Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.

Contraceptive use, menstrual resumption, and experience of pregnancy and birth among girls and young women in an internally displaced persons camp in Northeastern Nigeria.

In Northeastern Nigeria 600,000 internally displaced girls and women need sexual and reproductive health and rights (SRHR) services. We examined the relationships between contraceptive use, menstrual resumption, and pregnancy and birth experiences among girls (ages 15-19) and young women (ages 20-24) in an IDP camp. Data are from a cross-sectional survey collected using three-stage cluster sampling; the analytic sample is 480. Data were analyzed in Stata 14 using logistic regression models. Sixty-three percent of respondents had ever had sex and over half were currently sexually active. Current contraceptive use was 8% and 47% had ever been pregnant. Older respondents and those who had ever had sex were more likely to have heard of a contraceptive method and current use was higher for women with 5 or more births. These findings indicate a need for better contraceptive education for girls before sexual activity and promotion of contraception that accounts for fertility preferences in this setting.

Strengthening local capacity for abortion-related research in contexts with highly restrictive abortion laws: The case of STARS in Mali.

Strong local abortion research capacity is missing in many African countries. We report on the Strengthening Abortion Research Capacity in sub-Saharan Africa (STARS) program, an ongoing initiative to strengthen local capacity for abortion research in Mali, West Africa. We highlight the background, context, and methodology of the initiative as well as its achievements, challenges, and emerging lessons. Within a short time, STARS has initiated some key studies on abortion in Mali and created a much-needed platform for nurturing the country's next generation of abortion researchers, institutionalizing abortion research, increasing the quantity and quality of locally generated evidence on abortion, and facilitating evidence-informed abortion policy and programmatic action. The program's learning-by-doing approach has boosted the skills of individual researchers while also enhancing institution-based abortion and sexual and reproductive health and rights (SRHR) research expertise in Mali. Although STARS' capacity to deliver its mandate over time is evident, ultimate results will depend on the sustained commitment of funders to the program in the full realization that capacity building requires long-term investment and support for it to fully bear fruits.

Provision and uptake of sexual and reproductive health services during the COVID-19 pandemic: The case of Mali.

A qualitative study assessed the effects of the COVID-19 epidemic on Malian sexual and reproductive health services. Sexual and reproductive health (SRHR) providers in 25 purposively selected public health facilities in urban Bamako, rural Kita (western Mali) and Koutiala (southeast Mali) were interviewed. Disruptions within SRH supply, staffing, the prioritization of SRHR services, and patients' ability to seek, obtain and pay for services were reported across urban and rural settings at all levels of public health care, and by all cadres of SRHR providers. Most facilities in the study areas sustained some SRHR services at the height of the COVID-19 epidemic through innovative outreach and phone-based consultations. This study offers critical lessons for SRHR service provision during future waves of the pandemic or during periods of comparable emergency.

From nerve net to nerve ring, nerve cord and brain--evolution of the nervous system.

The puzzle of how complex nervous systems emerged remains unsolved. Comparative studies of neurodevelopment in cnidarians and bilaterians suggest that this process began with distinct integration centres that evolved on opposite ends of an initial nerve net. The 'apical nervous system' controlled general body physiology, and the 'blastoporal nervous system' coordinated feeding movements and locomotion. We propose that expansion, integration and fusion of these centres gave rise to the bilaterian nerve cord and brain.

Men's support for abortion in Upper East and Upper West Ghana.

The second leading cause of maternal mortality in Ghana is unsafe abortion. Research in Ghana shows that men's support influences women's use of safe abortion services. The aim of this study was to understand what men know about abortion, why they support their partners to seek an abortion, and to identify effective ways to reach men with abortion information. We conducted eleven focus groups and ten in-depth interviews with men of reproductive age in rural Ghana. Inclusion criteria were written consent, age and marital status. Focus groups and interviews were conducted in local languages using a semi-structured guide. Focus groups and interviews were audiotaped, transcribed, translated, computerised and coded for analysis. Most men knew the difference between unsafe and safe abortion and would support their partner to have an abortion. Messages that reinforce safe abortion as acceptable and that address fears of death or barrenness should be developed to educate men about safe abortion. Multiple channels to communicate these messages should be used and include pictures, video or audio for those men who cannot read. Through an intensive intervention inclusive of men, women's access to safe abortion services can be improved in Ghana.

Women's experiences with medication for menstrual regulation in Bangladesh.

Menstrual regulation has been legal in Bangladesh since 1974, but the use of medication for menstrual regulation is new. In this study, we sought to understand women's experiences using medication for menstrual regulation in Bangladesh. We conducted 20 in-depth interviews with rural and urban women between December 2013 and February 2014. All interviews were audiotaped, transcribed, translated, computer recorded and coded for analysis. The majority of women in our study had had positive experiences with medication for menstrual regulation and successful outcomes, regardless of whether they obtained their medication from medicine sellers/pharmacies, doctors or clinics. Women were strongly influenced by health providers when deciding which method to use. There is a need to educate not only women of reproductive age, but also communities as a whole, about medication for menstrual regulation, with a particular emphasis on cost and branding the medication. Continued efforts to improve counselling by providers about the dose, medication and side-effects of medication for menstrual regulation, along with education of the community about medication as an option for menstrual regulation, will help to de-stigmatise the procedure and the women who seek it.

Larval body patterning and apical organs are conserved in animal evolution.

BACKGROUND: Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data. RESULTS: To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3ß antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the 'apical plate'. We also unraveled a molecular signature of the apical organ--devoid of six3 but expressing foxj, irx, nkx3 and hox--that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla. CONCLUSIONS: The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ--comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells--was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis.

The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral-aboral (O-Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O-Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3ß inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O-Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal-vegetal patterning at earlier stages of anthozoan development.

Women's perceptions about abortion in their communities: perspectives from western Kenya.

Unsafe abortion in Kenya is a leading cause of maternal morbidity and mortality. In October 2012, we sought to understand the methods married women aged 24-49 and young, unmarried women aged ≤ 20 used to induce abortion, the providers they utilized and the social, economic and cultural norms that influenced women's access to safe abortion services in Bungoma and Trans Nzoia counties in western Kenya. We conducted five focus groups with young women and five with married women in rural and urban communities in each county. We trained local facilitators to conduct the focus groups in Swahili or English. All focus groups were audiotaped, transcribed, translated, computerized, and coded for analysis. Abortion outside public health facilities was mentioned frequently. Because of the need for secrecy to avoid condemnation, uncertainty about the law, and perceived higher cost of safer abortion methods, women sought unsafe abortions from community midwives, drug sellers and/or untrained providers at lower cost. Many groups believed that abortion was safer at higher gestational ages, but that there was no such thing as a safe abortion method. Our aim was to inform the design of a community-based intervention on safe abortion for women. Barriers to seeking safe services such as high cost, perceived illegality, and fear of insults and abuse at public facilities among both age groups must be addressed.

Abortion services for sex workers in Uganda: successful strategies in an urban clinic.

Sex workers' need for safe abortion services in Uganda is greater than that of the population of women of reproductive age because of their number of sexual contacts, the inconsistent use of contraception and their increased risk of forced sex, rape or other forms of physical and sexual violence. We sought to understand sex workers' experiences with induced abortion services or post-abortion care (PAC) at an urban clinic in Uganda. We conducted nine in-depth interviews with sex workers. All in-depth interviews were audiotaped, transcribed, translated, computer recorded and coded for analysis. We identified several important programmatic considerations for safe abortion services for sex workers. Most important is creating community-level interventions in which women can speak openly about abortion, creating a support network among sex workers, training peer educators, and making available a community outreach educator and community outreach workshops on abortion. At the health facility, it is important for service providers to treat sex workers with care and respect, allow sex workers to be accompanied to the health facility and guarantee confidentiality. These programmatic elements help sex workers to access safe abortion services and should be tried with all women of reproductive age to improve women's access to safe abortion in Uganda.

Postpartum family planning service provision in Durban, South Africa: client and provider perspectives.

Researchers in Sub-Saharan Africa have found that health facility factors influence client contraceptive use. We sought to understand how client-provider interactions, discussions of side effects, and HIV status influence women's contraceptive use postpartum. We conducted in-depth interviews with eight HIV negative clients and six HIV positive clients in Zulu, and with five nurses in English. Interviews were translated and transcribed into English. We created a codebook and coded all transcripts. Nurses and clients reported limited time to discuss contraception, side effects, and HIV. Nurses did not comply with national contraceptive policies and created unnecessary barriers to contraceptive use.

Single cell atlas of Xenoturbella bocki highlights limited cell-type complexity.

Phylogenetic analyses over the last two decades have united a few small, and previously orphan clades, the nematodermatids, acoels and xenoturbelids, into the phylum Xenacoelomorpha. Some phylogenetic analyses support a sister relationship between Xenacoelomorpha and Ambulacraria (Xenambulacraria), while others suggest that Xenacoelomorpha may be sister to the rest of the Bilateria (Nephrozoa). An understanding of the cell type complements of Xenacoelomorphs is essential to assessing these alternatives as well as to our broader understanding of bilaterian cell type evolution. Employing whole organism single-cell RNA-seq in the marine xenacoelomorph worm Xenoturbella bocki, we show that Xenambulacrarian nerve nets share regulatory features and a peptidergic identity with those found in cnidarians and protostomes and more broadly share muscle and gland cell similarities with other metazoans. Taken together, these data are consistent with broad homologies of animal gland, muscle, and neurons as well as more specific affinities between Xenoturbella and acoel gut and epidermal tissues, consistent with the monophyly of Xenacoelomorpha.

Functional roles of Notch signaling in the cnidarian Nematostella vectensis.

Notch signaling is among the oldest of known Metazoan signaling pathways and is used in a multitude of developmental contexts to effect cellular differentiation, specification and the maintenance of stem cell state. Here we report the isolation and expression of the canonical Notch signaling pathway in the early branching metazoan Nematostella vectensis (Anthozoa, Cnidaria) during embryonic and larval development. We have used pharmacological treatment, morpholino knockdown, and dominant negative misexpression experiments to demonstrate that Notch signaling acts to mediate cnidogenesis, the development of cnidarian-specific neural effecter cells. Notch signaling often results in the transcriptional activation of NvHes genes, a conserved family of bHLH transcription factors. A loss of Notch signaling through use of pharmacological inhibition or knock-down of the Notch effecter gene Suppressor of Hairless Su(H) similarly results in a loss of cnidocyte cell fate. We also provide evidence that Notch signaling is responsible for certain aspects of neurogenesis in developing N. vectensis planula in which disruption of Notch cleavage via the pharmacological agent DAPT results in increased expression of neural marker genes in vivo. This data suggests that Notch signaling acting on components of the developing nervous system is an ancient role of this pathway. The shared requirement of Notch signaling for the development of both cnidocytes and neurons further supports the hypothesis that cnidocytes and neurons share common origins as multifunctional sensory-effecter cells.

Shifting landscapes: the role of 3D genomic organizations in gene regulatory strategies.

3D genome folding enables the physical storage of chromosomes into the compact volume of a cell's nucleus, allows for the accurate segregation of chromatin to daughter cells, and has been shown to be tightly coupled to the way in which genetic information is converted into transcriptional programs [1-3]. Importantly, this link between chromatin architecture and gene regulation is a selectable feature in which modifications to chromatin organization accompany, or perhaps even drive the establishment of new regulatory strategies with enduring impacts on animal body plan complexity. Here, we discuss the nature of different 3D genome folding systems found across the tree of life, with particular emphasis on metazoans, and the relative influence of these systems on gene regulation. We suggest how the properties of these folding systems have influenced regulatory strategies employed by different lineages and may have catalyzed the partitioning and specialization of genetic programs that enabled multicellularity and organ-grade body plan complexity.

Epidemiological Profile of Intimate Partner Homicides of Sexual and Gender Minority Women in the United States, 2003 to 2017.

Sexual and gender minority (SGM) women are among the many victims killed by intimate partner homicide (IPH) each year, though the differences between different SGM groups (and how these groups compare to non-SGM IPH) have not been well established. The objective of this article was to identify practicable, correlated risk factors of IPH of SGM women that may have utility in prevention of future IPH among these populations in the U.S. Homicide data from the National Violent Death Reporting System spanning 2003 to 2017 were used to identify a profile of IPH specific to SGM women compared to women who were neither sexual nor gender minorities. Situational and individual characteristics significantly differentiated sexual minority (SM) women from non-SGM women victims of IPH, including substance abuse history (adjusted odds ratio [AOR] = 4.80 [2.42, 9.51]), having themselves used a weapon during the incident (AOR = 3.63 [1.44, 9.16]), and the type of weapon(s) used, such as firearms (AOR = 0.61 [0.40, 0.91]), with notably different differentiating characteristics for gender minority (GM) women (vs. non-SGM women) such as the likelihood that the victim was known to have experienced interpersonal violence victimization in the previous month (AOR = 0.50 [0.07, 3.67]). Lesbian and bisexual women homicide victims were far more likely to have been killed via IPH than non-SGM women (AOR for Black SM women = 7.84 [3.65, 16.88], AOR for White SM women = 2.30 [1.03, 5.17]). There was no corresponding difference for GM women victims, whose likelihood of being killed by an intimate partner was similar to that of non-SGM women. Based on these findings, actionable public health recommendations-centered around evidence that neither "all women" nor "all LGBTQ people" are appropriate intimate partner violence prevention umbrellas-are proposed.

A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering.

The gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals. In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors. We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell-cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies.

Specificity of associations between bacteria and the coral Pocillopora meandrina during early development.

Relationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coral Pocillopora meandrina was investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of the Alphaproteobacteria, a Pseudoalteromonas species of the Gammaproteobacteria, and a Synechococcus species of the Cyanobacteria phylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination of P. meandrina planulae by fluorescence in situ hybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains of Pseudoalteromonas and Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade and Synechococcus did not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.

Fertility intent and contraceptive decision-making among HIV positive and negative antenatal clinic attendees in Durban, South Africa.

We explored contraceptive decision-making among South African antenatal clinic attendees, fertility intent post-HIV diagnosis, and women's experiences at government health facilities. Data are from in-depth interviews with HIV negative and HIV positive women. We interviewed women in Zulu; interviews were recorded, transcribed, and translated. We conducted qualitative analyses of interviews. Women were the dominant decisionmakers about contraceptive use, whether they involved their partners or not. A majority of women obtained a contraceptive method at a government facility; however, several women were unable to attain sterilizations. Women were presented with limited contraceptive options and were not always able to access services.

Single-Cell Transcriptomic Analysis in the Regenerating Cnidarian Nematostella vectensis.

Cnidarians have historically served as excellent laboratory models for regenerative development given their capacity to regrow large portions of the adult organism. This capacity is notably absent or poorly developed in the powerful genetic laboratory models Drosophila, C. elegans, and mouse. Increasingly, development of genetic and genomic resources and the application of next-generation sequencing-based techniques in cnidarian systems has further expanded the potential of cnidarian regenerative models. Here, we present a workflow for the characterization of the regenerative response in the sea anemone Nematostella vectensis utilizing fluorescence-activated cell sorting and a plate-based single-cell RNA-sequencing pipeline. This approach can characterize the transcriptional response during regeneration in distinct populations of cells, thus providing a quantitative view of a whole organism process at cellular resolution.