Linking Angelman and dup15q data for expanded research (LADDER) database: a model for advancing research, clinical guidance, and therapeutic development for rare conditions.

Angelman syndrome (AS) and duplication 15q (dup15q) syndrome are rare neurogenetic conditions arising from a common locus on the long arm of chromosome 15. Individuals with both conditions share some clinical features (e.g. intellectual disability, epilepsy) and often require lifelong care. Disease-modifying therapies for both conditions are emerging, resulting in a significant need for a better understanding of the natural history of both AS and dup15q. Patient advocacy groups for both conditions recognized a need for a data repository that would link data on individuals from multiple sources to expand research, increase understanding of natural history, and accelerate the development of treatments, resulting in the Linking Angelman and Dup15q Data for Expanded Research (LADDER) Database. This paper describes the development and functionality of the LADDER Database - including challenges, lessons learned, and preliminary feasibility - and how it can be used as a model for other rare conditions.

The LADDER database: a model for advancing research, clinical guidance, and therapeutic development for rare conditions This paper describes the development and functionality of the Linking Angelman and Dup15q Data for Expanded Research (LADDER) Database, which is a data repository for two rare neurogenetic conditions: Angelman syndrome (AS) and duplication 15q (dup15q) syndrome. AS and dup15q syndrome arise from genetic abnormalities on chromosome 15 and share some clinical features (e.g. intellectual disability, epilepsy). LADDER was developed by patient advocacy organizations representing each condition in partnership with RTI International. LADDER links data on individuals from multiple sources to expand research, increase understanding of natural history, and accelerate the development of treatments for both AS and dup15q syndrome. The LADDER Database can be used as a model for expanding research and enhancing clinical trial readiness in other rare conditions.

Age of diagnosis for children with chromosome 15q syndromes.

OBJECTIVE: The objective of this study was to identify the age of diagnosis for children with one of three neurogenetic conditions resulting from changes in chromosome 15 (Angelman syndrome [AS], Prader-Willi syndrome [PWS], and duplication 15q syndrome [Dup15q]). METHODS: Data about the diagnostic process for each condition were contributed by the advocacy organizations. Median and interquartile ranges were calculated for each condition by molecular subtype and year. Comparison tests were run to explore group differences. RESULTS: The median age of diagnosis was 1.8 years for both AS and Dup15q. PWS was diagnosed significantly younger at a median age of 1 month. Deletion subtypes for both PWS and AS were diagnosed earlier than nondeletion subtypes, and children with isodicentric duplications in Dup15q were diagnosed earlier than those with interstitial duplications. CONCLUSION: Understanding variability in the age of diagnosis for chromosome 15 disorders is an important step in reducing the diagnostic odyssey and improving access to interventions for these populations. Results from this study provide a baseline by which to evaluate efforts to reduce the age of diagnosis for individuals with these conditions.

Systematic discovery of neoepitope-HLA pairs for neoantigens shared among patients and tumor types.

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.

Inclusion of postdoctoral trainees in a translational science training TL1 program was associated with greater diversification of research across the translational science continuum: a bibliometric analysis of TL1 trainee publications.

The NIH National Center for Advancing Translational Science (NCATS) was established to support translational research that spans the entire TS Continuum, with the goal of bridging the gap between preclinical biomedical research and real-world applications to advance treatments to patients more quickly. In 2018, the Translational Science Training (TST) TL1 Program at the University of Texas Health Science Center at San Antonio implemented new strategies to better include and encourage research more broadly across the TS Continuum, including the addition of postdoctoral scientists and a clinically trained Program Co-Director, expansion of team science and community engagement programming, and targeted trainee recruitment from schools of nursing, dentistry, and allied health, in addition to medicine. The objective of this bibliometric analysis was to determine if the program exhibited a more diverse mix of T-types after the adjustments made in 2018. The TST/TL1 Program experienced a shift in T-type, from mostly T0 (preclinical) to more T3/T4 (clinical implementation/public health) research, after new strategies were implemented. This supports the conclusion that strategic programmatic adjustments by an NCATS-funded predoctoral training program resulted in outcomes that better align with NCATS priorities to develop Trainees who contribute across the entire TS Continuum.

Durvalumab Treatment Patterns for Patients with Unresectable Stage III Non-Small Cell Lung Cancer in the Veterans Health Administration (VHA): A Nationwide, Real-World Study.

BACKGROUND: Durvalumab is approved for the treatment of adults with unresectable stage III non-small cell lung cancer (NSCLC) post-chemoradiotherapy (CRT). This real-world study describes patient characteristics and durvalumab treatment patterns (number of doses and therapy duration; treatment initiation delays, interruptions, discontinuations, and associated reasons) among VHA-treated patients. METHODS: This was a retrospective cohort study of adults with unresectable stage III NSCLC receiving durvalumab at the VHA between 1 January 2017 and 30 June 2020. Patient characteristics and treatment patterns were presented descriptively. RESULTS: A total of 935 patients were included (median age: 69 years; 95% males; 21% Blacks; 46% current smokers; 16% ECOG performance scores ≥ 2; 50% squamous histology). Durvalumab initiation was delayed in 39% of patients (n = 367). Among the 200 patients with recorded reasons, delays were mainly due to physician preference (20%) and CRT toxicity (11%). Overall, patients received a median (interquartile range) of 16 (7-24) doses of durvalumab over 9.0 (2.9-11.8) months. Treatment interruptions were experienced by 19% of patients (n = 180), with toxicity (7.8%) and social reasons (2.6%) being the most cited reasons. Early discontinuation occurred in 59% of patients (n = 551), largely due to disease progression (24.2%) and toxicity (18.2%). CONCLUSIONS: These real-world analyses corroborate PACIFIC study results in terms of the main reasons for treatment discontinuation in a VHA population with worse prognostic factors, including older age, predominantly male sex, and poorer performance score. One of the main reasons for durvalumab initiation delays, treatment interruptions, or discontinuations was due to toxicities. Patients could benefit from improved strategies to prevent, identify, and manage CRT and durvalumab toxicities timely and effectively.

Multimodal, broadly neutralizing antibodies against SARS-CoV-2 identified by high-throughput native pairing of BCRs from bulk B cells.

TruAB Discovery is an approach that integrates cellular immunology, high-throughput immunosequencing, bioinformatics, and computational biology in order to discover naturally occurring human antibodies for prophylactic or therapeutic use. We adapted our previously described pairSEQ technology to pair B cell receptor heavy and light chains of SARS-CoV-2 spike protein-binding antibodies derived from enriched antigen-specific memory B cells and bulk antibody-secreting cells. We identified approximately 60,000 productive, in-frame, paired antibody sequences, from which 2,093 antibodies were selected for functional evaluation based on abundance, isotype and patterns of somatic hypermutation. The exceptionally diverse antibodies included RBD-binders with broad neutralizing activity against SARS-CoV-2 variants, and S2-binders with broad specificity against betacoronaviruses and the ability to block membrane fusion. A subset of these RBD- and S2-binding antibodies demonstrated robust protection against challenge in hamster and mouse models. This high-throughput approach can accelerate discovery of diverse, multifunctional antibodies against any target of interest.

Positive Allosteric Modulator of SERCA Pump NDC-1173 Exerts Beneficial Effects in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects millions of people worldwide. AD does not have a cure and most drug development efforts in the AD field have been focused on targeting the amyloid pathway based on the "amyloid cascade hypothesis". However, in addition to the amyloid pathway, substantial evidence also points to dysregulated neuronal calcium (Ca2+) signaling as one of the key pathogenic events in AD, and it has been proposed that pharmacological agents that stabilize neuronal Ca2+ signaling may act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+ stabilizing agents. In the present study, we report the development of a novel SERCA PAM agent, compound NDC-1173. To test the effectiveness of this compound, we performed behavioral studies with the APP/PS1 transgenic AD mouse model. We also evaluated effects of this compound on expression of endoplasmic reticulum (ER) stress genes in the hippocampus of APP/PS1 mice. The results of this study support the hypothesis that the SERCA pump is a potential novel therapeutic drug target and that NDC-1173 is a promising lead molecule for developing disease-modifying agents in AD.

Health Equity in Patients Receiving Durvalumab for Unresectable Stage III Non-Small Cell Lung Cancer in the US Veterans Health Administration.

BACKGROUND: Real-world evidence is limited regarding the relationship between race and use of durvalumab, an immunotherapy approved for use in adults with unresectable stage III non-small cell lung cancer (NSCLC) post-chemoradiotherapy (CRT). This study aimed to evaluate if durvalumab treatment patterns differed by race in patients with unresectable stage III NSCLC in a Veterans Health Administration (VHA) population. MATERIALS AND METHODS: This was a retrospective analysis of White and Black adults with unresectable stage III NSCLC treated with durvalumab presenting to any VHA facility in the US from January 1, 2017, to June 30, 2020. Data captured included baseline characteristics and durvalumab treatment patterns, including treatment initiation delay (TID), interruption (TI), and discontinuation (TD); defined as CRT completion to durvalumab initiation greater than 42 days, greater than 28 days between durvalumab infusions, and more than 28 days from the last durvalumab dose with no new durvalumab restarts, respectively. The number of doses, duration of therapy, and adverse events were also collected. RESULTS: A total of 924 patients were included in this study (White = 726; Black = 198). Race was not a significant factor in a multivariate logistic regression model for TID (OR, 1.39; 95% CI, 0.81-2.37), TI (OR, 1.58; 95% CI, 0.90-2.76), or TD (OR, 0.84; 95% CI, 0.50-1.38). There were also no significant differences in median (interquartile range [IQR]) number of doses (White: 15 [7-24], Black: 18 [7-25]; P = .25) or median (IQR) duration of therapy (White: 8.7 months [2.9-11.8], Black: 9.8 months [3.6-12.0]; P = .08), although Black patients were less likely to experience an immune-related adverse event (28% vs. 36%, P = .03) and less likely to experience pneumonitis (7% vs. 14%, P < .01). CONCLUSION: Race was not found to be linked with TID, TI, or TD in this real-world study of patients with unresectable stage III NSCLC treated with durvalumab at the VHA.

S-nitroso-glutathione (GSNO) inhibits hydrogen peroxide production by alpha-ketoglutarate dehydrogenase: An investigation into sex and diet effects.

Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital sources of hydrogen peroxide (H2O2) and key sites for redox regulation. Here, we report KGDH is more sensitive to inhibition by S-nitroso-glutathione (GSNO) when compared to PDH and deactivation of both enzymes by nitro modification is affected by sex and diet. Liver mitochondria from male C57BL/6 N mice displayed a robust inhibition of H2O2 production after exposure to 500-2000 µM GSNO. H2O2 genesis by PDH was not significantly affected by GSNO. Purified KGDH of porcine heart origin displayed a ∼82% decrease in H2O2 generating activity at 500 µM GSNO, which was mirrored by a decrease in NADH production. By contrast, H2O2- and NADH-producing activity of purified PDH was only minimally affected by an incubation in 500 µM GSNO. Incubations in GSNO had no significant effect on the H2O2-generating activity of KGDH and PDH in female liver mitochondria when compared to samples collected from males, which was attributed to higher GSNO reductase (GSNOR) activity. High fat feeding augmented the GSNO-mediated inhibition of KGDH in liver mitochondria from male mice. Exposure of male mice to a HFD also resulted in a significant decrease in the GSNO-mediated inhibition of H2O2 genesis by PDH, an effect not observed in mice fed a control-matched diet (CD). Female mice displayed higher resistance to the GSNO-induced inhibition of H2O2 production, regardless of being fed a CD or HFD. However, exposure to a HFD did result in a small but significant decrease in H2O2 production by KGDH and PDH when female liver mitochondria were treated with GSNO. Although, the effect was less when compared to their male counterparts. Collectively, we show for the first time GSNO deactivates H2O2 production by α-keto acid dehydrogenases and we demonstrate that sex and diet are determinants for the nitro-inhibition of both KGDH and PDH.

ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.

The mechanisms underlying ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.

Health disparity in use of novel agents for first-line therapy in Black and White patients with chronic lymphocytic leukemia in the Department of Veterans Affairs.

BACKGROUND: Novel agents (NAs) (ibrutinib, idelalisib, and venetoclax) were first introduced in 2013 as therapeutic options to treat chronic lymphocytic leukemia (CLL). OBJECTIVES: To determine if the uptake of NAs for first-line treatment was similar in Black and White patients with CLL treated in the Department of Veterans Affairs (VA). METHODS: We conducted a retrospective cohort study including adults with CLL managed in the VA from October 1, 2013, to September 30, 2017. Descriptive statistics were used to summarize demographic data, and appropriate bivariable statistical tests were used to compare NA use, baseline characteristics, health outcomes, and complications. A multivariable logistic regression model was used to identify factors associated with uptake of NAs. The study included 565 patients; 86% were White and 14% were Black. Black patients were younger than White patients (median age [66 vs 69 years; P < 0.01]) but had similar median baseline Charlson comorbidity scores (4 vs 5). RESULTS: Overall, Black patients were less likely to receive NAs than White patients (14% vs 26%; P = 0.02). The gap narrowed over the study period: 4% vs 17% (2014), 13% vs 25% (2015), 17% vs 33% (2016), and 31% vs 33% (2017). Black race (P = 0.02) and fiscal year (P < 0.01) were the only variables significantly associated with NA use in the multivariable model. Health outcomes and most complications were similar for Black and White patients despite the difference in prescribing patterns. CONCLUSIONS: This is the first study to identify a potential health disparity with respect to use of NAs among Black and White patients with CLL treated in the VA. Fortunately, health outcomes and most complications were similar for Black and White patients despite the difference in prescribing patterns. DISCLOSURES: Funding for the study was provided by AstraZeneca as a research grant to the Foundation for Advancing Veterans' Health Research (FAVHR), a non-profit entity within the Audie L. Murphy Veterans Hospital, San Antonio, TX. Drs Nooruddin and Frei have received research grants (paid to FAVHR) from AstraZeneca in the last 3 years. Ms Ryan is an employee of AstraZeneca. The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs, the National Institutes of Health, or the authors' affiliated institutions.

Comparison of bibliometrics for predoctoral Translational Science Training (TST) TL1 Program participants and nonparticipants, male and female participants, and participants from underrepresented and well-represented backgrounds.

Research education and training in Translational Science develops and sustains a workforce to efficiently advance studies designed to improve human health. We evaluated the effectiveness of a Translational Science Training (TST) TL1 Program. Participants had significantly better publications/year, citations/year, h-index, and m-quotient than nonparticipants. Female and male participants, and participants from underrepresented and well-represented backgrounds, performed similarly on all bibliometric assessments. Finally, TST/TL1 Program participants outperformed students from other PhD programs at our institution. This analysis suggests that the TST/TL1 Program has been effective for participants, including those who are female and from underrepresented backgrounds.

Early outcomes from a national Train-the-Trainer Interprofessional Team Development Program.

The imperative need to train health professions faculty (educators and clinicians) to lead interprofessional education efforts and promote interprofessional team-based care is widely recognized. This need stems from a growing body of research that suggests collaboration improves patient safety and health outcomes. This short report provides an overview of a Train-the-Trainer Interprofessional Team Development Program (T3 Program) that equips faculty leaders with the skills to lead interprofessional education and interprofessional collaborative practice across the learning continuum. We also describe the history, approach, and early outcomes of this innovative program.

Rewriting the script: How COVID-19 affected the relation between intrinsic aspirations and depressive symptoms.

Self-determination theory proposes that intrinsic aspirations protect against negative mental health outcomes by satisfying people's basic psychological needs of autonomy, relatedness, and competence. The present study investigated this relationship using two four-wave prospective longitudinal studies which followed undergraduate students across the Canadian academic calendar (September to May). The first was conducted across 2018-19 and the second across 2019-20. By comparing these two samples, we examined whether baseline levels of intrinsic aspirations moderated the impact of the COVID-19 pandemic on the development of depressive symptoms. Three main findings emerged, the first being that students reported higher levels of depressive symptoms in Spring 2020 than in Spring 2019. Second, students with more intrinsic aspirations in the pre-pandemic sample (2018-19) experienced fewer depressive symptoms from December to May while students with more intrinsic aspirations in the pandemic sample (2019-20) experienced more depressive symptoms during this period. Lastly, the latter relationship was mediated by need frustration, whereby students with higher levels of intrinsic aspirations experienced greater need frustration during the pandemic year. Together, these findings suggest that although intrinsic aspirations typically protect against negative psychological outcomes, the unique need frustrating context of the pandemic made them a risk factor for depression.

Coping methods and college adjustment in a sample of U.S. Undergraduate students during the COVID-19 pandemic.

Objective: To examine the associations between coping methods and college adjustment among a sample of U.S. undergraduate students during the COVID-19 pandemic. Participants: We used a sample of 117 undergraduate students between the age of 18-25 years old. Approximately 76% of the sample identified as women and 58% identified as White. Methods: Participants completed online questionnaires that assessed the use of forward-focused coping, trauma-focused coping, and several domains of college adjustment (i.e., academic adjustment, social adjustment, personal-emotional adjustment, and attachment). We used multiple regression to identify the association between coping methods and college adjustment, using race and gender as control variables. Results: Forward-focused coping methods were significantly and positively related to academic adjustment, social adjustment, and attachment, while and trauma-focused coping methods were significantly and negatively related to personal-emotional adjustment. Conclusions: The use of forward-focused coping methods may be beneficial for undergraduate students during the COVID-19 pandemic.

Conditions Conducive to the Glutathionylation of Complex I Subunit NDUFS1 Augment ROS Production following the Oxidation of Ubiquinone Linked Substrates, Glycerol-3-Phosphate and Proline.

Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O2●-/H2O2) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O2●-/H2O2 production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50-1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies.

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

Feasibility of establishing a veterinary marker to total residue in edible tissues with non-radiolabeled study using high-resolution mass spectrometry.

Traditionally, in vivo metabolism and total residue studies in veterinary drug research were conducted using radiolabeled drug where information on metabolite profiles and marker residue to total residue ratio is obtained. The Veterinary International Conference on Harmonisation (VICH) guideline GL46 indicates that the metabolism and residue kinetics in food-producing animals may be documented by an alternative approach, one other than the traditional radiolabeled study. High-resolution mass spectrometry (HRMS) has been widely used in human pharmaceutical R&D from metabolite profiling and identification in early drug discovery to first-in-human (FIH) studies in development. Recent advances in data mining tools have greatly improved the metabolite profiling capability with HRMS. It is now routine to study metabolism using non-radiolabeled samples without missing any major metabolites. In the current paper, we explored the feasibility of conducting non-radiolabeled marker residue studies to obtain metabolism information using HRMS. Metabolite profiles of gamithromycin in edible tissues of sheep treated with 6 mg/kg body weight subcutaneous injections were obtained with HRMS. The semi-quantitative relationship between the level of gamithromycin and the total treatment-related residues was established by determining the percentages of extracted ion chromatograms for metabolites and parent compound residues in each tissue. Major components (gamithromycin and its metabolite, declad) were measured quantitatively using a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Metabolite profiles in excreta were also obtained and the major components measured quantitatively with a LC-MS/MS method to ensure no major metabolite was missing. Combining previous knowledge of marker residue studies in cattle and swine, as well as an in vitro comparative metabolism study with metabolite data across various species, gamithromycin was designated as the marker residue in sheep edible tissues. The marker to total residue ratios were established using a combination of the semi-quantitative HRMS results and quantitative results with the major components: the marker residue and declad. The pros and cons of the HRMS method as well as the appropriate use of the method for marker residue studies are discussed.