Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

The cellular landscape of the endochondral bone during the transition to extrauterine life.

The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison with the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis and hematopoiesis. Our data also suggest dynamic inter- and intra-compartment interactions, as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

Pathogenic Aß production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1.

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-ß peptides (Aß42 and/or Aß43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aß production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aß42/Aß40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aß42/Aß40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aß production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.

Transgender Women's Experiences Using SMARTtest, a Smartphone Application to Facilitate Self- and Partner-HIV/Syphilis Testing Using the INSTI Multiplex.

We present experiences of transgender women (TW) who have sex with men with SMARTtest, a smartphone app to accompany the INSTI Multiplex®, a one-minute, dual blood-based HIV/syphilis rapid test. TW participants (N = 11) received 10 INSTI Multiplex® tests to take home for self- and/or partner-testing and installed the SMARTtest app on their phones. The SMARTtest app aimed to support INSTI Multiplex users in correctly performing the test, interpreting the results, and connecting with care following a positive HIV or syphilis screening. After 3 months, users completed in-depth interviews on their experiences. A total of 9 TW used SMARTtest with partners. App feedback was positive, but refining is necessary. Specifically, TW reported that SMARTtest is easy to use and convenient; instructions on how to use the INSTI Multiplex presented on the app were helpful to complete procedures correctly; the most frequently used feature on SMARTtest was the information on clinics that offered confirmatory testing; and participants and their partners were not concerned about app privacy but reported that this could change if INSTI Multiplex detected an HIV-positive result. Further, participants provided recommendations on how to improve SMARTtest, and changes were mostly related to features, content, functionality, navigation, and overall "look" of the app. SMARTtest is promising to facilitate INSTI Multiplex® use in TW. User feedback should be integrated in future versions.

Topologically associating domain boundaries are required for normal genome function.

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1-3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

Rapid or Immediate ART, HIV Stigma, Medical Mistrust, and Retention in Care: An Exploratory Mixed Methods Pilot Study.

Rapid or immediate antiretroviral therapy (iART) after HIV diagnosis improves linkage to care and time to viral suppression. However, iART may affect or be affected by HIV-related stigma and medical mistrust. In this mixed-methods pilot study, we examined the bi-directional role of HIV stigma, medical mistrust, and visit adherence (VA) in the context of iART in a diverse, newly diagnosed patient population. Participants were recruited from an HIV clinic in New York City and we utilized a convergent parallel design integrating quantitative data from demographic surveys, the HIV Stigma Survey (HIVSS), the Medical Mistrust Index (MMI) and electronic medical records, and qualitative data from in-depth interviews. Among the sample (N = 30), 26% (N = 8) initiated ART same-day or within 3 days, while the majority (N = 17) initiated between 4 and 30 days, and 17% (N = 5) initiated ART > 30 days. The median (range) age was 35, and most were English-speaking, Black or Hispanic men and identified as gay. Time to ART initiation was associated with time to linkage to care and time to viral suppression. Day 0-3 group's major theme was iART as stigma prevention, and they had the highest mean HIVSS, lowest MMI score, and a visit adherence of 0.86. Day 4-30 group's major theme was alleviation of internalized stigma, and they had the lowest mean HIVSS score, and highest visit adherence of 0.91. Day > 30 group's major theme was exacerbation of perceived or anticipated stigma, had the highest MMI score and a visit adherence of 0.85. iART implementation requires equitable strategies that address HIV-stigma and mistrust.

The little skate genome and the evolutionary emergence of wing-like fins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Engagement in Care Among Newly Diagnosed HIV-Positive Gay, Bisexual, and Other Men Who Have Sex With Men in the United States: Results From the Together 5,000 Study.

One-quarter of gay, bisexual, and other men who have sex with men (GBMSM) with diagnosed HIV are not engaged in HIV care. Between 2018 and 2019, 50 GBMSM completed qualitative interviews 3 months after receiving an HIV-positive result. Interviews explored barriers to and facilitators of engagement and retention in HIV testing and care. Thematic analysis revealed five major themes: (1) reason for HIV testing (e.g., self-testing), (2) linkage to care (e.g., appointment/logistic issues and social support as encouragement), (3) barriers to engagement in care (e.g., financial burden, competing priorities, and fear/stigma), (4) facilitators of engagement (e.g., financial assistance, patient-provider relationships, auxiliary support services, and health agency), and (5) PrEP as a missed prevention opportunity. Addressing individual-, social-, and policy-level barriers could improve GBMSM's engagement in HIV care. Further, capitalizing on GBMSM's health agency through partnerships with local agencies and fostering better patient-provider relationships could optimize HIV care continuity.

Negotiating Use of a Blood-Based, Dual HIV and Syphilis Test with Potential Sexual Partners Among a Sample of Cisgender Men and Transgender Women Who Have Sex with Men in New York City.

Cisgender men who have sex with men (cMSM) and transgender women (TGW) are disproportionally burdened by HIV. Among these populations, HIV partner-testing is a highly acceptable harm reduction tool. Particularly, cMSM and TGW report a stronger preference for blood-based tests that include assays for multiple STIs. However, no existing research has explored how these populations negotiate blood-based testing with sexual partners. In the SMARTtest study, 48 sexually active cMSM and TGW took home dual, blood-based HIV/Syphilis kits for self- and partner-testing. After 3 months, they completed a follow-up assessment and in-depth interviews about their experiences initiating testing. Of the 42 responding participants, 27 (64%) reported that it had been "fairly" or "very easy" to raise the idea of testing with partners. Participants predominantly employed partner-conscious communication strategies, including framing the testing proposal as a mandatory, non-personal component of their participation in a research study, gradually incorporating testing mentions into discussions about sexual health, and using the kits to facilitate joint testing. Yet, 21 (44%) participants reported having sex with at least one partner they did not ask to test. Concern regarding partner reactions emerged as a significant barrier to discussing test use; similarly, many partners were averse to taking a blood-based test in the context of a casual sexual encounter. Nonetheless, these findings suggest that dual, blood-based HIV/STI rapid tests may represent acceptable harm reduction tools among similar populations of cMSM and TGW, particularly if future partner-testing research is broadened to consider key couples' dynamics that may impact test usage.

Infrequent STI Testing in New York City Among High Risk Sexual and Gender Minority Individuals Interested In Self- and Partner-Testing.

Testing for sexually transmitted infections (STIs) remains low among sexual and gender minority populations. We assessed STI testing history using a retrospective survey among 129 HIV-negative cisgender men who have sex with men (cMSM) and transgender women who have sex with men (tWSM) who were at high risk for STI acquisition. All participants were enrolled in a parent study on self- and partner-testing for HIV and syphilis, and reported condomless anal intercourse with multiple partners during the prior 3 months. We additionally used bivariate tests to evaluate participants' STI testing by their history of using pre-exposure prophylaxis (PrEP). One-in-seven respondents (n = 18) reported having never tested for an STI, one-quarter (n = 33) had not tested in the past year, and two-thirds (n = 83) had never used PrEP. PrEP-naïve respondents were less likely to report recent STI testing (47% vs. 85%). "Routine doctor's visit" was the most prevalent reason for testing, but was less common among PrEP-naïve respondents (83% vs. 100%). Testing was remarkably low given the sample's high risk of HIV and STI infection. Findings suggest that STI testing is more frequent among those who have ever used PrEP, but the risk of selection bias warrants evaluation in a larger probability sample.

Experiences of Transgender Women Who Used a Dual HIV/Syphilis Rapid Self-test to Screen Themselves and Potential Sexual Partners (the SMARTtest Study).

HIV/syphilis self- and partner-testing may be especially appropriate for transgender women, since they shoulder a disproportionate burden of HIV, other STIs (e.g., syphilis), and report high levels of medical mistrust. The SMARTest study enrolled N = 50 sexual and gender minority individuals. The present analysis aims to understand the experiences (via in-depth interviews) of a subset of n = 11 transgender women who used INSTI Multiplex®, a combination HIV/syphilis rapid self-test, on themselves and potential sex partners. Participants reported that many partners were willing to test, and reported no testing-related violence. Most participants completed tests successfully, though gaining comfort with blood collection took time. There were no HIV-positive tests in this study; one participant and two partners reported a positive syphilis screening. All sought care. Our sample was small and results should be interpreted with caution, but indicate potential future directions for conducting research on self- and partner-testing among transgender women.

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Transgender Women's Barriers, Facilitators, and Preferences on Tailored Injection Delivery Strategies to Administer Long-Acting Injectable Cabotegravir (CAB-LA) for HIV Pre-exposure Prophylaxis (PrEP).

Long-acting injectable cabotegravir (CAB-LA) is in advanced stages of clinical trials. Under the standard protocol, CAB-LA is injected into the gluteal muscle by a healthcare provider every eight weeks. To explore transgender women's barriers and facilitators to tailored delivery strategies-including self-injection and injection in "drop-in" centers-we completed in-depth interviews with N = 15 transgender women in New York City. Participants endorsed the alternative delivery methods and the corresponding features we proposed, and expressed likes and dislikes about each. These fell into the following categories: competence (e.g., the person delivering CAB-LA must have skills to do so), convenience (e.g., CAB-LA must be easy to obtain), and privacy or fear of judgement (e.g., participants did not want to feel judged for using CAB-LA by providers or other service consumers). Findings suggest the need to offer CAB-LA to transgender women through multiple delivery protocols.

Attitudes and Perceptions About Disclosing HIV and Syphilis Results Using Smarttest, a Smartphone App Dedicated to Self- and Partner Testing.

We explored interest in disclosing test results through a smartphone app dedicated to self- and partner testing for HIV/syphilis. Fifty-nine cisgender men and transgender women each participated in an in-person survey and interview. We examined their interests in sharing test results by audience (e.g., partners, physicians) and by positive versus negative test result. Participants wanted the ability to share results, with notable interest in disclosing negative results to sexual partners and on social media and forwarding positive results to physicians. Participants envisioned smartphone sharing as a means to normalize testing, to notify partners of results, and to expedite linkage to care. Some questioned the authenticity of results shared by smartphone, while others voiced optimism that a personalized, authenticated app could ensure the security and veracity of results. Smartphone testing apps for HIV/syphilis may facilitate disclosure, partner notification, and linkage to care, but need to address concerns about the security and veracity of results.

Insurance- and medical provider-related barriers and facilitators to staying on PrEP: results from a qualitative study.

Pre-exposure prophylaxis (PrEP) is a highly effective biobehavioral strategy for preventing HIV acquisition. Although PrEP uptake has increased steadily, discontinuation rates are high among members of key populations like gay and bisexual men (GBM). Understanding the challenges that arise for PrEP users is key to better PrEP implementation and sustained use over time. We report on barriers that arose for PrEP-using GBM, as well as facilitating factors that aided PrEP persistence, with the goal of informing PrEP implementation efforts. In 2015-2016, 103 PrEP-using GBM in NYC completed qualitative interviews about their engagement with PrEP, including their experiences navigating PrEP-related medical care. Interviews were transcribed verbatim, coded, and analyzed thematically. Over half of participants (53%) received their PrEP-related care from their primary care provider (PCP), one-third (33%) from a community-based health clinic, and 13% from multiple medical providers. Emergent themes regarding the barriers and facilitators to PrEP persistence fell into two categories: insurance- and medical appointment-related barriers and facilitators to continued PrEP use. The experiences of PrEP-using GBM can provide useful insights for providers, program developers, and policymakers aiming to improve the implementation of PrEP. To support PrEP persistence, reliable insurance coverage, cost-assistance, and easy appointment scheduling are key to maintenance. Removing insurance- and appointment-related barriers to persistence may prove essential for sustaining use among GBM.

Experiences Receiving HIV-Positive Results by Phone: Acceptability and Implications for Clinical and Behavioral Research.

Improving HIV testing rates and increasing early detection among men who have sex with men (MSM) are critical strategies for enhancing overall health and decreasing HIV transmission. Remote testing and phone delivery of HIV test results may reduce barriers such as geographic isolation or HIV-related stigma. In 2018-19, 50 MSM completed qualitative interviews about their experience receiving a positive HIV test result via phone through their participation in a research study that included remote HIV testing. Interview topics included the acceptability of, and concerns about, phone delivery of HIV results, as well as suggestions for improvement. Interviews were transcribed, coded, and analysed using an inductive thematic approach. Overall, participants reported high acceptability of phone delivery of HIV-positive results. Participants praised the support and information provided by study staff. Benefits identified included increased convenience compared to in-person medical visits, allowing participants to emotionally process their test results privately, as well as receiving the results from supportive and responsive staff members. A few participants indicated drawbacks to phone-based HIV test result delivery, such as logistical concerns about receiving a phone call during the day (e.g., while at work), reduced confidentiality, and the lack of in-person emotional support. Overall, participants described phone delivery of positive HIV-results as acceptable. At-home testing with phone delivery has the potential to increase HIV testing access, especially to geographically isolated or medically underserved patients.

Willingness to Distribute HIV Self-Testing Kits to Recent Sex Partners Among HIV-Negative Gay and Bisexual Men and an Examination of Free-Response Data from Young Men Participating in the Nationwide Cohort.

Sexual minority men (SMM)-and young SMM in particular-are disproportionately affected by HIV. Secondary distribution of HIV self-testing (HIVST) kits-wherein patients deliver kits to partners-is a novel strategy to increase HIV testing access. Using quantitative data, we assessed willingness to distribute HIVST kits to recent sex partners among a U.S. national sample of HIV-negative SMM (n = 786). A thematic analysis was then conducted to identify barriers and facilitators of kit distribution to partners among young SMM (M age = 25.75 years; range: 20-29; n = 165). Overall, 93.5% of SMM (and 97.0% of young SMM) were willing to deliver HIVST kits to recent sex partners. Among young SMM, main barriers and facilitators included concerns about their partners' reaction, availability and cost, protection beliefs for others, HIV stigma and perceived infidelity, packaging and support, communication skill needs, inability to contact partners, requests for anonymity, and dyadic self-testing with their partners. The findings highlight the need for supportive intervention strategies such as informational content for HIVST, using motivational interviewing when providing the testing kits to index clients and providing skills-based training through role-playing exercises. Secondary distribution of HIVST kits through index patients is a potentially acceptable approach that could be used to expand access to HIV testing and aid in efforts to end the HIV epidemic in the U.S.

Using an HIV Self-test Kit to Test a Partner: Attitudes and Preferences Among High-Risk Populations.

For those most at risk of contracting HIV, new strategies for preventing transmission and increasing testing are needed. As part of a multi-site, randomized, controlled trial, we explored attitudes and preferences among 272 HIV-negative men who have sex with men and HIV-negative transgender women using an HIV self-testing (HIVST) kit to test partners. Less than one quarter had previously self-tested with HIVST kits (21.7%) and few had partner-tested (4.8%). Most preferred gum swab (96%) over fingerprick tests (69%), but would prefer a blood test if it gave results for other sexually transmitted infections (STIs) (86%). Five percent reported difficulties performing the test, four percent with storage, and 26% with portability. Ninety-three percent reported likelihood of using HIVST to test partners in future, but only 3% were willing to pay the current price. Efforts to improve HIVST uptake should focus on incorporating testing for other STIs, reducing test kit size, and reducing cost.

RESUMEN: Se necesitan nuevas estrategias para prevenir la transmisión del VIH e incrementar el testeo para aquellos con mayor riesgo al contagio. Como parte de un ensayo controlado aleatorio (ECA) multicentrico, exploramos las actitudes y preferencias entre 272 hombres VIH-negativos que tienen sexo con hombres y mujeres transgenero VIH-negativos que utilizaron kits del auto-test para el VIH (HIVST) para testear a sus parejas. Menos de una cuarta parte de los participantes había utilizado HIVST para auto-testearse (21.7%), y solo pocos para testear a sus parejas (4.8%) antes del comienzo del estudio. El hisopo bucal fue preferido (96%) sobre una prueba con un pinchazo de sangre (69%) por la mayoría de los participantes pero estos preferirían la prueba de sangre si puedieran obtener resultados para otras enfermedades de transmisión sexual (ETS) (86%). Cinco por ciento reportó problemas con el uso de la prueba, 4% con el almacenamiento, y el 26% con la portabilidad. Noventa y tres por ciento reportó que utilizarían HIVST para testear a sus parejas en el futuro, pero solo 3% estaba dispuesto a pagar el precio actual. Los futuros esfuerzos para mejorar la adopción del HIVST deberían enfocarse en incorporar otras pruebas de ETS, en reducir el tamaño del kit y en reducir los costos.