CRISPR-Cas9-mediated barcode insertion into Bacillus thuringiensis for surrogate tracking.

The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded B. thuringiensis strains for use in future studies on surrogate genomes. IMPORTANCE: The use of Bacillus anthracis as a biothreat agent poses significant challenges for public health and national security. Bacillus anthracis surrogates, like Bacillus thuringiensis, are invaluable tools for safely understanding Bacillus anthracis properties without the safety concerns that would arise from using a virulent strain of Bacillus anthracis. We report a simple method for barcode insertion into Bacillus thuringiensis using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in Bacillus thuringiensis suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded Bacillus thuringiensis surrogate strains, among other biotechnological applications in Bacillus species.

Identification of Amino Acids in Trm734 Required for 2'-O-Methylation of the tRNAPhe Wobble Residue.

All organisms methylate their nucleic acids, and this methylation is critical for proper gene expression at both the transcriptional and translational levels. For proper translation in eukaryotes, 2'-O-methylation of C32 (Cm32) and G34 (Gm34) in the anticodon loop of tRNAPhe is critical, with defects in these modifications associated with human disease. In yeast, Cm32 is formed by an enzyme that consists of the methyltransferase Trm7 in complex with the auxiliary protein Trm732, and Gm34 is formed by an enzyme that consists of Trm7 in complex with Trm734. The role of Trm732 and Trm734 in tRNA modification is not fully understood, although previous studies have suggested that Trm734 is important for tRNA binding. In this report, we generated Trm734 variants and tested their ability to work with Trm7 to modify tRNAPhe. Using this approach, we identified several regions of amino acids that are important for Trm734 activity and/or stability. Based on the previously determined Trm7-Trm734 crystal structure, these crucial amino acids are near the active site of Trm7 and are not directly involved in Trm7-Trm734 protein-protein interactions. Immunoprecipitation experiments with these Trm734 variants and Trm7 confirm that these residues are not involved in Trm7-Trm734 binding. Further experiments should help determine if these residues are important for tRNA binding or have another role in the modification of the tRNA. Furthermore, our discovery of a nonfunctional, stable Trm734 variant will be useful in determining if the reported roles of Trm734 in other biological processes such as retromer processing and resistance to Ty1 transposition are due to tRNA modification defects or to other bona fide cellular roles of Trm734.

The Relationship between Vaping Cannabis and Frequency of Cannabis Use and Cannabis-Related Problems among Urban High School Students.

Background: Vaping, including vaping cannabis, is increasing among adolescents. In this longitudinal study, we examined the relationship between vaping cannabis and frequency of cannabis use and related problems over 6 months among adolescents. Material and Methods: Data were from 233 participants (46.8% male, 93.1% African American, mean age = 16.4 years) reporting cannabis use. The Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) assessed frequency of past 30-day cannabis use and cannabis-related problems at baseline, 3- and 6-months post-baseline. We used latent growth curve modeling to compare vaping to non-vaping adolescents on trends in cannabis use frequency and ASSIST cannabis scores. Results: Adolescents who vaped cannabis (11.7%) had higher past 30-day frequency (mean = 17.89 days, SD = 10.49) of cannabis use at baseline compared to adolescents who had not vaped (mean = 12.1 days, SD = 10.93), but reported a significantly sharper decline in frequency of cannabis use (b = -0.34, p = 0.017). A significantly steeper decrease existed in the mean cannabis ASSIST scores for the vaping group than for the non-vaping group (b = -0.34, p = 0.014). Mean ASSIST scores on the cannabis subscale between the two groups were significantly different at 6-month follow-up (Vape mean = 6.00, SD = 8.12 vs. Non-vape mean = 9.6, SD = 9.39; p < 0.021). Conclusions: In a sample of cannabis-using adolescents, adolescents with experience vaping cannabis, compared to adolescents without vaping experience, on average reported sharper decreases in frequency of cannabis use and cannabis-related problems such as health or social problems.

Mapping prescription drug monitoring program data to self-report measures of non-medical prescription opioid use in community pharmacy settings.

BACKGROUND: Community pharmacists are well-positioned to identify patients engaged in non-medical prescription opioid use (NMPOU) through Prescription Drug Monitoring Program (PDMP) databases. Integrating patient-reported outcomes with PDMP data may improve the interpretability of PDMP information to support clinical decision-making. OBJECTIVE: This study linked patient-reported clinical measures of substance use with PDMP data to examine relationships between average daily opioid dose in morphine milligram equivalents (MME) and visits to multiple pharmacies/prescribers with self-reported NMPOU. METHODS: Data from a cross-sectional health assessment given to patients aged ≥18 years filling opioid prescriptions were linked to PDMP records. NMPOU in the past three months was assessed on a continuous scale (range 0-39) using an adapted version of the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST). PDMP measures included average daily MME and number of distinct pharmacies/prescribers visited in the past 180 days. Univariable and multivariable zero-inflated negative binomial models estimated associations between PDMP measures and any NMPOU and severity of use. RESULTS: The sample included 1421 participants. In multivariable models adjusted for sociodemographic, mental health, and physical health characteristics, any NMPOU was associated with higher average daily MME (adjusted OR = 1.22, 95% CI = 1.05-1.39) and number of distinct prescribers visited (adjusted OR = 1.15, 95% CI = 1.01-1.30). Higher average daily MME (adjusted mean ratio (MR) = 1.12, 95% CI = 1.08-1.15), number of distinct pharmacies visited (adjusted MR = 1.11, 95% CI = 1.04-1.18), and number of distinct prescribers visited (adjusted MR = 1.07, 95% CI = 1.02-1.11) were associated with increased NMPOU severity. CONCLUSIONS: We observed significant, positive associations between average daily MME and visits to multiple pharmacies/prescribers with any NMPOU and severity of use. This study demonstrates self-report clinical measures of substance use can be cross-walked to PDMP data and translated into clinically interpretable information.

"It Just Depends on Their Stability": A Qualitative Examination of Patient Factors Influencing Providers' Contraceptive Counseling Approaches for Persons With Substance Use Disorders.

OBJECTIVES: This qualitative study examined how patient-related factors influence providers' contraceptive counseling for persons with substance use disorders (SUDs). Specifically, we explored individual behavior and social factors that contribute to providers modifying their contraceptive counseling approaches and described how providers alter their counseling recommendations and communication strategies in the presence of such factors. METHODS: In 2019, we purposively recruited a national sample of contraceptive providers (N = 24) and conducted semistructured phone interviews to inquire about their contraceptive counseling practices for women with SUDs. Interviews were audio recorded, transcribed verbatim, and analyzed using thematic analysis with inductive codes. RESULTS: Participants included 10 medical doctors, 8 nurse practitioners, and 6 certified nurse-midwives. We found that providers modify their contraceptive counseling provision when their patients are actively using substances or have unstable living conditions, such as intimate partner violence or homelessness. With patients experiencing these instabilities, providers reported postponing contraceptive discussions until patients are stabilized in treatment, recommending long-active reversible contraceptive methods, and varying communication styles according to their own perceptions of patients' communication needs. Providers perceived that individuals in long-term recovery have increased stability and fewer barriers to contraceptive access and adherence and therefore reported increased willingness to provide greater autonomy during contraceptive decision making and shift the counseling focus to short-acting contraceptive methods. CONCLUSIONS: This study highlights that substance use and social "stability" of patients contributes to how providers approach their contraceptive counseling and make methods recommendations for their patients with SUDs. More research is needed to understand strategies that individuals with SUDs use to overcome barriers to contraceptive access and adherence in the context of active substance use and social instability.

Racial discrimination, John Henryism coping, and behavioral health conditions among predominantly poor, urban African Americans: Implications for community-level opioid problems and mental health services.

The current study examined the relationship between John Henryism Active Coping (JHAC), experiences of racial discrimination, and behavioral health outcomes in a community sample of 319 Black adults. Assessments included primary health care screenings as well as self-reported survey questions to assess JHAC, experiences of discrimination, and self-reported behavioral health. Logistic regression models, adjusted for control variables, found a significant relationship between JHAC and having an opioid problem (odds ratio [OR] = 0.95, p = 0.003) and needing mental health services (OR = 0.95, p < 0.001), such that higher levels of coping were associated with lower odds of reporting an opioid problem and needing mental health services. Notably, racial discrimination was not significantly independently associated with behavioral health. Implications for interventions and community programming are discussed.