Cytokinin-CLAVATA cross-talk is an ancient mechanism regulating shoot meristem homeostasis in land plants.

SignificancePlants grow from their tips. The gametophore (shoot-like organ) tip of the moss Physcomitrium patens is a single cell that performs the same functions as those of multicellular flowering plants, producing the cells that make leaves and regenerating new stem cells to maintain the shoot tip. Several pathways, including CLAVATA and cytokinin hormonal signaling, regulate stem cell abundance in flowering plants and in mosses, although the mechanisms whereby these pathways regulate stem cell abundance and their conservation between these plant lineages is poorly understood. Using moss, we investigated how PpCLAVATA and cytokinin signaling interact. Overall, we found evidence that PpCLAVATA and cytokinin signaling interact similarly in moss and flowering plants, despite their distinct anatomies, life cycles, and evolutionary distance.

Genetic analyses of embryo homology and ontogeny in the model grass Zea mays subsp. mays.

The homology of the single cotyledon of grasses and the ontogeny of the scutellum and coleoptile as the initial, highly modified structures of the grass embryo are investigated using leaf developmental genetics and targeted transcript analyses in the model grass Zea mays subsp. mays. Transcripts of leaf developmental genes are identified in both the initiating scutellum and the coleoptile, while mutations disrupting mediolateral leaf development also disrupt scutellum and coleoptile morphology, suggesting that these grass-specific organs are modified leaves. Higher-order mutations in WUSCHEL-LIKE HOMEOBOX3 (WOX3) genes, involved in mediolateral patterning of plant lateral organs, inform a model for the fusion of coleoptilar margins during maize embryo development. Genetic, RNA-targeting, and morphological evidence supports models for cotyledon evolution where the scutellum and coleoptile, respectively, comprise the distal and proximal domains of the highly modified, single grass cotyledon.

Plant homeodomain proteins provide a mechanism for how leaves grow wide.

The mechanisms whereby leaf anlagen undergo proliferative growth and expansion to form wide, flat leaves are unclear. The maize gene NARROWSHEATH1 (NS1) is a WUSCHEL-related homeobox3 (WOX3) homolog expressed at the margins of leaf primordia, and is required for mediolateral outgrowth. To investigate the mechanisms of NS1 function, we used chromatin immunoprecipitation and laser-microdissection RNA-seq of leaf primordial margins to identify gene targets bound and modulated by NS1. Microscopic analyses of cell division and gene expression in expanding leaves, and reverse genetic analyses of homologous NS1 target genes in Arabidopsis, reveal that NS1 controls mediolateral outgrowth by repression of a growth inhibitor and promotion of cell division at primordial leaf margins. Intriguingly, homologous WOX gene products are expressed in stem cell-organizing centers and traffic to adjoining cells to activate stem-cell identity non-autonomously. In contrast, WOX3/NS1 does not traffic, and stimulates cell divisions in the same cells in which it is transcribed.

Integrating GWAS and TWAS to elucidate the genetic architecture of maize leaf cuticular conductance.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

The ratio of auxin to cytokinin controls leaf development and meristem initiation in Physcomitrium patens.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, to those in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens, especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin-insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

Multi-media teacher training and HIV-related stigma among primary and secondary school teachers in Western Kenya.

ABSTRACTHIV stigma is associated with delayed HIV disclosure and worse clinical outcomes for adolescents living with HIV (ALWH). Teachers critically influence school environments, but are understudied in terms of HIV stigma. We implemented a school-level, cluster-randomized trial to assess the impact of a one-day multi-media training on the knowledge, attitudes and beliefs (K/A/B) of school teachers in western Kenya. Teachers' K/A/B were evaluated at baseline and six months. Additionally, we assessed stigma with ALWH enrolled in the included schools to explore the impact of the training. Teachers (N = 311) and ALWH (N = 19) were enrolled from 10 primary and 10 secondary schools. The intervention and control groups did not significantly differ in overall stigma score (mean 1.83 vs. 1.84; adjusted difference, 0.18 [95% CI, -0.082 to 0.045]) at six months; however, we found a trend towards improvement in overall stigma score and a significant difference in the community discrimination sub-scale among secondary school teachers (mean 3.02 vs. 3.19; adjusted difference, -0.166 [95% CI, -0.310 to -0.022]). ALWH reported few experiences of discrimination, but emphasized keeping their HIV status secret (84%). The teacher-training reduced secondary school teacher perceptions of community-level stigma, but did not impact individual attitudes or beliefs..

"It is like an umbrella covering you, yet it does not protect you from the rain": a mixed methods study of insurance affordability, coverage, and financial protection in rural western Kenya.

Countries in Sub-Saharan Africa are increasingly adopting mandatory social health insurance programs. In Kenya, mandatory social health insurance is being implemented through the national health insurer, the National Hospital Insurance Fund (NHIF), but the level of coverage, affordability and financial risk protection provided by health insurance, especially for rural informal households, is unclear. This study provides as assessment of affordability of NHIF premiums, the need for financial risk protection, and the extent of financial protection provided by NHIF among rural informal workers in western Kenya.Methods We conducted a mixed methods study with a cross-sectional household survey (n = 1773), in-depth household interviews (n = 36), and 6 focus group discussions (FGDs) with community stakeholders in rural western Kenya. Health insurance status was self-reported and households were categorized into insured and uninsured. Using survey data, we calculated the affordability of health insurance (unaffordability was defined as the monthly premium being > 5% of total household expenditures), out of pocket expenditures (OOP) on healthcare and its impact on impoverishment, and incidence of catastrophic health expenditures (CHE). Logistic regression was used to assess household characteristics associated with CHE.Results Only 12% of households reported having health insurance and was unaffordable for the majority of households, both insured (60%) and uninsured (80%). Rural households spent an average of 12% of their household budget on OOP, with both insured and uninsured households reporting high OOP spending and similar levels of impoverishment due to OOP. Overall, 12% of households experienced CHE, with uninsured households more likely to experience CHE. Participants expressed concerns about value of health insurance given its cost, availability and quality of services, and financial protection relative to other social and economic household needs. Households resulted to borrowing, fundraising, taking short term loans and selling family assets to meet healthcare costs.Conclusion Health insurance coverage was low among rural informal sector households in western Kenya, with health insurance premiums being unaffordable to most households. Even among insured households, we found high levels of OOP and CHE. Our results suggest that significant reforms of NHIF and health system are required to provide adequate health services and financial risk protection for rural informal households in Kenya.

Plant stem-cell organization and differentiation at single-cell resolution.

Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing (scRNA-seq) to achieve an unbiased characterization of the transcriptional landscape of the maize shoot stem-cell niche and its differentiating cellular descendants. Stem cells housed in the SAM tip are engaged in genome integrity maintenance and exhibit a low rate of cell division, consistent with their contributions to germline and somatic cell fates. Surprisingly, we find no evidence for a canonical stem-cell organizing center subtending these cells. In addition, trajectory inference was used to trace the gene expression changes that accompany cell differentiation, revealing that ectopic expression of KNOTTED1 (KN1) accelerates cell differentiation and promotes development of the sheathing maize leaf base. These single-cell transcriptomic analyses of the shoot apex yield insight into the processes of stem-cell function and cell-fate acquisition in the maize seedling and provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis at the cellular level.

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves.

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

A high-resolution gene expression atlas links dedicated meristem genes to key architectural traits.

The shoot apical meristem (SAM) orchestrates the balance between stem cell proliferation and organ initiation essential for postembryonic shoot growth. Meristems show a striking diversity in shape and size. How this morphological diversity relates to variation in plant architecture and the molecular circuitries driving it are unclear. By generating a high-resolution gene expression atlas of the vegetative maize shoot apex, we show here that distinct sets of genes govern the regulation and identity of stem cells in maize versus Arabidopsis. Cell identities in the maize SAM reflect the combinatorial activity of transcription factors (TFs) that drive the preferential, differential expression of individual members within gene families functioning in a plethora of cellular processes. Subfunctionalization thus emerges as a fundamental feature underlying cell identity. Moreover, we show that adult plant characters are, to a significant degree, regulated by gene circuitries acting in the SAM, with natural variation modulating agronomically important architectural traits enriched specifically near dynamically expressed SAM genes and the TFs that regulate them. Besides unique mechanisms of maize stem cell regulation, our atlas thus identifies key new targets for crop improvement.

A Pilot Study of a Mobile Intervention to Support Mental Health and Adherence Among Adolescents Living with HIV in Western Kenya.

Mobile technologies represent potentially novel and scalable intervention delivery platforms for adolescents living with HIV (ALWH) in low- and middle-income countries. We conducted a prospective, mixed methods pilot study to evaluate the acceptability and feasibility of the WhatsApp® platform to deliver individual counseling services and facilitate peer support for ALWH in western Kenya. Thirty ALWH (17 female, mean age 15.4) on ART, engaged in HIV care and aware of their status, were enrolled. After 6 months, participants described their experiences with the intervention. Treatment adherence, stigma, and mental and behavioral health were assessed prospectively. Participants reported overall positive experiences and indicated that the platform encouraged peer network development. They endorsed potential benefits for treatment adherence, stigma reduction, and mental and behavioral health. All participants supported intervention expansion. In western Kenya, WhatsApp® was an acceptable and feasible platform for mobile counseling and peer support for ALWH.

A systematic review of interventions to reduce HIV-related stigma among primary and secondary school teachers.

HIV/AIDS-related stigma (HIV stigma) affects every aspect of adolescents' HIV management. Adolescents living with HIV (ALWH) are particularly vulnerable in schools where they have described experiencing HIV stigma. Teachers play a significant role in their students' lives. Stigmatizing attitudes or behaviors by teachers not only impact ALWH directly, but may influence the attitudes and behaviors of their peers. There is a dearth of literature exploring interventions to address HIV stigma in school-based settings. The objective of this review is to examine interventions to reduce HIV stigma among teachers globally. To conduct this systematic review, we used the PRISMA guidelines. Two articles met the inclusion criteria. Both eligible studies aimed to reduce HIV stigma among teachers or teachers in training through teacher training interventions in sub-Saharan Africa. The interventions included an interactive CD-ROM, a 2-day workshop, and peer facilitated workshops. Both studies demonstrated a significant decrease in HIV stigma in at least one study measure. Findings from this review are inconclusive. There is evidence to suggest that interventions can successfully decrease HIV stigma among teachers, but it is very limited. More research is needed in order to develop, implement, and evaluate stigma reducing interventions in the classroom.

Network analyses identify a transcriptomic proximodistal prepattern in the maize leaf primordium.

The formation of developmental boundaries is a common feature of multicellular plants and animals, and impacts the initiation, structure and function of all organs. Maize leaves comprise a proximal sheath that encloses the stem, and a distal photosynthetic blade that projects away from the plant axis. An epidermally derived ligule and a joint-like auricle develop at the blade/sheath boundary of maize leaves. Mutations disturbing the ligule/auricle region disrupt leaf patterning and impact plant architecture, yet it is unclear how this developmental boundary is established. Targeted microdissection followed by transcriptomic analyses of young leaf primordia were utilized to construct a co-expression network associated with development of the blade/sheath boundary. Evidence is presented for proximodistal gradients of gene expression that establish a prepatterned transcriptomic boundary in young leaf primordia, before the morphological initiation of the blade/sheath boundary in older leaves. This work presents a conceptual model for spatiotemporal patterning of proximodistal leaf domains, and provides a rich resource of candidate gene interactions for future investigations of the mechanisms of blade/sheath boundary formation in maize.

'It was hell in the community': a qualitative study of maternal and child health care during health care worker strikes in Kenya.

BACKGROUND: Health care workers in Kenya have launched major strikes in the public health sector in the past decade but the impact of strikes on health systems is under-explored. We conducted a qualitative study to investigate maternal and child health care and services during nationwide strikes by health care workers in 2017 from the perspective of pregnant women, community health volunteers (CHVs), and health facility managers. METHODS: We conducted in-depth interviews and focus group discussions (FGDs) with three populations: women who were pregnant in 2017, CHVs, and health facility managers. Women who were pregnant in 2017 were part of a previous study. All participants were recruited using convenience sampling from a single County in western Kenya. Interviews and FGDs were conducted in English or Kiswahili using semi-structured guides that probed women's pregnancy experiences and maternal and child health services in 2017. Interviews and FGDs were audio-recorded, translated, and transcribed. Content analysis followed a thematic framework approach using deductive and inductive approaches. RESULTS: Forty-three women and 22 CHVs participated in 4 FGDs and 3 FGDs, respectively, and 8 health facility managers participated in interviews. CHVs and health facility managers were majority female (80%). Participants reported that strikes by health care workers significantly impacted the availability and quality of maternal and child health services in 2017 and had indirect economic effects due to households paying for services in the private sector. Participants felt it was the poor, particularly poor women, who were most affected since they were more likely to rely on public services, while CHVs highlighted their own poor working conditions in response to strikes by physicians and nurses. Strikes strained relationships and trust between communities and the health system that were identified as essential to maternal and child health care. CONCLUSION: We found that the impacts of strikes by health care workers in 2017 extended beyond negative health and economic effects and exacerbated fundamental inequities in the health system. While this study was conducted in one County, our findings suggest several potential avenues for strengthening maternal and child health care in Kenya that were highlighted by nationwide strikes in 2017.

The role of community health workers in the surgical cascade: a scoping review.

BACKGROUND: Community health workers (CHWs) can increase access to various primary healthcare services; however, their potential for improving surgical care is under-explored. We sought to assess the role of CHWs in the surgical cascade, defined as disease screening, linkage to operative care, and post-operative care. Given the well-described literature on CHWs and screening, we focused on the latter two steps of the surgical cascade. METHODS: We conducted a scoping review of the peer-reviewed literature. We searched for studies published in any language from January 1, 2000 to May 1, 2020 using electronic literature databases including Pubmed/MEDLINE, Web of Science, SCOPUS, and Google Scholar. We included articles on CHW involvement in linkage to operative care and/or post-operative surgical care. Narrative and descriptive methods were used to analyze the data. RESULTS: The initial search identified 145 articles relevant to steps in the surgical cascade. Ten studies met our inclusion criteria and were included for review. In linkage to care, CHWs helped increase surgical enrollment, provide resources for vulnerable patients, and build trust in healthcare services. Post-operatively, CHWs acted as effective monitors for surgical-site infections and provided socially isolated patients with support and linkage to additional services. The complex and wide-ranging needs of surgical patients illustrated the need to view surgical care as a continuum rather than a singular operative event. CONCLUSION: While the current literature is limited, CHWs were able to maneuver complex medical, cultural, and social barriers to surgical care by linking patients to counseling, education, and community resources, as well as post-operative infection prevention services. Future studies would benefit from more rigorous study designs and larger sample sizes to further elucidate the role CHWs can serve in the surgical cascade.

Perspectives of education sector stakeholders on a teacher training module to reduce HIV/AIDS stigma in Western Kenya.

BACKGROUND: For adolescents living with HIV (ALWH), school may be the most important but understudied social sphere related to HIV stigma. Teachers are role models in the classroom and within the community, and their attitudes and behavior towards people living with HIV may have critical psychosocial and treatment ramifications. Altering teachers' knowledge, attitudes and beliefs (K/A/B) about HIV could reduce the stigmatizing content within their teaching, classrooms and school, improving the environment for ALWH. METHODS: We developed a one-day teacher training module to enrich teacher K/A/B that included lecture presentations, HIV films and educational animation, structured instructions for teacher role play scenarios, and a question-and-answer session facilitated by a trained ALWH peer educator. We also conducted key informant interviews with education sector subject matter experts (SMEs), including education officers, county commissioners and head teachers to review and provide feedback on the teacher training module. RESULTS: We assembled an adolescent community advisory board and recruited 50 SMEs to review the training module and provide feedback. All SME participants stressed the importance and need for interventions to reduce stigma in the classroom, highlighting their own experiences observing stigmatizing behaviors in the community. The participants perceived the training as culturally relevant and easy to understand and had minor suggestions for improvement, including using image-based resources and brighter colors for ease of reading. All participants thought that the training should be expanded outside of the schools, as all people in a community have a role in the reduction of HIV stigma, and offered suggestions for other settings for implementation. CONCLUSION: Data from interviews with education sector stakeholders demonstrate that our process for developing a culturally appropriate multi-media intervention to reduce HIV stigma in the schools was feasible.

A retrospective study of the impact of health worker strikes on maternal and child health care utilization in western Kenya.

BACKGROUND: There have been dozens of strikes by health workers in Kenya in the past decade, but there are few studies of their impact on maternal and child health services and outcomes. We conducted a retrospective survey study to assess the impact of nationwide strikes by health workers in 2017 on utilization of maternal and child health services in western Kenya. METHODS: We utilized a parent study to enroll women who were pregnant in 2017 when there were prolonged strikes by health workers ("strike group") and women who were pregnant in 2018 when there were no major strikes ("control group"). Trained research assistants administered a close-ended survey to retrospectively collect demographic and pregnancy-related health utilization and outcomes data. Data were collected between March and July 2019. The primary outcomes of interest were antenatal care (ANC) visits, delivery location, and early child immunizations. Generalized estimating equations were used to estimate risk ratios between the strike and control groups, adjusting for socioeconomic status, health insurance status, and clustering. Adjusted risk ratios (ARR) were calculated with 95% confidence intervals (95%CI). RESULTS: Of 1341 women recruited in the parent study in 2017 (strike group), we re-consented 843 women (63%) to participate. Of 924 women recruited in the control arm of the parent study in 2018 (control group), we re-consented 728 women (79%). Women in the strike group were 17% less likely to attend at least four ANC visits during their pregnancy (ARR 0.83, 95%CI 0.74, 0.94) and 16% less likely to deliver in a health facility (ARR 0.84, 95%CI 0.76, 0.92) compared to women in the control group. Whether a child received their first oral polio vaccine did not differ significantly between groups, but children of women in the strike group received their vaccine significantly longer after birth (13 days versus 7 days, p = 0.002). CONCLUSION: We found that women who were pregnant during nationwide strikes by health workers in 2017 were less likely to receive WHO-recommended maternal child health services. Strategies to maintain these services during strikes are urgently needed.

Correction: Mendelian and Non-Mendelian Regulation of Gene Expression in Maize.

[This corrects the article DOI: 10.1371/journal.pgen.1003202.].

Genomic prediction of maize microphenotypes provides insights for optimizing selection and mining diversity.

Effective evaluation of millions of crop genetic stocks is an essential component of exploiting genetic diversity to achieve global food security. By leveraging genomics and data analytics, genomic prediction is a promising strategy to efficiently explore the potential of these gene banks by starting with phenotyping a small designed subset. Reliable genomic predictions have enhanced selection of many macroscopic phenotypes in plants and animals. However, the use of genomicprediction strategies for analysis of microscopic phenotypes is limited. Here, we exploited the power of genomic prediction for eight maize traits related to the shoot apical meristem (SAM), the microscopic stem cell niche that generates all the above-ground organs of the plant. With 435 713 genomewide single-nucleotide polymorphisms (SNPs), we predicted SAM morphology traits for 2687 diverse maize inbreds based on a model trained from 369 inbreds. An empirical validation experiment with 488 inbreds obtained a prediction accuracy of 0.37-0.57 across eight traits. In addition, we show that a significantly higher prediction accuracy was achieved by leveraging the U value (upper bound for reliability) that quantifies the genomic relationships of the validation set with the training set. Our findings suggest that double selection considering both prediction and reliability can be implemented in choosing selection candidates for phenotyping when exploring new diversity is desired. In this case, individuals with less extreme predicted values and moderate reliability values can be considered. Our study expands the turbocharging gene banks via genomic prediction from the macrophenotypes into the microphenotypic space.

Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves.

BACKGROUND AND AIMS: Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. METHODS: We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. KEY RESULTS: The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. CONCLUSIONS: Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.