Meaningfully Characterizing Cannabis Use for Research and Clinical Settings: A Comprehensive Review of Existing Measures and Proposed Future Directions.

Cannabis use is increasingly common. There is a need for validated tools to meaningfully assess recreational, medical, and disordered cannabis use in both research and clinical contexts. Cannabis assessments were considered against pre-determined inclusion criteria within a comprehensive review. Measures were categorized as either (i) evaluating use frequency or quantity, (ii) measuring symptoms of disordered use and withdrawal, or (iii) assessing use motives, effects, and perceptions. The applications and validations for each assessment are summarized. Finally, recommendations for refining of existing measures or development of new measures are presented. The literature review resulted in 289 publications that were reviewed in detail, yielding 21 assessments that met inclusion criteria. The applications of these assessments are described here, in addition to the information about the validation studies of each assessment. Based on the complication of these tools, 5 areas of potential development are highlighted to guide future research, including (i) sensitivity to the mode of cannabis administration as well as sensitivity to (ii) potency of cannabis products alongside frequency and quantity, (iii) unit equivalence, (iv) aligning clinical measures consistently with cannabis use disorder (CUD) diagnostic criteria, and (v) creating measures specific to medical users, their motives for use, and their perceptions of therapeutic benefits or side effects. Clinicians and researchers can pragmatically benefit from this summary of validated measures of cannabis use, and future work could improve the study of and clinical care for cannabis use and CUD by pursuing one or more key areas of development described here.

Shifting From Best Practice to Standard Practice: Implementing Measurement-Based Care in Health Systems.

There is a high prevalence of untreated depression in adults and youth observed at the population level in the United States, and many who would benefit from treatment do not receive it. One proposed effort to increase access to care is the use of measurement-based care (MBC; repeated use of symptom measures for screening and treatment guidance) by primary care physicians to treat non-complex cases of depression. MBC has been shown to improve patient outcomes compared to care as usual, but there are barriers that need to be addressed at the health system level for effective implementation to occur. Herein we provide an overview of MBC and detail benefits and barriers of MBC implementation. Relevant considerations and guidance for implementing MBC are presented, and a case example of a health system implementing MBC is included. Though issues of reimbursement, limited human and technological resources, and resistance to systemic change are barriers to implementing MBC, effective strategies exist to overcome these barriers. In addition to helping health systems align with changes to value-based care models, effective implementation of MBC can likely improve patient outcomes and result in net financial benefits.

Characterizing Measurement-Based Care in the Texas Youth Depression and Suicide Research Network (TX-YDSRN).

Integration of measurement-based care (MBC) into clinical practice has shown promise in improving treatment outcomes for depression. Yet, without a gold standard measure of MBC, assessing fidelity to the MBC model across various clinical settings is difficult. A central goal of the Texas Youth Depression and Suicide Research Network (TX-YDSRN) was to characterize MBC across the state of Texas through the development of a standardized tool to assess the use of MBC strategies when assessing depression, anxiety, side effects, and treatment adherence. A chart review of clinical visits indicated standardized depression measures (71.2%) and anxiety measures (64%) were being utilized across sites. The use of standardized measures to assess medication adherence and side effects was limited to less than six percent for both, with the majority utilizing clinical interviews to assess adherence and side effects; yet medication was changed in nearly half. Rates of utilization of standardized measures for participants with multiple MBC forms were similar to those who only provided one form.

Integration of Measurement-Based Care for Youth Depression and Suicidality Using VitalSign6.

Depression and suicidality are prevalent in youth and are associated with a range of negative outcomes. The current study aimed to evaluate a measurement-based care (MBC) software (VitalSign6) tool to improve the screening and treatment of depression and suicidality in youth aged 8-17 years within a rural, underserved population. To assess for depression and suicidality, the Patient Health Questionnaire-2 was administered as an initial screen, and the Patient Health Questionnaire-9 Modified for Adolescents (PHQ-9-A) was administered if the initial screen was positive. Data were collected at medical clinics over one year, and descriptive statistics and t-tests or Wilcoxon-Mann-Whitney tests were conducted. A total of 1,984 youth were initially screened (mean age of 13 years; 51.6% female); 24.2% screened positive for depression, and 14.9% endorsed suicidality. Of those who screened positive, the mean PHQ-9-A score was 12.8; 66.9% had PHQ-9-A scores in the moderate to severe range, and 44.2% endorsed suicidality. Almost half of the youth who screened positive for depression had at least one follow-up assessment, and about one quarter achieved remission 4 months after initial screening. Adolescents (12-17 years) had higher PHQ-9-A scores, higher suicidality, and more follow-up assessments than younger youth (8-11 years). Younger youth had higher rates of remission. The widespread use of MBC was feasible in this setting. It is important to utilize MBC to identify and treat youth with depression and suicidality and to do so in younger populations to improve their trajectory over time; VitalSign6 is one tool to help achieve these goals.

The clinical presentation of major depressive disorder in youth with co-occurring obsessive-compulsive disorder.

BACKGROUND: Major depressive disorder (MDD) is common in youth and among the most frequent comorbid disorders in pediatric obsessive-compulsive disorder (OCD), but it is unclear whether the presence of OCD affects the symptom presentation of MDD in youth. METHODS: A sample of youth with OCD and MDD (n = 124) and a sample of youth with MDD but no OCD (n = 673) completed the Patient Health Questionnaire for Adolescents (PHQ-A). The overall and symptom-level presentation of MDD were examined using group comparisons and network analysis. RESULTS: Youth with MDD and OCD, compared to those with MDD and no OCD, had more severe MDD (Cohen's d = 0.39) and more reported moderate to severe depression (75 % vs 61 %). When accounting for demographic variables and the overall severity of MDD, those with comorbid OCD reported lower levels of anhedonia and more severe difficulties with psychomotor retardation/agitation. No significant differences in the interconnections among symptoms emerged. LIMITATIONS: Data were cross-sectional and self-reported, gold standard diagnostic tools were not used to assess OCD, and the sample size for the group with MDD and OCD was relatively small yielding low statistical power for network analysis. CONCLUSIONS: Youth with MDD and OCD have more severe MDD than those with MDD and no OCD and they experience more psychomotor issues and less anhedonia, which may relate to the behavioral activation characteristic of OCD.

Treatment of Adolescent Depression: Comparison of Psychiatric and Pediatric Settings at an Academic Medical Center Using the VitalSign6 Application.

Background: Similar outcomes and remission rates have been found for the treatment of depression in adults in primary and psychiatric care settings. However, comparatively little is known about how pediatric depression is managed across different settings. This study aims to address this gap by comparing depression treatment in pediatric and psychiatric settings. We hypothesized that pediatric care settings would be more likely to treat individuals with lower depression severity and would select pharmacotherapy less frequently as a treatment option. Methods: Patients (n = 3498) were screened for depression at a children's hospital from May 2017 to May 2022 as part of the VitalSign6 project, a web-based application for depression management. The two-item patient health questionnaire (PHQ) was used for screening, and the data set contains patient-reported measures and provider-reported diagnoses and treatment selections at each clinic visit. Patients with nine-item PHQ (PHQ-9) scores ≥10 at baseline were included in the analysis to compare diagnosis and treatment recommendations between pediatric and psychiatric settings. Results: Among the 1323 patients who screened positive for depression, those in psychiatric settings had higher PHQ-9 scores (15.9 ± 5.0 vs. 12.1 ± 5.5; p < 0.0001). Patients with PHQ-9 ≥ 10 in psychiatric settings were more likely to be diagnosed with major depressive disorder (60.6% vs. 24.7%, p < 0.0001) and receive pharmacotherapy (54.8% vs. 6.6%) than those in pediatric settings. Pediatric setting patients were more likely to receive nonpharmacological treatment alone (36.3% vs. 4.3%) or an outside referral (27.7% vs. 5.7%). Remission rates did not significantly differ between the two settings. Conclusions: Youth in psychiatric settings are more likely to screen positive for depression and to have greater depression severity than those in pediatric settings. Both settings provide treatment recommendations for moderate-to-severe depression, but treatment types vary substantially. Yet, remission rates remain similar. Further research is needed to understand the nuances of treatment differences and their implications.

Linking trauma to mental health in the statewide Texas Youth Depression and Suicide Research Network (TX-YDSRN).

Rates of youth depression and suicide are rising worldwide and represent public health crises. The present study examined the relationship between trauma history and symptoms of depression, suicidal ideation, and anxiety among suicidal and depressed youth. A diverse group of 1000 8-20-year-olds enrolled in the statewide Texas Youth Depression and Suicide Research Network (TX-YDSRN) reported their trauma history (Traumatic Events Screening Inventory for Children) and symptoms of depression (Patient Health Questionnaire for adolescents; PHQ-A), anxiety (Generalized Anxiety Disorder scale; GAD-7), and suicidality (Concise Health Risk Tracking scale; CHRT-SR). Nearly half of the sample reported exposure to multiple categories of traumatic experiences. Number of trauma exposure categories significantly predicted PHQ-A and GAD-7 scores. Exposure to interpersonal trauma and to sexual trauma were significantly associated with PHQ-A, GAD-7, and CHRT-SR scores. The number of trauma exposure categories was associated with increased levels of anxiety and depression; however, only exposure to interpersonal or sexual trauma was associated with more suicidality. Clinicians should assess trauma exposure in patients seeking psychiatric care, especially for interpersonal and sexual trauma, which may be predictive of increased risk for suicidality in depressed youth. Future work should disentangle the effects of specific trauma types from multiple trauma exposure.

Baseline affective symptomatology moderates acute subjective effects of high potency THC and CBD cannabis concentrates.

Highly potent cannabis concentrates are widely available and associated with affective disturbance and cannabis use disorder. Little is known about the effects of concentrated Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) and their relationship to long-term affect. We explored how baseline affective symptoms (anxiety and depression) relate to acute (i.e., immediate or short-term) subjective mood and intoxication effects during naturalistic use of cannabis concentrates. Fifty-four cannabis users (48% female; Mage = 29.87) were assigned to ad libitum use of either a THC-dominant (84.99% THC and THCa, < 1% CBD) or CBD-dominant (74.7% CBD, 4.1% CBDa, 4.5% THC and THCa) concentrate. Individuals were assessed at baseline and before, immediately after, and 1 hr after naturalistic use of their assigned product. Models regressed each outcome on time, product condition, baseline affective symptoms, and their interactions. An interaction emerged between condition and baseline depression symptoms on positive mood (F = 9.47, p < .005); higher depression symptom level was associated with higher positive mood with THC-dominant product use. There was an interaction between condition, baseline depression symptoms, and time on negative mood (F = 5.55, p < .01); negative mood decreased with CBD-dominant product use for all depression symptom levels but increased with THC-dominant product use at high levels. Finally, there was an interaction between condition and time on intoxication (F = 3.72, p = .03); the THC-dominant condition was more intoxicated postuse than the CBD-dominant condition. This novel exploratory study suggests that baseline affect moderates the acute effects of ad libitum use of THC and CBD concentrates such that preexisting affective symptoms modulate the intensity of subjective drug experiences. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

Brain Concentrations of MDPV and its Metabolites in Male Rats: Relationship to Pharmacodynamic Effects.

BACKGROUND: MDPV (3,4-methylenedioxypyrovalerone) is a synthetic stimulant that blocks transmitter uptake at transporters for dopamine and norepinephrine. Less is known about MDPV pharmacokinetics, especially with respect to brain concentrations of the drug and its metabolites. OBJECTIVES: The goal of the present study was: 1) to determine brain concentrations of MDPV and its metabolites, 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxy-pyrovalerone (4-OH-3-MeOPV), after administration of MDPV, and 2) to relate brain pharmacokinetic measures to pharmacodynamic endpoints in the same subjects. METHODS: Male Sprague-Dawley rats (300-400 g) received s.c. MDPV injection (1, 2, or 4 mg/kg) or its saline vehicle. Groups of rats were decapitated at 40 min and 240 min postinjection. Locomotor behavior was rated before decapitation, and the core temperature was obtained. Plasma and frontal cortex were analyzed to quantitate MDPV and its metabolites. Striatal samples were analyzed to measure dopamine, serotonin (5-HT), and their metabolites. RESULTS: MDPV displayed brain-to-plasma ratios greater than 1 (range 8.8-12.1), whereas 3,4-catechol-PV and 4-OH-3-MeO-PV showed ratios less than 1 (range 0-0.3). MDPV increased behavioural scores reflective of locomotor stimulation at 40 and 240 min and produced slight hyperthermia at 240 min. MDPV had no effect on striatal dopamine but produced an increase in the metabolite homovanillic acid (HVA). Brain MDPV concentrations were positively correlated with behavioural scores and striatal HVA but not with other endpoints. CONCLUSION: The behavioural effects of MDPV are related to brain concentrations of the parent drug and not its metabolites. The modest effects of MDPV on monoamine systems suggest that other non-monoamine mechanisms may contribute to the effects of the drug in vivo.

Online survey of medicinal cannabis users: Qualitative analysis of patient-level data.

Aim: To characterize perceived benefits and challenges experienced by medicinal cannabis users. Methods: An anonymous online survey collected demographics, health information, and open-ended responses from medicinal cannabis users regarding perceptions, motivations, and experience of treatment. Qualitative open-ended responses were thematically analyzed. Results: Respondents (N = 808) were predominantly White (79%), female (63%), with a mean (SD) age of 38 (20). Two hundred eighty-four (35%) respondents provided data on a dependent family member (e.g., child; 22% of total sample). Most used cannabidiol (CBD)-dominant products (58%), primarily for neurological disorders (38%) or pain (25%). Primary motivations for medicinal cannabis use were based on beliefs that traditional treatments were ineffective and/or had intolerable side effects (51%), positive scientific or media portrayals of the safety/efficacy of cannabis as a therapeutic (29%), or preference for "natural" treatments over pharmaceuticals (21%). A majority of respondents (77%) attributed positive effects to the medicinal use of cannabis/cannabinoids. These included physical symptom improvements such as reduced pain (28%), improved sleep (18%), and seizure reduction (18%), and mental health improvements including reduced anxiety (22%) and improved mood (11%). Additionally, respondents reported reduced use of other medications (e.g., opioids) (12%), and improved quality of life (14%). Problems associated with use were cited by 41% of respondents, and included unwanted side effects (16%), lack of information or medical support (16%), prohibitive costs (12%), and legal concerns (10%). Conclusion: Most participants reported benefits from cannabis use for a variety of conditions where traditional treatments were ineffective or unacceptable. Concerns regarding cannabis side effects, legality, lack of information, and cost were raised. Data indicate greater research and education on the safety and efficacy of medicinal cannabis/cannabinoid use is warranted.

Engineering Citrobacter freundii using CRISPR/Cas9 system.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to ß-ketoadipic acid by Pseudomonas putida KT2440.

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, ß-ketoadipic acid (ßKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L ßKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to ßKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Brain Concentrations of Methylone and Its Metabolites after Systemic Methylone Administration: Relationship to Pharmacodynamic Effects.

3,4-Methylenedioxy-N-methylcathinone (methylone) is a new psychoactive substance with stimulant properties and potential for abuse. Despite its popularity, limited studies have examined relationships between brain concentrations of methylone, its metabolites, and pharmacodynamic effects. The goal of the present study was 2-fold: 1) to determine pharmacokinetics of methylone and its major metabolites-4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-dihydroxy-N-methylcathinone (HHMC), and 3,4-methylenedioxycathinone (MDC)-in rat brain and plasma and 2) to relate brain pharmacokinetic parameters to pharmacodynamic effects including locomotor behavior and postmortem neurochemistry. Male Sprague-Dawley rats received subcutaneous methylone (6, 12, or 24 mg/kg) or saline vehicle (n = 16/dose), and subgroups were decapitated after 40 or 120 minutes. Plasma and prefrontal cortex were analyzed for concentrations of methylone and its metabolites by liquid chromatography-tandem mass spectrometry. Frontal cortex and dorsal striatum were analyzed for dopamine, 5-HT, and their metabolites by high-performance liquid chromatography-electrochemical detection. Brain and plasma concentrations of methylone and its metabolites rose with increasing methylone dose, but brain methylone and MDC concentrations were greater than dose-proportional. Brain-to-plasma ratios for methylone and MDC were ≥ 3 (range 3-12), whereas those for HHMC and HMMC were ≤ 0.2 (range 0.01-0.2). Locomotor activity score was positively correlated with brain methylone and MDC, whereas cortical 5-HT was negatively correlated with these analytes at 120 minutes. Our findings show that brain concentrations of methylone and MDC display nonlinear accumulation. Behavioral and neurochemical effects of systemically administered methylone are related to brain concentrations of methylone and MDC but not its hydroxylated metabolites, which do not effectively penetrate into the brain. SIGNIFICANCE STATEMENT: Behavioral and neurochemical effects of methylone are related to brain concentrations of methylone and its metabolite MDC but not its hydroxylated metabolites, 4-hydroxy-3-methoxy-N-methylcathinone and 3,4-dihydroxy-N-methylcathinone, which do not effectively penetrate into the brain. Methylone and MDC display nonlinear accumulation in the brain, which could cause untoward effects on serotonin neurons in vulnerable brain regions, including the frontal cortex.

Rewiring of gene expression in circulating white blood cells is associated with pregnancy outcome in heifers (Bos taurus).

Infertility is a challenging phenomenon in cattle that reduces the sustainability of beef production worldwide. Here, we tested the hypothesis that gene expression profiles of protein-coding genes expressed in peripheral white blood cells (PWBCs), and circulating micro RNAs in plasma, are associated with female fertility, measured by pregnancy outcome. We drew blood samples from 17 heifers on the day of artificial insemination and analyzed transcript abundance for 10,496 genes in PWBCs and 290 circulating micro RNAs. The females were later classified as pregnant to artificial insemination, pregnant to natural breeding or not pregnant. We identified 1860 genes producing significant differential coexpression (eFDR < 0.002) based on pregnancy outcome. Additionally, 237 micro RNAs and 2274 genes in PWBCs presented differential coexpression based on pregnancy outcome. Furthermore, using a machine learning prediction algorithm we detected a subset of genes whose abundance could be used for blind categorization of pregnancy outcome. Our results provide strong evidence that transcript abundance in circulating white blood cells is associated with fertility in heifers.

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid.

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h-1 and 0.38 h-1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.

Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design.

The development of Pseudomonas strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in Pseudomonas putida KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.