The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.

Impact of a Web-based Information and Referral Tool on Access to Federal and State Programs for Older Adults.

Federal and state governments provide a plethora of benefits programs intended to help older Americans, but take-up rates for the programs is low. BenefitsCheckUp® is an online tool intended to increase enrollment in these programs. To evaluate the impact of this national online screening tool providing individualized benefit information, we conducted a web survey of individuals who screened potentially eligible for programs including Supplemental Security Income, Medicaid, Medicare Savings Programs, Supplemental Nutrition Assistance Program, and energy assistance. Thirty-six percent of those surveyed applied for at least one benefit at an annualized, estimated average value of $2,865, and 20.5% enrolled, representing about 7% of the approximately 2 million site visitors age 60+. These results indicate that an online screening tool is a promising strategy for increasing benefit take-up rates among older adults with the value of benefits received far exceeding investments.

Overcoming Language Barriers Using an Information Video on Spinal Anesthesia for Cesarean Delivery: Implementation and Impact on Maternal Anxiety.

BACKGROUND: It is unknown whether the implementation of an information video on spinal anesthesia for cesarean delivery, narrated in a patient's first language, reduces anxiety, increases satisfaction, and improves doctor-patient communication if there is a language barrier. In South Africa, most doctors speak English, and patients speak Xhosa, with educational and cultural disparities existing in many doctor-patient interactions. METHODS: One hundred seventy-five Xhosa patients scheduled for elective cesarean delivery were enrolled in the study. The first 92 patients received "usual care" verbal explanations of the spinal anesthesia procedure (control group); the next 83 patients watched a spinal anesthesia information video (intervention group), narrated in Xhosa. Videos were displayed using smartphones. Maternal anxiety was assessed before and after spinal explanation, using a Numerical Visual Analog Anxiety Scale (NVAAS). A difference in postexplanation NVAAS score of 1.5 points between intervention and control groups was regarded as clinically significant. Patient satisfaction was assessed using the Maternal Satisfaction Scale for Cesarean Section (MSSCS). RESULTS: The mean (standard deviation [SD]) age (31.5 years [5.2 years] and 32.1 years [5.4 years]) and preexplanation NVAAS score (4.2 [3.2] and 4.0 [3.0]) of the intervention and control groups, respectively, showed no difference at baseline. The mean (SD) postexplanation decrease in NVAAS score was greater in the intervention than in the control group (1.6 [3.5] vs 0.7 [2.3]; P = .046; unadjusted mean difference, 0.9 points [95% confidence interval {CI}, 0.02-1.8]). A linear regression model for the postexplanation NVAAS score showed that the intervention effect was significantly associated with the preexplanation score (P = .002), adjusted for age and English fluency. Patients with preexplanation NVAAS scores ≥5 showed a statistically significant intervention effect. There was no significant difference in patient satisfaction between the intervention and control groups. The smartphone was an accessible and convenient display medium for the video. Ninety-nine percent of patients exposed to the intervention would recommend watching the video before the procedure. CONCLUSIONS: In this pilot study, lower NVAAS scores were observed in anxious patients, when a Xhosa information video was used to ameliorate challenges posed by a doctor-patient language barrier. It is easily implemented and demonstrates a novel use of mobile health technology. The study provides baseline data to inform sample size calculations for future studies. A high level of patient recommendation for the video suggests that this is an agreeable practice.

Implementation of a CMS Nursing Facility Initiative: Differences by Racial Minority Resident Population.

Objectives: The CMS Initiative to Reduce Avoidable Hospitalizations Among Nursing Facility Residents: Payment Reform (NFI 2) provided billing opportunities to incentivize participating facilities to keep long-stay residents onsite for acute care, rather than hospitalizing them. We examined cross-facility differences in NFI 2 implementation by racial composition of facility resident populations. Methods: We analyzed Medicare claims in conjunction with in-person and telephone interviews among facility staff to assess NFI 2 engagement in relation to racial minority resident population. Results: Participating facilities with larger racial minority resident populations faced additional barriers to NFI 2 implementation. These facilities submitted fewer NFI 2 claims, reported more challenges engaging resident families, and experienced greater facility staff and leadership instability, compared to facilities with predominantly white resident populations. Discussion: Addressing structural differences within facilities with larger populations of racial minority residents may encourage future development of targeted programs to support diverse nursing facilities.

Treatment strategies for hyperkalemia secondary to urethral obstruction in 50 male cats: 2002-2017.

OBJECTIVES: The aims of this study were: to describe the potassium-lowering treatment strategies used to manage moderate-to-severe hyperkalemia in male cats with urethral obstruction (UO); to determine how much dextrose was required per unit of insulin to prevent hypoglycemia; to determine whether early initiation of a dextrose continuous rate infusion (CRI) prevented hypoglycemia; and to determine whether in-hospital mortality was associated with presenting plasma potassium concentration ([K+]). METHODS: The medical records of male cats presenting with a [K+] ⩾7.0 mEq/l due to UO that had another [K+] measured within 6 h were reviewed retrospectively. All [K+] values within the first 6 h, blood glucose concentrations, treatments for hyperkalemia and survival to discharge were recorded. Analyses were performed to test for associations between dextrose:insulin ratios or method of dextrose administration and the development of hypoglycemia; and for presenting [K+] and mortality. Normally distributed groups of continuous data were compared with a t-test and categorical data were compared with a Fisher's exact test. RESULTS: Fifty cats were included. Mean presenting [K+] was 8.9 ± 1.0 mEq/l, while the mean final [K+] within 6 h was 6.6 ± 1.4 mEq/l. Forty-two (84%) cats were treated with intravenous fluids and 40 (80%) were treated with dextrose and insulin. Median dextrose:insulin ratio was 2 g/u (range 0.4-100). No dextrose:insulin ratio was found to protect against hypoglycemia, and 3/8 cats that became hypoglycemic had received ⩾2 g dextrose per unit of insulin. There was no association between the early initiation of a dextrose-containing CRI and avoidance of hypoglycemia. No association was found between presenting [K+] and mortality. CONCLUSIONS AND RELEVANCE: While no specific dextrose:insulin ratio was found to protect against hypoglycemia, there is evidence that the commonly recommended dextrose:insulin ratio of 2 g/u may be inadequate in preventing hypoglycemia in every cat. Severity of hyperkalemia was not associated with mortality.

Lack of Association Between Sex Hormones, MDSCs, LDGs and pDCs in Males and Females With Systemic Lupus Erythematosus.

Plasmacytoid dendritic cells (pDCs) and low-density granulocytes (LDGs) are interferon-alpha producing cells that create a pro-inflammatory response in Systemic Lupus Erythematosus (SLE) leading to auto antibody production and organ damage. Both pDCs and LDGs have been shown to be dysfunctional in patients with active SLE. Myeloid-derived suppressor cells (MDSCs) have the capacity to control T and B cell activation and differentiation, and have recently been identified as cells of interest in SLE as well. While not fully understood, previous studies have suggested that pDCs are regulated in part by both X chromosome inactivation and estradiol. Whether sex chromosomes or sex hormones regulate MDSCs and LDGs remain to be determined. We aimed to explore the relative role of sex and sex hormones on pDC, MDSC and LDG frequency and function in SLE patients. We recruited patients with SLE as defined by ACR or SLICC classification criteria and healthy controls in conjunction with the Cleveland Clinic Lupus Cohort and Clinical Research Unit. We analyzed serum sex hormone levels by ELISA, and frequencies of pDCs, MDSCs, and LDGs among PBMCs and serum cytokine levels by flow cytometry. PBMCs were further analyzed for expression of genes involved in or induced by toll-like receptor (TLR)7 or TLR9 stimulation. In all SLE patients, the serum estradiol/testosterone ratio and levels of granulocytic MDSCs and LDGs were increased, while levels of pDCs were decreased. Furthermore, pDCs from active SLE patients expressed lower levels of TLR7 and TLR9 and showed diminished production of TLR9-induced IFNα and TNFα as compared to healthy controls. LDGs from healthy controls and SLE patients expressed very low levels of TLR7 and TLR9 and largely failed to respond to TLR9 stimulation. Thus, regardless of sex and sex-hormone levels, frequencies of pDCs, MDSCs and LDGs, TLR7 and TLR9 expression, and TLR9-driven cytokine production were similarly altered in male and female SLE patients.

Chronic paternal morphine exposure increases sensitivity to morphine-derived pain relief in male progeny.

Parental history of opioid exposure is seldom considered when prescribing opioids for pain relief. To explore whether parental opioid exposure may affect sensitivity to morphine in offspring, we developed a "rat pain scale" with high-speed imaging, machine learning, and mathematical modeling in a multigenerational model of paternal morphine self-administration. We find that the most commonly used tool to measure mechanical sensitivity in rodents, the von Frey hair, is not painful in rats during baseline conditions. We also find that male progeny of morphine-treated sires had no baseline changes in mechanical pain sensitivity but were more sensitive to the pain-relieving effects of morphine. Using RNA sequencing across pain-relevant brain regions, we identify gene expression changes within the regulator of G protein signaling family of proteins that may underlie this multigenerational phenotype. Together, this rat pain scale revealed that paternal opioid exposure increases sensitivity to morphine's pain-relieving effects in male offspring.

A Mixed Methods Exploration of Factors That Influence Student Participation in Optional Formative Review Quizzes.

Introduction: Weekly formative Review Quizzes are an integral feature of the Georgetown University School of Medicine assessment program. The Quizzes offer students an opportunity to test themselves in a low-stakes setting and then discuss their answers with peers in small groups; faculty are also present to help the groups with difficult problems. Methods: We conducted a mixed methods study in which we monitored quiz attendance over the course of the first four curricular blocks, deployed a study specific survey, and held focus groups to determine the factors that influenced quiz participation and how students perceived that the quiz contributed to their learning. Results: We observed that Quiz attendance, while initially robust, dropped steadily over the course of the year. Nearly all students reported that the practice questions along with faculty explanations contributed strongly to their learning. Fewer students felt that discussion with their peers was valuable, but those who valued peer discussion were significantly more likely to attend the quiz in person. The two things cited most often as barriers to quiz attendance were inconvenience and lack of adequate preparation. Many students reported that they saved questions and did not attempt to answer them until they had completed study of that subject. Discussion: Our results indicate that while there is ample evidence that early review and discussion with peers can contribute to learning, learners do not always recognize the value in this practice.

COVID-19 Outcomes in Patients Undergoing B Cell Depletion Therapy and Those with Humoral Immunodeficiency States: A Scoping Review.

BACKGROUND: The role of humoral immunity has been well established in reducing infection risk and facilitating viral clearance in patients with COVID-19. However, the relationship between specific antibody responses and severity of COVID-19 is less well understood. METHODS: To address this question and identify gaps in knowledge, we utilized the methodology of a scoping review to interrogate risk of infection and clinical outcomes of COVID-19 in patients with iatrogenic and inborn humoral immunodeficiency states based on existing literature. RESULTS: Among patients with iatrogenic B-cell depletion, particularly with agents targeting CD20, our analysis found increased risk of severe COVID-19 and death across a range of underlying disease states. Among patients with humoral inborn errors of immunity with COVID-19, our synthesis found that patients with dysregulated humoral immunity, predominantly common variable immunodeficiency (CVID), may be more susceptible to severe COVID-19 than patients with humoral immunodeficiency states due to X-linked agammaglobulinemia and other miscellaneous forms of humoral immunodeficiency. There were insufficient data to appraise the risk of COVID-19 infection in both populations of patients. CONCLUSIONS: Our work identifies potentially significant predictors of COVID-19 severity in patients with humoral immunodeficiency states and highlights the need for larger studies to control for clinical and biologic confounders of disease severity.

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

Androgen-Mediated Anti-inflammatory Cellular Processes as Therapeutic Targets in Lupus.

Systemic Lupus Erythematosus (SLE), among many other auto-immune diseases, is known to be more prevalent in females than in males. This observation has served as the foundation for studies into how sex hormones may interact with the immune system to either drive or inhibit immune activation. Early studies using castration in lupus mouse models showed the potential protective effect of testosterone against lupus development. These studies were later corroborated by observational studies in lupus patients, who upon treatment with testosterone therapy, displayed decreased disease burden. However, there are numerous limitations to treating (especially female) lupus patients with testosterone. Thus, identification of testosterone-targeted cellular and molecular mechanisms affecting immune activation is an attractive target for lupus treatment in the future. Recent studies have examined the effects of androgens on the activation of anti-inflammatory processes. As such, immunoregulatory cell types including myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells have been shown to be susceptible to manipulation by sex hormones. Here, we review studies of SLE and lupus-like disease in which testosterone or testosterone-derivatives were used to skew an ongoing immune reaction toward an anti-inflammatory state. Via evaluation of both clinical studies and immunologic models we propose new areas for research with the goal of identifying testosterone-driven anti-inflammatory mediators suitable for therapeutic targeting in patients with lupus and other autoimmune diseases.

A machine-vision approach for automated pain measurement at millisecond timescales.

Objective and automatic measurement of pain in mice remains a barrier for discovery in neuroscience. Here, we capture paw kinematics during pain behavior in mice with high-speed videography and automated paw tracking with machine and deep learning approaches. Our statistical software platform, PAWS (Pain Assessment at Withdrawal Speeds), uses a univariate projection of paw position over time to automatically quantify seven behavioral features that are combined into a single, univariate pain score. Automated paw tracking combined with PAWS reveals a behaviorally divergent mouse strain that displays hypersensitivity to mechanical stimuli. To demonstrate the efficacy of PAWS for detecting spinally versus centrally mediated behavioral responses, we chemogenetically activated nociceptive neurons in the amygdala, which further separated the pain-related behavioral features and the resulting pain score. Taken together, this automated pain quantification approach will increase objectivity in collecting rigorous behavioral data, and it is compatible with other neural circuit dissection tools for determining the mouse pain state.

Correlation between recombinase activating gene 1 ubiquitin ligase activity and V(D)J recombination.

The really interesting new gene (RING) finger ubiquitin ligase domain of the recombinase activating gene 1 (RAG1) V(D)J recombinase protein adopts a standard cross-brace architecture but co-ordinates three zinc ions as opposed to the canonical two. We demonstrated previously that disruption of the conserved zinc co-ordination sites resulted in loss of structural integrity and ubiquitin ligase (E3) activity and interfered with the ability of full-length RAG1 to support recombination. Here we present evidence that amino acids surrounding the third, non-canonical site also contribute to functional interaction with the ubiquitin conjugating (E2) enzyme CDC34, while certain residues on the RING domain's surface important for interaction between other E2-E3 pairs are less critical to the functional RAG1-CDC34 interaction in this assay. Partial reduction of ubiquitin ligase activity was significantly correlated with reduction in the ability of RAG1 to support recombination of extra-chromosomal substrates (r = 0.805, P = 0.009). While poly-ubiquitin chains could be generated, RAG1 did not promote rapid chain extension following mono-ubiquitylation of substrate, regardless of the E2 enzyme used. No single ubiquitin lysine mutant disrupted the ability of CDC34 to form ubiquitin chains on RAG1, and mass spectrometric analysis of the poly-ubiquitylated products indicated ubiquitin chain linkages through lysines 48 and 11. These data suggest that RAG1 promotes a mono-ubiquitylation reaction that is required for optimal levels of V(D)J recombination.

A 2.5-Year Longitudinal Assessment of Naturalistic Driving in Preclinical Alzheimer's Disease.

BACKGROUND: Emerging evidence shows that cognitively normal older adults with preclinical Alzheimer's disease (AD) make more errors and are more likely to receive a marginal/fail rating on a standardized road test compared to older adults without preclinical AD, but the extent to which preclinical AD impacts everyday driving behavior is unknown. OBJECTIVE: To examine self-reported and naturalistic longitudinal driving behavior among persons with and without preclinical AD. METHOD: We prospectively followed cognitively normal drivers (aged 65 + years) with (n = 10) and without preclinical AD (n = 10) for 2.5 years. Preclinical AD was assessed using amyloid positron emission tomography (PET) with Pittsburgh Compound B. The Driving Habits Questionnaire assessed self-reported driving outcomes. Naturalistic driving was captured using a commercial GPS data logger plugged into the on-board diagnostics II port of each participant's vehicle. Data were sampled every 30 seconds and all instances of speeding, hard braking, and sudden acceleration were recorded. RESULTS: Preclinical AD participants went to fewer places/unique destinations, traveled fewer days, and took fewer trips than participants without preclinical AD. The preclinical AD group reported a smaller driving space, greater dependence on other drivers, and more difficulty driving due to vision difficulties. Persons with preclinical AD had fewer trips with any aggression and showed a greater decline across the 2.5-year follow-up period in the number of days driving per month and the number of trips between 1-5 miles. CONCLUSION: Changes in driving occur even during the preclinical stage of AD.

Theoretical Explanation for Reduced Body Mass Index and Obesity Rates in Cannabis Users.

Introduction: Obesity is treatment-resistant, and is linked with a number of serious, chronic diseases. Adult obesity rates in the United States have tripled since the early 1960s. Recent reviews show that an increased ratio of omega-6 to omega-3 fatty acids contributes to obesity rates by increasing levels of the endocannabinoid signals AEA and 2-AG, overstimulating CB1R and leading to increased caloric intake, reduced metabolic rates, and weight gain. Cannabis, or THC, also stimulates CB1R and increases caloric intake during acute exposures. Goals: To establish the relationship between Cannabis use and body mass index, and to provide a theoretical explanation for this relationship. Results: The present meta-analysis reveals significantly reduced body mass index and rates of obesity in Cannabis users, in conjunction with increased caloric intake. Theoretical explanation: We provide for the first time a causative explanation for this paradox, in which rapid and long-lasting downregulation of CB1R following acute Cannabis consumption reduces energy storage and increases metabolic rates, thus reversing the impact on body mass index of elevated dietary omega-6/omega-3 ratios.

Continued Effectiveness of Relapse Prevention Cognitive-Behavioral Therapy Following Fluoxetine Treatment in Youth With Major Depressive Disorder.

OBJECTIVE: To evaluate the continued effect of a sequential treatment strategy (fluoxetine followed by continued medication plus relapse prevention cognitive-behavioral therapy [RP-CBT]) on relapse prevention beyond the treatment phase. METHOD: Youth (aged 8-17 years) with major depressive disorder (MDD) were treated with fluoxetine for 6 weeks. Responders (≥50% reduction on the Children's Depression Rating Scale-Revised [CDRS-R]) were randomized to continued medication management alone (MM) or continued medication management plus RP-CBT (MM+CBT) for an additional 6 months. Long-term follow-up assessments were conducted at weeks 52 and 78. RESULTS: Of 144 youth randomized to MM (n = 69) or MM+CBT (n = 75), 67% had at least 1 follow-up assessment, with equal rates in the 2 groups. Remission rates were high, although most had remitted during the 30-week treatment period. Only 6 additional participants remitted during long-term follow-up, and there were no differences on time to remission between MM+CBT and MM. The MM+CBT group had a significantly lower risk of relapse than the MM group throughout the 78-week follow-up period (hazard ratio = 0.467, 95% CI = 0.264 to 0.823; χ(2) = 6.852, p = .009). The estimated probability of relapse during the 78-week period was lower with MM+CBT than MM only (36% versus 62%). Mean time to relapse was also significantly longer with MM+CBT compared to MM alone by approximately 3 months (p = .007). CONCLUSION: The addition of RP-CBT after acute response to medication management had a continued effect on reducing risk of relapse even after the end of treatment. Clinical trial registration information-Sequential Treatment of Pediatric MDD to Increase Remission and Prevent Relapse; http://clinicaltrials.gov/; NCT00612313.

Sequential treatment with fluoxetine and relapse--prevention CBT to improve outcomes in pediatric depression.

OBJECTIVE: The authors evaluated a sequential treatment strategy of fluoxetine and relapse-prevention cognitive-behavioral therapy (CBT) to determine effects on remission and relapse in youths with major depressive disorder. METHOD: Youths 8-17 years of age with major depression were treated openly with fluoxetine for 6 weeks. Those with an adequate response (defined as a reduction of 50% or more on the Children's Depression Rating Scale-Revised [CDRS-R]) were randomly assigned to receive continued medication management alone or continued medication management plus CBT for an additional 6 months. The CBT was modified to address residual symptoms and was supplemented by well-being therapy. Primary outcome measures were time to remission (with remission defined as a CDRS-R score of 28 or less) and rate of relapse (with relapse defined as either a CDRS-R score of 40 or more with a history of 2 weeks of symptom worsening, or clinical deterioration). RESULTS: Of the 200 participants enrolled in acute-phase treatment, 144 were assigned to continuation treatment with medication management alone (N=69) or medication management plus CBT (N=75). During the 30-week continuation treatment period, time to remission did not differ significantly between treatment groups (hazard ratio=1.26, 95% CI=0.87, 1.82). However, the medication management plus CBT group had a significantly lower risk of relapse than the medication management only group (hazard ratio=0.31, 95% CI=0.13, 0.75). The estimated probability of relapse by week 30 was lower with medication management plus CBT than with medication management only (9% compared with 26.5%). CONCLUSIONS: Continuation-phase relapse-prevention CBT was effective in reducing the risk of relapse but not in accelerating time to remission in children and adolescents with major depressive disorder.

Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking.

Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.

Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats.

Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105% of the target value. Variability was 4.0-8.9% for all analytes within the range of 5-200 ng/mL for heroin, 5-1000 ng/mL for 6-MAM, and 10-200 ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4 mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80°C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3% over one week in rat plasma under these conditions, and by <1-4.7% in bovine serum or human plasma.