform presentation in a regional research symposium. In addition, it provides a great opportunity of problem solving for the faculty and the students. As we mentioned earlier, we encountered a number of unexpected issues. However, we worked together to improve our protocols and methodologies. For example, we tried six differentpre-column derivatization methods. Pre-column derivatization with 2-Hydrazinoquinoline (HQ) Pre-column derivatization with 4-Acetamido-7-mercapto-2,1,3 benzoxadiazole (AABD-SH) Pre-column derivatization with 4-methylbenylamine (4-BMBA) Pre-column derivatization with 4-bromo-N-methylanline (4-BMA) Pre-column derivatization with 3-picolyamine (3-PA) Pre-column derivatization with 3-nitrophenylhydrazine (3-NPH)- the successful reaction All the derivatization reactions worked, however, reactions 1-5 had poor recovery percentage lower than 70% or the method was only qualitative not quantitative. Using the method 6 we successfully isolated GHB and it was quantitative. We determined the retention time. However, to successfully identify our compound of interested was produced, we would need the chemical synthesize derivatized GHB. We reached out to various national synthetic chemistry compounds. However, a few disagreed that the synthesize would not be able to be possible. Luckily, a company in Canada was able to synthesize our compound of interest. The product actually was actually synthesized in Ukraine. We ran the synthesized derivatized GHB on the UPLC with our developed method, but the retention was different. The previous retention identified another product. We have been optimizing the method for an incorrect analyte. Finally, with more method development we were able to successfully isolate and validate derivatized GHB via 3-nitrophenylhydrazine (3-NPH) pre-column derivatization on UPLC-MRM-MS. How have the results been disseminated to communities of interest?We have some interesting preliminary results. The student trainee gave an oral presentation in a regional research symposium. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will improve our methodology with more controls. We will perform experiments at different time points. We will start writing two manuscripts for publication.

Impacts
What was accomplished under these goals? We have finally established a validated protocol after many attempts usingUPLC-MRM-MS to detect organic compound triggered by infection. Our preliminary data suggested that Hypothesis 2 that GHB appeared to be a key cellular pathway intermediate. We have tried many methods such as GC/MS direct injection, GC/MS SPME, HPLC UV detection, LC/MS direct detection, LC/MSpre-column derivatization, etc. All have issues in obtaining satisfactory results with accuraaacy and sensitivity. Our current method is well established and validated and we are very confident it will be an invaluable tool for the scientific community.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: QUANTIFICATION AND DETECTION OF VOLATILE ORGANIC COMPOUND, GAMMA-BUTYROLACTONE RELEASE UPON HERPES SIMPLEX VIRUS TYPE-1 INFECTION