Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Mitochondrial Dysfunction in PCOS: Insights into Reproductive Organ Pathophysiology.

Polycystic ovary syndrome (PCOS) is a complex, but relatively common endocrine disorder associated with chronic anovulation, hyperandrogenism, and micro-polycystic ovaries. In addition to reduced fertility, people with PCOS have a higher risk of obesity, insulin resistance, and metabolic disease, all comorbidities that are associated with mitochondrial dysfunction. This review summarizes human and animal data that report mitochondrial dysfunction and metabolic dysregulation in PCOS to better understand how mitochondria impact reproductive organ pathophysiology. This in-depth review considers all the elements regulating mitochondrial quantity and quality, from mitochondrial biogenesis under the transcriptional regulation of both the nuclear and mitochondrial genome to the ultrastructural and functional complexes that regulate cellular metabolism and reactive oxygen species production, as well as the dynamics that regulate subcellular interactions that are key to mitochondrial quality control. When any of these mitochondrial functions are disrupted, the energetic equilibrium within the cell changes, cell processes can fail, and cell death can occur. If this process is ongoing, it affects tissue and organ function, causing disease. The objective of this review is to consolidate and classify a broad number of PCOS studies to understand how various mitochondrial processes impact reproductive organs, including the ovary (oocytes and granulosa cells), uterus, placenta, and circulation, causing reproductive pathophysiology. A secondary objective is to uncover the potential role of mitochondria in the transgenerational transmission of PCOS and metabolic disorders.

Assessment of modifications to evidence-based psychotherapies using administrative and chart note data from the US department of veterans affairs health care system.

Background: The US Department of Veterans Affairs (VA) has over 15 years of experience in delivery of evidence-based psychotherapies (EBPs). This paper describes strategies for using clinical documentation and administrative data to understand adherence and modifications to EBPs for Posttraumatic Stress Disorder (PTSD). Methods: This study focused on two EBPs for PTSD, Cognitive Processing Therapy (CPT) and Prolonged Exposure (PE). The sample included VA therapists from across the US who provided CPT and PE and the patients they treated over a 1-year period. The data sources for this study were templated EBP chart notes and VA administrative data. We used a manual review of note content and administrative data rules to code therapy adherence and modifications in 7,297 EBP sessions for 1,257 patients seen by 182 therapists. Two trained coders rated each therapy note and resolved discrepancies through consensus. To contextualize and explain variation in adherence and modifications, we conducted brief 30-45-min semi-structured interviews with a purposive subsample of these therapists (n = 32). Findings: Combining manual chart review and administrative data allowed for identification of 11 types of modifications. Raters disagreed on adherence for 30% of notes. The disagreement stemmed from the presence of therapy modifications that were not clearly documented, necessitating the development of decision rules and strategies for modification coding. Both therapists and patients contributed to the variance in the extent to which different modifications occurred. Therapist interviews demonstrated therapist awareness of modifying the protocols in the ways identified through chart review. Conclusion: Healthcare systems can use data collected as part of routine care to understand how and when EBPs are modified but need to develop scalable strategies to document adaptations and modifications to EBPs in routine care.

Development and Evaluation of a Sound-Swapped Video Database for Misophonia.

Misophonia has been characterized as intense negative reactions to specific trigger sounds (often orofacial sounds like chewing, sniffling, or slurping). However, recent research suggests high-level, contextual, and multisensory factors are also involved. We recently demonstrated that neurotypicals' negative reactions to aversive sounds (e.g., nails scratching a chalkboard) are attenuated when the sounds are synced with positive attributable video sources (PAVS; e.g., tearing a piece of paper). To assess whether this effect generalizes to misophonic triggers, we developed a Sound-Swapped Video (SSV) database for use in misophonia research. In Study 1, we created a set of 39 video clips depicting common trigger sounds (original video sources, OVS) and a corresponding set of 39 PAVS temporally synchronized with the OVS videos. In Study 2, participants (N = 34) rated the 39 PAVS videos for their audiovisual match and pleasantness. We selected the 20 PAVS videos with best match scores for use in Study 3. In Study 3, a new group of participants (n = 102) observed the 20 selected PAVS and 20 corresponding OVS and judged the pleasantness or unpleasantness of each sound in the two contexts accompanying each video. Afterward, participants completed the Misophonia Questionnaire (MQ). The results of Study 3 show a robust attenuating effect of PAVS videos on the reported unpleasantness of trigger sounds: trigger sounds were rated as significantly less unpleasant when paired with PAVS with than OVS. Moreover, this attenuating effect was present in nearly every participant (99 out of 102) regardless of their score on the MQ. In fact, we found a moderate positive correlation between the PAVS-OVS difference and misophonia severity scores. Overall our results provide validation that the SSV database is a useful stimulus database to study how misophonic responses can be modulated by visual contexts. Here, we release the SSV database with the best 18 PAVS and 18 OVS videos used in Study 3 along with aggregate ratings of audio-video match and pleasantness (https://osf.io/3ysfh/). We also provide detailed instructions on how to produce these videos, with the hope that this database grows and improves through collaborations with the community of misophonia researchers.

A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity.

Hepatic cell lines serve as economical and reproducible alternatives for primary human hepatocytes. However, the utility of hepatic cell lines to examine bile acid homeostasis and cholestatic toxicity is limited due to abnormal expression and function of bile acid-metabolizing enzymes, transporters, and the absence of canalicular formation. We discovered that culturing HuH-7 human hepatoma cells with dexamethasone (DEX) and 0.5% dimethyl sulfoxide (DMSO) for two weeks, with Matrigel overlay after one week, resulted in a shorter and improved differentiation process. These culture conditions increased the expression and function of the major bile acid uptake and efflux transporters, sodium taurocholate co-transporting polypeptide (NTCP) and the bile salt export pump (BSEP), respectively, in two-week cultures of HuH-7 cells. This in vitro model was further characterized for expression and function of bile acid-metabolizing enzymes, transporters, and cellular bile acids. Differentiated HuH-7 cells displayed a marked shift in bile acid composition and induction of cytochrome P450 (CYP) 7A1, CYP8B1, CYP3A4, and bile acid-CoA: amino acid N-acyltransferase (BAAT) mRNAs compared to control. Inhibition of taurocholate uptake and excretion after a 24-h treatment with prototypical cholestatic drugs suggests that differentiated HuH-7 cells are a suitable model to examine cholestatic hepatotoxicity.

Support person interventions to increase use of quitline services among racially diverse low-income smokers: A pilot study.

INTRODUCTION: Social support from nonsmokers may have a role in prompting smokers to use evidence-based cessation treatment. Prior studies found that an intervention for nonsmoking support persons (SPs) was effective for promoting smokers' use of free, state quitline services. This pilot study adapted and assessed feasibility of this intervention for a racially diverse, low-income population. METHODS: Single group, non-randomized design enrolling SP-smoker dyads with low income status enrolled in one of three study "waves" of 10 pairs each. Participants were recruited using flyers and in-person outreach methods. The SP intervention included a 1-session coaching call and written materials; study waves 2 and 3 also included text messaging and a monetary incentive for smokers who used quitline services. Using content analysis, the intervention was iteratively adapted based on SP feedback. Baseline measures assessed socio-demographics, dyad and tobacco use characteristics. Follow-up assessments were conducted among SPs at 1-month follow-up and among smokers at 3-months follow-up. Feasibility indicators were recruitment, retention, and SP intervention acceptability and adherence. Secondary outcomes were smokers' use of any quitline service verified by quitline staff and 7-day, point prevalence, biochemically verified smoking abstinence at 3 months. RESULTS: Recruitment of 30 dyads was feasible; in-person recruitment methods were the most successful. SPs who completed follow-up assessments found the intervention acceptable, suggesting only minor content modifications, and they perceived the quitline information as novel. But the study had some feasibility challenges (e.g., SP coaching call completion: 60% and SP study retention: 53%). At 3 months, 2 smokers (7%) had used any quitline service and 13% were biochemically confirmed smoking abstinent. CONCLUSIONS: This pilot study demonstrated feasibility of recruiting SP-smoker dyads from diverse, low-income communities. While the intervention was well received, its delivery was not feasible in this population. Results suggest that further consumer adaptation of the intervention is needed among both SPs and smokers.