Clavulanic Acid-Mediated Increases in Anterior Cingulate Glutamate Levels are Associated With Decreased Cocaine Craving and Brain Network Functional Connectivity Changes.

Background: There is an urgent need for pharmacological treatment for cocaine (COC) use disorder (CUD). Glutamatergic transmission in the prefrontal cortex is affected by addictive behaviors. Clavulanic acid (CLAV), a glutamate transporter GLT-1 (excitatory amino acid transporter) activator, is a clinical-stage medication that has potential for treating CUD. Methods: In a pilot study, nine participants with CUD received 500 mg CLAV with dose escalations to 750 mg and 1000 mg over 10 days. In 5 separate magnetic resonance imaging (MRI) sessions, brain anterior cingulate cortex (ACC) glutamate level and resting state network (RSN) functional connectivity (FC) were assessed using MR spectroscopy and functional MRI. Craving was assessed at the same time points, between baseline (before CLAV), 6 days, and 10 days of CLAV. Independent component analysis with dual regression was used to identify RSN FC changes from baseline to Days 6 and 10. Relationships among glutamate, craving, and resting state FC values were analyzed. Results: Participants who achieved high ACC glutamate levels after CLAV treatment had robust decreases in COC craving (r = -0.90, P = 0.0009, n = 9). The salience network (SN) and executive control network (ECN) demonstrated an association between increased FC after CLAV treatment and low baseline ACC Glu levels (SN CLAV 750 mg, r = -0.82, P = 0.007) (ECN CLAV 1000 mg, r = -0.667, P = 0.050; n = 9). Conclusions: Glutamate associated changes in craving and FC of the salience and executive control brain networks support CLAV as a potentially efficacious pharmacological treatment for CUD.

The developing toolkit of continuous directed evolution.

Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.

Molecular Markers in Maternal Blood Exosomes Allow Early Detection of Fetal Alcohol Spectrum Disorders.

Prenatal alcohol exposure can cause developmental abnormalities (fetal alcohol spectrum disorders; FASD), including small eyes, face and brain, and neurobehavioral deficits. These cannot be detected early in pregnancy with available imaging techniques. Early diagnosis could facilitate development of therapeutic interventions. Banked human fetal brains and eyes at 9−22 weeks' gestation were paired with maternal blood samples, analyzed for morphometry, protein, and RNA expression, and apoptotic signaling. Alcohol (EtOH)-exposed (maternal self-report) fetuses were compared with unexposed controls matched for fetal age, sex, and maternal race. Fetal brain-derived exosomes (FB-E) were isolated from maternal blood and analyzed for protein, RNA, and apoptotic markers. EtOH use by mothers, assessed by self-report, was associated with reduced fetal eye diameter, brain size, and markers of synaptogenesis. Brain caspase-3 activity was increased. The reduction in eye and brain sizes were highly correlated with amount of EtOH intake and caspase-3 activity. Levels of several biomarkers in FB-E, most strikingly myelin basic protein (MBP; r > 0.9), correlated highly with morphological abnormalities. Reduction in FB-E MBP levels was highly correlated with EtOH exposure (p < 1.0 × 10−10). Although the morphological features of FAS appear long before they can be detected by live imaging, FB-E in the mother's blood may contain markers, particularly MBP, that predict FASD.

Virtual/Remote Labs for Fluorescent Immunocytochemistry or Western Blotting: The Next Best Thing to Being There.

The SARS CoV-2 pandemic forced many college courses to convert to remote instruction almost overnight in the middle of the spring 2020 teaching semester. This article presents two molecular biology labs formerly performed in person by students but converted into virtual labs. The virtual immunocytochemistry experiment teaches the specificity of antibody staining, principles of fluorescent microscopy, diversity of brain cell types and morphologies, and journal article Figure construction skills. The virtual Western blotting experiment teaches sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the specificity of antibody binding, and graph creation and interpretation skills. Both virtual experiments use professionally-produced web-based videos of scientists conducting the lab procedures. Students must answer questions about the techniques and analyze real experimental data generated by past students to take a quiz and write a journal article-style lab report. At the whole-class level, student quiz and lab report scores from these virtual labs were not statistically different from those from the in-person versions of the same labs from a previous semester, using t tests with the Bonferroni correction. On the virtual Western blot quiz, students who did the virtual version actually scored higher than students who did the in-person version. These results were significant when the 2020 data were analyzed by within-student paired t tests for in-person labs done before COVID-19 versus those done virtually after dismissal for all-remote instruction. The students learned the laboratory concepts and data analysis skills just as well virtually as their predecessors had in person. However, the students trained virtually reported that they could not enter the lab and actually do Western blotting or fluorescent immunocytochemistry with their own hands without extensive additional training. These virtual experiments can be done with data included in the supplemental materials or can easily be adapted for any micrographs or Western blotting images available from previous lab experiments, or in the published literature. When COVID-19 or other public health emergencies necessitate remote instruction and we can't use the best practice of hands-on lab work, virtual labs can be the next best thing to being there.

Double-blind, placebo-controlled, proof-of-concept trial of a kappa-selective opioid receptor antagonist augmentation in treatment-resistant depression.

BACKGROUND: Kappa-opioid antagonism may possess antidepressant properties. We assessed, in a proof-of-concept pilot trial among patients with major depressive disorder with inadequate response to antidepressants, the efficacy of adjunctive CERC-501 (formerly LY2456302), a kappaselective opioid receptor antagonist. METHODS: In a Sequential Parallel Comparison Design study, patients were pre-randomized to: a) 10 mg/d of CERC-501 for 6 days, b) 20 mg/d of CERC-501 for 6 days, c) placebo for 3 days followed by 10 mg/d of CERC- 501 for 3 days, d) placebo for 3 days followed by 20 mg/d of CERC-501 for 3 days, or e) placebo for 6 days. RESULTS: The study was terminated early by the National Institute of Mental Health due to slow enrollment (N = 8). The weighted mean difference of changes (drug vs placebo) in the 6-item Hamilton Depression Rating Scale (HAMD-6) (primary outcome measure) (1.28), Montgomery-Åsberg Depression Rating Scale (MADRS) (2.33), Perceived Stress Scale (1.01), Symptoms of Depression Questionnaire (9.17), Positive Affect Scale (PAS) (6.39), Symptom Questionnaire (SQ) Depression scale (2.94), SQ Anger- Hostility scale (1.67), and Patient-Reported Outcomes Measurement Information System Satisfaction with Participation in Discretionary Social Activities (4.67) scores were all numerically but not statistically greater for CERC-501 than for placebo. CONCLUSIONS: Although the small sample size limits the ability to draw conclusions, results suggest that CERC-501 may have antidepressant effects. Additional studies are necessary to further explore these effects of CERC-501.

Integrating Research into the Undergraduate Curriculum: 1. Early Research Experiences and Training.

Undergraduate research experiences have emerged as some of the most beneficial high-impact practices in education, providing clear benefits to students that include improved critical thinking and scientific reasoning, increased academic performance, and enhanced retention both within STEM majors and in college overall. These benefits extend to faculty members as well. Several disciplines, including neuroscience, have implemented research as part of their curriculum, yet many research opportunities target late stage undergraduates, despite evidence that early engagement can maximize the beneficial nature of such work. A 2019 Society for Neuroscience professional development workshop provided multiple examples of integrating research into an undergraduate curriculum, including early engagement (Fernandes, 2020). This article is the first in a series of three that expands upon the information presented in those workshop discussions, focusing on ways to promote early research opportunities. The benefits and challenges associated with early research engagement suggest thoughtful consideration of the best mechanisms for implementation are warranted; some options might include apprenticeship models or course-based approaches. Regardless of mechanism, early research can serve to initiate more prolonged, progressive, scaffolded experiences that span the academic undergraduate career.

Integrating Research into the Undergraduate Curriculum: 2. Scaffolding Research Skills and Transitioning toward Independent Research.

Undergraduate research experiences are widely regarded as high-impact practices that foster meaningful mentoring relationships, enhance retention and graduation, and stimulate postbaccalaureate enrollment in STEM graduate and professional programs. Through immersion in a mentored original research project, student develop and apply their skills in critical thinking, problem solving, intellectual independence, communication, collaboration, project ownership, innovation, and leadership. These skills are readily transferable to a wide array of future careers in and beyond STEM that are well-served by evidence-based approaches. The 2019 Society for Neuroscience meeting included a well-attended workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). This article is the second of three articles that summarize, analyze, and expand the workshop discussions. In this second article, we specifically describe approaches to transitional research courses that prepare students for independent research experiences such as undergraduate research theses. Educators can intentionally scaffold research experience and skills across the curriculum, to foster participation in scientific research and enhance diversity, equity, and inclusivity in research training. This article provides an overview of important goals and considerations for intermediate undergraduate research experiences, specific examples from several institutions of transitional courses that scaffold research preparation using different structures, and a summary of lessons learned from these experiences.

Integrating Research into the Undergraduate Curriculum: 3. Research Training in the Upper-level Neuroscience Curriculum.

The benefits of undergraduate training in research are significant. Integration of such training into the undergraduate experience, however, can be challenging at institutions without extensive research programs, and may inadvertently exclude some populations of students. Therefore, inclusion of research into the academic curriculum ensures all students can access this important training. The 2019 annual meeting of the Society for Neuroscience included a workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). In this last article of a three-part series, we describe models for integrating research into advanced stages of the undergraduate curriculum, specifically for juniors and seniors. First, we describe multiple models of faculty-mentored group-based research. Second, we detail a peer-mentored research system, in which seniors mentor groups of first through third year students. Third, we describe multiple examples of integrating research into "capstone" courses for seniors. Fourth, we describe models in which a senior thesis is a graduation requirement for all students. Lastly, we describe several models of implementing an optional honors thesis for students. Although similarities exist across these programs, their differences allow for specific secondary objectives to be met, which are often unique to institutions and/or departments. Therefore, for each of these examples, we describe the context, specific design, and required student assessments. We conclude by discussing some of the key successes and challenges of developing programs that facilitate undergraduate research by upper-level students, and suggest a number of concepts that should be considered by individuals developing and assessing new programs.

Lack of ethnic differences in the pharmacokinetics and pharmacodynamics of inclacumab in healthy Japanese and Caucasian subjects.

PURPOSE: Inclacumab, a novel monoclonal antibody against P-selectin, is in development for the treatment and prevention of atherosclerotic cardiovascular diseases. This study was conducted to investigate potential differences in the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single intravenous doses of inclacumab between Japanese and Caucasian healthy volunteers. METHOD: Sixty-two subjects (31 Japanese and 31 Caucasian) were enrolled in a single-center, open-label, parallel, three dose groups (0.3, 3.0, and 20 mg/kg), single-dose study in Japanese and Caucasian healthy volunteers. Inclacumab concentrations, platelet-leukocyte aggregates (PLA), free/total soluble P-selectin (sP-selectin) ratio, and antibody formation were measured along with routine safety monitoring during the conduct of the study. RESULTS: The PK profiles of inclacumab in Caucasian and Japanese subjects were similar following single-dose intravenous infusion. The statistical analysis of peak (C max) and total exposure (AUClast) indicated that bioavailability was similar for both races when corrected for body weight. The geometric mean ratios for AUClast and C max in the Japanese versus Caucasian cohort were 101 and 111%, respectively, in 0.3 mg/kg dose group, 108 and 107%, respectively, in 3.0 mg/kg dose group, and 97 and 96%, respectively, in 20 mg/kg dose group. No differences were observed in the level of PLA inhibition and mean free/total soluble P-selectin ratio between Japanese and Caucasian subjects. PK/PD relationship between the free/total sP-selectin ratio or PLA and plasma concentration of inclacumab appeared to be similar in both Japanese and Caucasian populations. The effect of race as a covariate was explored on both PK/PD models for PLA and free/total sP-selectin ratio and did not have a significant effect over the reduced model without race as a covariate. CONCLUSIONS: Ethnicity had no clinically relevant influence on inclacumab pharmacokinetics or pharmacodynamics. No dose adjustment of inclacumab is required for differences in race.

Smoking and Cellular Telephone Use Among Pregnant Women Seeking Prenatal Care: Opportunities for Intervention.

OBJECTIVES: Although smoking during pregnancy is associated with poor pregnancy outcomes, many women continue to smoke throughout pregnancy. Behavioral interventions for smoking cessation yield modest benefits, particularly in lower socioeconomic groups. Pharmacotherapy, a first-line option for smoking cessation, has not shown clear benefits for pregnant smokers, partly due to limited adherence. We evaluated the feasibility of conducting a pharmacotherapy trial for smoking cessation in pregnant women, using text messaging to enhance medication adherence. METHODS: We surveyed 724 predominantly minority pregnant women to examine the prevalence and correlates of smoking and the use of cellular telephones and text messaging. RESULTS: Nearly 18% of the respondents were current smokers, with a majority (67.7%) expressing interest in participating in a smoking cessation trial. Only about 6% of women with a smoking history ever received nicotine dependence treatment. Smokers were significantly more likely to be depressed than non-smokers. The vast majority of respondents (92.1%) owned cell phones, with 93.2% having an unlimited text-messaging plan. CONCLUSIONS: These data support the feasibility of conducting a pharmacotherapy smoking cessation trial and using text messaging to enhance medication adherence in a predominantly minority population of pregnant smokers.

Measuring change in somatosensation across the lifespan.

OBJECTIVE: The study aim was to determine natural variability in somatosensation across age groups using brief measures. We validated measures in a community-dwelling population as part of the National Institutes of Health (NIH) Toolbox for Assessment of Neurological and Behavioral Function (NIH Toolbox; http://www.nihtoolbox.org). METHOD: Participants included community-dwelling children and adults (N=367, ages 3-85 yr) across seven sites. We tested haptic recognition, touch detection-discrimination, and proprioception using brief affordable measures as required by the NIH Toolbox. RESULTS: Accuracy improved from young children to young adults; from young to older adults, the pattern reversed slightly. We found significant differences between adults and older adults. One proprioception test (kinesthesia; p=.003) showed gender differences (females more accurate). We provide expected score ranges for age groups as a basis for understanding age-related expectations for somatosensory perception. CONCLUSION: The age-related patterns of somatosensory perception from this study refine decision making about performance.

Clavulanic Acid Decreases Cocaine Cue Reactivity in Addiction-Related Brain Areas, a Randomized fMRI Pilot Study.

Background: Preclinical studies show that clavulanic acid (CLAV) inhibits cocaine self-administration. This study investigates the effect of CLAV on regions of brain activation in response to cocaine cues during functional magnetic resonance imaging (fMRI) in participants with cocaine use disorder (CUD). Methods: A double-masked, placebo-controlled clinical trial with thirteen individuals with severe CUD who were randomized to treatment with CLAV (N = 10, 9 completers) 500 mg/day or matched placebo (PBO) (N = 3) for 3 days. fMRI was used to assess brain reactivity to 18 alternating six-second video clips of cocaine or neutral scenes. In this paradigm, participants were exposed to three different stimulus conditions: NEUTRAL, WATCH (passive watching), and DOWN (actively inhibiting craving while watching). Results: Participants who received CLAV demonstrated a significant reduction in brain activity in the anterior cingulate gyrus (p = 0.009) and the caudate (p = 0.018) in response to DOWN cocaine cues. There was a trend toward lessened cue reactivity in other regions implicated in CUD. Conclusion: CLAV reduced the response of the brain regions associated with motivation and emotional response during the DOWN condition compared to PBO, suggesting CLAV may strengthen voluntary efforts to avoid cocaine use. This pilot data supports the use of CLAV for CUD. (Trial registered in ClinicalTrials.gov NCT04411914).

Cross-Cutting mHealth Behavior Change Techniques to Support Treatment Adherence and Self-Management of Complex Medical Conditions: Systematic Review.

Background: Mobile health (mHealth) interventions have immense potential to support disease self-management for people with complex medical conditions following treatment regimens that involve taking medicine and other self-management activities. However, there is no consensus on what discrete behavior change techniques (BCTs) should be used in an effective adherence and self-management-promoting mHealth solution for any chronic illness. Reviewing the extant literature to identify effective, cross-cutting BCTs in mHealth interventions for adherence and self-management promotion could help accelerate the development, evaluation, and dissemination of behavior change interventions with potential generalizability across complex medical conditions. Objective: This study aimed to identify cross-cutting, mHealth-based BCTs to incorporate into effective mHealth adherence and self-management interventions for people with complex medical conditions, by systematically reviewing the literature across chronic medical conditions with similar adherence and self-management demands. Methods: A registered systematic review was conducted to identify published evaluations of mHealth adherence and self-management interventions for chronic medical conditions with complex adherence and self-management demands. The methodological characteristics and BCTs in each study were extracted using a standard data collection form. Results: A total of 122 studies were reviewed; the majority involved people with type 2 diabetes (28/122, 23%), asthma (27/122, 22%), and type 1 diabetes (19/122, 16%). mHealth interventions rated as having a positive outcome on adherence and self-management used more BCTs (mean 4.95, SD 2.56) than interventions with no impact on outcomes (mean 3.57, SD 1.95) or those that used >1 outcome measure or analytic approach (mean 3.90, SD 1.93; P=.02). The following BCTs were associated with positive outcomes: self-monitoring outcomes of behavior (39/59, 66%), feedback on outcomes of behavior (34/59, 58%), self-monitoring of behavior (34/59, 58%), feedback on behavior (29/59, 49%), credible source (24/59, 41%), and goal setting (behavior; 14/59, 24%). In adult-only samples, prompts and cues were associated with positive outcomes (34/45, 76%). In adolescent and young adult samples, information about health consequences (1/4, 25%), problem-solving (1/4, 25%), and material reward (behavior; 2/4, 50%) were associated with positive outcomes. In interventions explicitly targeting medicine taking, prompts and cues (25/33, 76%) and credible source (13/33, 39%) were associated with positive outcomes. In interventions focused on self-management and other adherence targets, instruction on how to perform the behavior (8/26, 31%), goal setting (behavior; 8/26, 31%), and action planning (5/26, 19%) were associated with positive outcomes. Conclusions: To support adherence and self-management in people with complex medical conditions, mHealth tools should purposefully incorporate effective and developmentally appropriate BCTs. A cross-cutting approach to BCT selection could accelerate the development of much-needed mHealth interventions for target populations, although mHealth intervention developers should continue to consider the unique needs of the target population when designing these tools.

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH.

INTRODUCTION: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers' blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. METHODS: Fetal brain tissues and maternal blood were collected at 9-23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. CONCLUSIONS: Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

Association Among Chronic Obstructive Pulmonary Disease Severity, Exacerbation Risk, and Anxiety and Depression Symptoms in the SPIROMICS Cohort.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common, progressive lung disease that often manifests with psychiatric symptoms. Despite this, patients with COPD are not routinely screened for anxiety and depression, which substantially contribute to COPD-related morbidity. OBJECTIVE: To determine the relationship among COPD symptom severity, exacerbation risk, and clinically significant anxiety and depression symptoms in ever smokers with COPD. METHODS: We used baseline data from the Subpopulations and Intermediate Outcome Measures In COPD Study (SPIROMICS) cohort to examine ever smokers with COPD across Global Initiative for Obstructive Lung Disease (GOLD) disease severity groups. Multivariable logistic regression models were used to calculate odds ratios for clinically significant anxiety and depression for each GOLD group, which was compared to the control group of ever smokers without COPD. Odds ratios were adjusted for subject demographics, medical comorbidities, and substance use covariates, and comparisons were completed using 2-tailed tests. RESULTS: Of the 2664 subjects studied, 784 (29.4%) had clinically significant anxiety, and 497 (18.7%) had clinically significant depression. In the multivariable analysis, high pulmonary symptom groups, groups B and D, had increased adjusted odds of clinically significant anxiety (group B: adjusted odds ratios [AOR] 1.28, P = 0.03; group D: AOR 1.95, P < 0.0001) and depression (group B: AOR 2.09, P < 0.0001; group D: AOR 3.04, P < 0.0001). GOLD group D, the group with high pulmonary symptoms and high COPD exacerbation risk, had the greatest risk of both anxiety and depression among the GOLD groups. CONCLUSIONS: High COPD symptom severity, even in the absence of elevated COPD exacerbation risk, is associated with clinically significant anxiety and depression. Our separate analyses of anxiety and depression symptoms in a large, multisite, national cohort are unique within the literature and have important treatment implications for COPD patients. Our findings also highlight the utility of screening patients with high COPD symptom severity for anxiety and depression.

The GLT-1 enhancer clavulanic acid suppresses cocaine place preference behavior and reduces GCPII activity and protein levels in the rat nucleus accumbens.

The ß-lactam antibiotic ceftriaxone (CTX) is a glutamate transporter subtype 1 (GLT-1) enhancer that reduces cocaine reinforcing efficacy and relapse in rats, but pharmacokinetic liabilities limit translational utility. An attractive alternative is clavulanic acid (CLAV), a structurally related ß-lactamase inhibitor and component of FDA-approved Augmentin. CLAV retains the GLT-1 enhancing effects of CTX but displays greater oral bioavailability, brain penetrability and negligible antibacterial activity. CLAV reduces morphine conditioned place preference (CPP) and ethanol consumption in rats, but knowledge about the efficacy of CLAV in preclinical models of drug addiction remains sparse. Here, we investigated effects of CLAV (10 mg/kg, IP) on the acquisition, expression, and maintenance of cocaine CPP in rats, and on two glutamate biomarkers associated with cocaine dependence, GLT-1 and glutamate carboxypeptidase II (GCPII). CLAV administered during cocaine conditioning (10 mg/kg, IP x 4 d) did not affect the development of cocaine CPP. However, a single CLAV injection, administered after the conditioning phase, reduced the expression of cocaine CPP. In rats with established cocaine preference, repeated CLAV administration facilitated extinction of cocaine CPP. In the nucleus accumbens, acute CLAV exposure reduced GCPII protein levels and activity, and a 10-d CLAV treatment regimen enhanced GLT-1 levels. These results suggest that CLAV reduces expression and maintenance of cocaine CPP but lacks effect against development of CPP. Moreover, the ability of a single injection of CLAV to reduce both GCPII activity and protein levels, as well as expression of cocaine CPP, points toward studying GCPII as a therapeutic target of CLAV.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.