Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

A Content Analysis of Reddit Users' Posts about Challenges to Contraceptive care-seeking during COVID-19-related Restrictions in the United States.

The COVID-19 pandemic in the United States caused disruptions in care seeking and delivery during the spring of 2020, including for contraceptive care. We examined how some individuals experienced and responded to barriers to accessing contraceptive care by conducting a content analysis of relevant Reddit posts. We collected 2666 posts by scraping relevant subreddits from February 1, 2020, to April 15, 2020, and filtering by selected keywords. Among the 101 posts on contraception and the COVID-19 pandemic, we explored three main themes: barriers to accessing general healthcare during the early pandemic, problems and concerns specific to contraceptive use, and attempts to navigate the obstacles to contraceptive care or use-related concerns. The Reddit posts demonstrated the disruptive force the early pandemic had on contraceptive care and provided a unique window into the concerns posters expressed on Reddit during this time. Many posters asked questions related to accessing contraception and side effects and sought reassurance from these online forums. Our results suggest that there were barriers to accessing reliable, high-quality, and evidence-based information about contraception during this disruption in care. The findings also underscore that conversational and interactive means of seeking out information are important modes for learning about and discussing contraception for some and may be especially helpful during clinic closures and other restrictions on access.

Contraceptive and abortion practices of young Ghanaian women aged 15-24: evidence from a nationally representative survey.

BACKGROUND: Young Ghanaian women experience high rates of unmet need for contraception and unintended pregnancy, and face unique barriers to accessing sexual and reproductive health services. This study provides a comprehensive national analysis of young women's contraceptive and abortion practices and needs. METHODS: In 2018, we conducted a nationally representative survey of women aged 15-49, including 1039 women aged 15-24. We used descriptive statistics, multivariable logistic and multinomial regression to compare young versus older (25-49 year-old) women's preferred contraceptive attributes, reasons for discontinuing contraception, quality of counseling, use of Primolut N-tablet, method choice correlates, and friends' and partners' influence. We also examined youth's self-reported abortion incidence, abortion methods, post-abortion care, and barriers to safe abortion. RESULTS: Among Ghanaian 15-24 year-olds who had ever had sex, one-third (32%) were using contraception. Compared to older women, they had higher desires to avoid pregnancy, lower ever use of contraception, more intermittent sexual activity, and were more likely to report pregnancies as unintended and to have recently ended a pregnancy. Young contraceptors most commonly used condoms (22%), injectables (21%), withdrawal (20%) or implants (20%); and were more likely than older women to use condoms, withdrawal, emergency contraception, and N-tablet. They valued methods for effectiveness (70%), no risk of harming health (31%) nor future fertility (26%), ease of use (20%), and no effect on menstruation (19%). Infrequent sex accounted for over half of youth contraceptive discontinuation. Relative to older women, young women's social networks were more influential on contraceptive use. The annual self-reported abortion rate among young women was 30 per thousand. Over half of young women used abortion methods obtained from non-formal providers. Among the third of young women who experienced abortion complications, 40% did not access treatment. CONCLUSIONS: Young people's intermittent sexual activity, desire for methods that do not harm their health, access barriers and provider bias, likely contribute to their greater use of coital-dependent methods. Providers should be equipped to provide confidential, non-discriminatory counseling addressing concerns about infertility, side effects and alternative methods. Use of social networks can be leveraged to educate around issues like safe abortion and correct use of N-tablet.

Young Ghanaian women can experience difficulties accessing sexual and reproductive health services, and many are not using contraception despite wanting to avoid pregnancy. To better understand their needs, we describe their preferences and behaviors around contraception and abortion. We surveyed a nationally representative sample of women aged 15­49, and compared young (15­24) versus older (25­49) women's contraceptive preferences, reasons for stopping contraception, quality of counseling, friends' and partners' influence on contraceptive use, and use of abortion. One-third of 15­24 year-olds who ever had sex were using contraception. Compared to older women, young women's pregnancies were more likely to be unintended and to end in abortion. Young women most commonly used condoms, injectables, withdrawal or implants; and were more likely than older women to use condoms, withdrawal, emergency contraception, and Primolut N-tablet. They preferred methods that were effective, did not harm their health or future fertility, were easy to use, and did not disrupt their menstrual cycle. Over half of young women who stopped contraception did so because they were not having sex regularly. Friends had more influence on contraceptive use among young women than older women. Each year on average, there were 30 abortions per 1000 young women. Over half of young women who had abortions used methods from non-formal providers, and 40% of those who had complications did not get treated. Providers should be equipped to provide confidential, non-discriminatory counseling about contraceptive side effects and options. Social networks can be used to educate women about safe abortion.

Transcriptome profiling in preadipocytes identifies long noncoding RNAs as Sam68 targets.

The KH-type RNA binding protein Sam68 is required for adipogenesis. We have previously shown that Sam68-deficient mice have a lean phenotype and are protected against dietary-induced obesity due to defects in mTOR and S6K1 alternative splicing. Herein we profiled the transcriptome of Sam68 wild type and deficient 3T3-L1 mouse preadipocytes. We identified 652 protein-coding genes and 9 ncRNAs that were significantly altered with the loss of Sam68. As expected, downregulated genes were significantly associated with GO terms linked to cell migration, motility, and fat cell differentiation, while upregulated genes were mostly associated with GO terms linked to neurogenesis. Of the lncRNAs, we identified Hotair, Mir155hg, as well as two new lncRNAs (SR-lncRNA-1 and SR-lncRNA-2) that were regulated by Sam68, and contained consensus Sam68 binding sites. RNA stability assays showed that Sam68-deficiency decreased the half-life of Hotair, and increased the half-lives of Mir155hg and SR-lncRNA-2, while the stability of SR-lncRNA-1 was unaffected. Depletion of Hotair and SR-lncRNA-1 in wild type 3T3-L1 cells led to defects in adipogenesis, whereas depletion of SR-lncRNA-2 in Sam68-deficient 3T3-L1 cells partially rescued the adipogenesis defect observed in these cells. Collectively, our findings define a new role for Sam68 as a regulator of lncRNAs during adipogenic differentiation.

The p53 status can influence the role of Sam68 in tumorigenesis.

The expression and activities of RNA binding proteins are frequently dysregulated in human cancer. Their roles, however, appears to be complex, with reports indicating both pro-tumorigenic and tumor suppressive functions. Here we show, using two classical mouse cancer models, that the role of KH-type RNA binding protein, Sam68, in tumor development can be influenced by the status of the p53 tumor suppressor. We demonstrate that in mice expressing wild type p53, Sam68-deficiency resulted in a higher incidence and malignancy of carcinogen-induced tumors, suggesting a tumor suppressive role for Sam68. In marked contrast, Sam68-haploinsufficiency significantly delayed the onset of tumors in mice lacking p53 and prolonged their survival, indicating that Sam68 accelerates the development of p53-deficient tumors. These findings provide considerable insight into a previously unknown relationship between Sam68 and the p53 tumor suppressor in tumorigenesis.

Sam68 functions as a transcriptional coactivator of the p53 tumor suppressor.

Sam68 is a known sequence-specific RNA binding protein that regulates alternative splicing events during the cell cycle and apoptosis. Sam68 has also been shown to influence transcription, but the molecular mechanism remains undefined. Herein we identify Sam68 as a transcriptional coactivator of the p53 tumor suppressor in response to DNA damage. Using CRISPR/Cas9 generated isogenic HCT116 Sam68-/- cell lines wild type or deficient for p53, we show that Sam68 is required for the efficient transactivation of p53 target genes. Consistently, Sam68 depletion caused defects in DNA damage-induced cell cycle arrest and apoptosis mediated by p53. Mechanistically, we demonstrate that Sam68 physically interacted with p53 in an RNA-dependent manner, and that this interaction was essential for the coactivator function of Sam68. Furthermore, we show that both Sam68 and p53 were recruited to promoters of p53-responsive genes, suggesting interdependence. Finally, Sam68 acted in concert with the p53 long noncoding RNA (lncRNA) target PR-lncRNA-1 for p53 recruitment, implicating a positive-feedback mechanism in which lncRNAs induced by the Sam68/p53 complex can enhance p53 transcriptional activity. These findings define a hitherto novel mechanism of action for Sam68 in governing p53 transcriptional activation, and represent the first report of Sam68 in the regulation of tumor suppressor activities.