Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

Cdc42 reactivation at growth sites is regulated by local cell-cycle-dependent loss of its GTPase-activating protein Rga4 in fission yeast.

In fission yeast, polarized cell growth stops during division and resumes after cytokinesis completes and cells separate. It is unclear how growth reactivation is timed to occur immediately after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth during septation, displaying a polar elongation simultaneous with septation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, during septation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. Although the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that, for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis in fission yeast.

Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. Why the cell needs two GEFs is unclear, given that they are partially redundant and activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis through recruitment of the scaffold protein Scd2, via a Cdc42 feedforward pathway. Similarly, during interphase Gef1 promotes Scd1 recruitment at the new end to enable the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation, and to maintain cell shape during interphase. Our findings reveal an elegant regulatory pattern in which Gef1 primes Cdc42 activation at new sites to initiate Scd1-dependent polarized growth, while Scd1 restricts Gef1 to sites of polarization. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes.This article has an associated First Person interview with the first author of the paper.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

QSAR models for prediction of chromatographic behavior of homologous Fab variants.

While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R2 > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc.

Evaluating human-disturbed habitats for recovery planning of endangered plants.

The recovery potential of endangered species is limited by the high prevalence of human-modified habitats, while effective in situ conservation strategies to identify and restore disturbed habitat within species ranges are lacking. Our goal was to determine the impact of human disturbance on the endangered endemic Barrens willow (Salix jejuna) to provide science-based protocols for future restoration of disturbed habitats; a key component of conservation and recovery plans for many rare plant species. Our study examined differences in substrate (e.g., % total plant cover, % species cover, substrate type) and vegetation in naturally- (via frost activity) vs human-disturbed limestone barrens (Newfoundland, Canada), across the entire species range of the endangered Barrens willow. There were distinct differences in substrate conditions and vegetation community structure between naturally- and human-disturbed limestone barrens habitat throughout the narrow range of this endemic willow. Human-disturbed sites are more homogeneous and differ significantly from the naturally-disturbed sites having a much coarser substrate (30% more gravel) with less fine grained sands, less exposed bedrock, decreased soil moisture, increased nitrogen content, and reduced phosphorus content. Substrate differences can inhibit return to the natural freeze-thaw disturbance regime of the limestone barrens, negatively affecting long-term persistence of this, and other rare plants. The structure of associated vegetation (specifically woody species presence) negatively affected willow abundance but was not linked to disturbance type. Human-disturbed sites are potential candidates for endangered plant recovery habitat if natural ecosystem processes, vegetation community structure, and habitat heterogeneity are restored, thereby supporting the establishment of long term viable populations.

Mindfulness and integrative self-knowledge: relationships with health-related variables.

This study examined how mindfulness and integrative self-knowledge were related to health-related issues. Men in general population (n = 103) and coronary heart disease samples (n = 101) completed the Mindful Attention Awareness Scale, the Integrative Self-knowledge Scale, the Type 2 subscale of the Interpersonal Reactions Inventory, the Perceived Stress Scale, and the Anxiety and Depression Scales. In both samples, there was a moderate positive correlation between mindfulness and integrative self-knowledge and they were negatively correlated with all health-related variables. However, only integrative self-knowledge explained independent variance in health-related variables. Specifically, in both samples, the relationship between mindfulness and health-related variables was mediated by integrative self-knowledge. Mindfulness and integrative self-knowledge are related domains of self-awareness that are associated with a range of health-related variables. These relationships are robust across samples drawn from general population and patients with coronary heart disease. The finding that integrative self-knowledge explained additional variance in the health-related variables after the contribution of mindfulness had been accounted for suggests that reflective self-awareness in integrative self-knowledge may make a unique contribution to the explanation of individual differences in health variables.

Persistence and fading of the cognitive and socio-emotional benefits of preschool education in a low-resource setting: Group differences and dose-dependent associations in longitudinal data from Vietnam.

Introduction: Four analytic approaches examined the effectiveness of preschool education in Vietnam, which provides a context in which national curricula and teaching standards for preschools and schools, high levels of preschool attendance, and fee subsidies for disadvantaged children, limit the heterogeneity in children's experiences that often obscure the outcomes associated with preschool attendance. Methods: The Young Lives Study provided longitudinal data on children's receptive vocabulary, mathematics, and life satisfaction at 5, 8, 12, and 15 years of age, and on their self-concept and relationships at 12 and 15 years. Results: The first analysis found that children who attended preschool (n = 1,562 at 5 years of age) had larger vocabularies at 5, 8, 12, and 15 years, greater mathematics knowledge at 5, 8, and 12 years, and higher life satisfaction at 5 and 12 years of age than the small number of children who did not attend preschool (n = 164 at 5 years of age). The second, found that the dose of preschool education (hours per week × 4 × months) received by children who attended preschool was positively associated with their receptive vocabulary and mathematics scores at 5, 8, 12, and 15 years of age, and with their life satisfaction at 5 and 15 years of age. Although the magnitude of the effect for vocabulary declined over time, it remained stable for mathematics. The third analysis found that a high dose of preschool education allowed disadvantaged rural children to achieve comparable or better scores than their urban peers for receptive vocabulary at 8, 12, and 15 years, mathematics at 12 years, and life satisfaction at all ages. The final analysis found that even a low dose of preschool education improved rural children's receptive vocabulary at 5, 8, and 15 years, and their numeracy/mathematics scores at 5, 8, and 12 years. Discussion: Together, the results suggest that preschool attendance had a small but meaningful positive association with Vietnamese children's cognitive skills and life satisfaction that persisted for at least 10 years. These findings provide insights into the scale, scope, and longevity of effects that can be achieved from scaled-up preschool programs under resource-constrained conditions.

Development of a NASA roadmap for planetary protection to prepare for the first human missions to Mars.

As part of planning for future space exploration, COSPAR (The Committee on Space Research) together with participating space agencies, organized and held interdisciplinary meetings to consider next steps in addressing knowledge gaps for planetary protection for future human missions to Mars. Beginning with the results of these meetings and earlier work by NASA, ESA, and COSPAR (e.g., Criswell et al., 2005; Hogan et al., 2006; Rummel et al., 2008) as a base the authors of this paper carried out a follow-on NASA planning activity to identify the necessary steps to be accomplished to close knowledge gaps. We identified significant overlap between the planetary protection needs and other sets of Mars preparation roadmaps (1) microbial monitoring requirements for crew health and medical systems, (2) studies of the microbiome of the built environment, (3) environmental control and life support systems (ECLSS), (4) waste management, and (5) planetary surface operations. In many cases, efforts to mature exploration class systems for Mars that are occurring in other domains can be leveraged with minor changes to address planetary protection gaps as well. In other cases, work planned for testing on the International Space Station (ISS) as an analog for crew Mars transit, or on the lunar surface as an analog for Mars surface operations can be used to close planetary protection technology and knowledge gaps. An overall strategic framework that combines these domains has the advantage of being more comprehensive, efficient, and timely for closing gaps. This approach has led to the development of a NASA roadmap for addressing planetary protection integrated with other related roadmaps. NASA's development and execution of the planetary protection is now viewed in an integrated way with related technology development and testing. Key features of the integrated capabilities roadmap include.

m 6 A RNA methylation orchestrates transcriptional dormancy during developmental pausing.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that m 6 A RNA methylation by Mettl3 is required for developmental pausing in mice by maintaining dormancy of paused embryonic stem cells and blastocysts. Mettl3 enforces transcriptional dormancy via two interconnected mechanisms: i) it promotes global mRNA destabilization and ii) suppresses global nascent transcription by specifically destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a critical anti-pausing factor. Our findings reveal Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during pausing, with implications for dormancy in stem cells and cancer.

m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N6-methyladenosine (m6A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3-/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3-/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer.

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes.

This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 µm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min.

Urban-rural differences in the relationship between stunting, preschool attendance, home learning support, and school readiness: A study in Côte d'Ivoire.

Background: Stunted physical growth during early childhood is a marker of chronic undernutrition, and the adverse life circumstances that underlie it. These have the potential to disrupt normal brain development and the acquisition of foundational cognitive, language, social and motor skills. Stunting is prevalent in most low-and middle-income countries. Because the prevention of stunting requires large-scale structural and attitudinal changes, several psycho-educational interventions have been developed to mitigate the adverse association between early stunting and skill development. However, the resource-intensive nature of custom-designed interventions limit their sustainability and scalability in resource-limited settings. This study explored the possibility that available resources that promote positive development (existing preschool education programs, and no- or low-cost home-based learning activities and resources) may protect against any negative association between stunting and the acquisition of foundational skills required for academic learning and adaptation at school. Method: Data for 36-to 59-month-old children (n = 3,522; M = 46.7 months; 51.2% male; 74.1% rural) were drawn from the most recent Multiple Indicator Cluster Survey conducted in Côte d'Ivoire (MICS5, 2016). Stunting was assessed using the WHO Child Growth Standards. Preschool attendance and home learning activities and resources were assessed by maternal report. School readiness was assessed using the 8-item form of the Early Child Development Index (ECDI). Results: A high percentage of children met the criteria for stunting (28.5%; 19.7% moderate; 8.8% severe). There were marked urban-rural differences in the prevalence of stunting, rates of preschool attendance, home learning activities and resources, children's school readiness scores, and the relationships between stunting, the protective factors and school readiness scores. These urban-rural differences in ECDI scores could be fully explained by differences between these settings in stunting and the protective factors. However, only two protective factors (access to books and home-based activities that promote learning) made independent contributions to variance in ECDI scores. There was tentative evidence that stunted children whose homes provided highly diverse learning activities and multiple types of learning resources were more likely than those who did not to have a high level of school readiness. Conclusion: Capitalizing on the existing practices of families that show positive deviance in caregiving may provide a basis for culturally appropriate, low-cost interventions to improve school readiness among children in low- and middle-income countries, including children with stunted growth.

Recurrence of childhood absence epilepsy as pyknolepsy in adolescence.

A developmentally normal adolescent boy with a history of childhood absence epilepsy presented with recurrence of pyknolepsy after a seven-year period of remission. The characteristics of his EEG showed the same 3-Hz generalised spike-wave discharge as in his previous EEG in childhood. To our knowledge, this is the first case report describing recurrence of childhood absence epilepsy as pyknolepsy in an adolescent. [Published with video sequences].

Cdc42 promotes Bgs1 recruitment for septum synthesis and glucanase localization for cell separation during cytokinesis in fission yeast.

Cytokinesis in fission yeast involves actomyosin ring constriction concurrent to septum synthesis followed by septum digestion resulting in cell separation. A recent report indicates that endocytosis is required for septum synthesis and cell separation. The conserved GTPase Cdc42 is required for membrane trafficking and promotes endocytosis. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). Cdc42 GEFs have been shown to promote timely initiation of septum synthesis and proper septum morphology. Here we show that Cdc42 promotes the recruitment of the major primary septum synthesizing enzyme Bgs1 and consequent ring constriction. Cdc42 is also required for proper localization of the septum digesting glucanases at the division site. Thus, Cdc42 is required to promote multiple steps during cytokinesis.

Use of electronic group method in assessing food safety training needs and delivery methods among international college students in the U.S.

The purpose of this study was to determine the types of unfamiliar foods international students in the U.S. encounter and to assess food safety information that international students would like to receive for mitigating risks associated with handling and preparing unfamiliar foods. The study identified preferred instructional delivery methods and media for receiving food safety training or information. An electronic group method was used for this study. The electronic group method was chosen to maximize group efficiency by allowing participants to share ideas simultaneously and anonymously with minimal use of time and resources.Types of different (unfamiliar) foods were grouped into major categories. Fast and ready-to-eat foods, and processed and frozen foods constituted a major change for some international students, who were accustomed to homemade and fresh foods in their countries. Participants were interested in receiving information about how to safely handle and prepare unfamiliar foods in their new environment. Preferred methods for receiving food safety information included written materials, online publications, presentations, and materials provided during student orientation. Food packages, websites, and television programs were other preferred methods of receiving food safety information.

Do palliative patients and carers agree about patients' psychological functioning?

OBJECTIVE: Palliative care clinicians and researchers often seek information about patients from informants. This research examines the extent of agreement between information from patients and family caregivers who were asked to serve as collateral sources of information about the patient. METHOD: Sixty-six patients with advanced cancer and their family caregivers participated in the study. Two measurement contexts were examined: Direct observation of patients' cognitive performance (Mini-Mental State Examination) was compared with carers' subjective reports about patients' everyday cognition (Cognitive Decline subscale of the Psychogeriatric Assessment Scale), and subjective reports about patient depression were compared between patients and carers who completed parallel forms of the same scale (Geriatric Depression Scale and Geriatric Depression Scale-Collateral Source, respectively). The relationship between patient-completed and carer-completed measures was examined in four ways: the correlation between total scores in the sample, agreement about the prevalence of impaired functioning and of specific symptoms in the sample, agreement concerning whether particular patients showed impaired functioning, and agreement about the presence or absence of a specific symptom for a particular patient. RESULTS: Although most measures of agreement showed that information provided by patients and carers was related, the magnitude of discrepancies was substantial. SIGNIFICANCE OF RESULTS: There was no empirical justification for substituting information from a collateral source for information provided by the patient for any of the measures of agreement for either cognition or depression. The use of information from collateral sources is most appropriate when information from the patient is likely to be incomplete or inaccurate, when insight into caregivers' needs or understanding is sought, and when using a multi-informant approach to assessment.

The effect of pH on antibody retention in multimodal cation exchange chromatographic systems.

The present paper builds upon previous work on mAb domain contributions to multimodal (MM) chromatography by examining how pH can impact mAb surface properties and retention in these systems. Linear salt gradient experiments were carried out between pH 5-7 for several mAbs with different pI and surface hydrophobicities in four different MM CEX resins at two ligand densities. mAb retention showed an inverse, non-linear correlation with pH. Changing pH affected the elution order, creating unique windows of selectivity in each of the MM CEX resins. One mAb showed a pH-dependent spectrum of domain contributions, demonstrating that pH can be used to tune the relative importance of the (Fab)2 and Fc domains for some mAbs in MM systems. Positive, negative, and hydrophobic patches were calculated between pH 5-7 for the mAbs. Visualizing these patches on the protein surface demonstrated that each mAb showed a unique distribution of surface charge and hydrophobicity that changed with pH. The sum of patch areas was tracked across this pH range to quantitatively understand how pH impacted these important surface properties. The quantitative analysis then was narrowed to consider only patches in the CDR loops, which were hypothesized to be an important interaction site for some mAbs in these systems. Interestingly, differences in the titration of CDR loop patches for each mAb were shown to be a result of Histidine titrations and patches in this region were qualitatively correlated with experimental trends including the observed elution order reversals. These results indicate that pH potentially can be employed as a lever for the strategic design of multimodal steps to create flow through, bind and elute, or weak partitioning operations with important implications for the design of integrated and/or continuous downstream purification processes. Furthermore, the ability to tune domain contributions in MM separations using pH creates intriguing possibilities for current downstream challenges such as the removal of product-related impurities, as well as the purification of bispecific mAbs.

Relationship between the Length of Sperm Tail Mitochondrial Sheath and Fertility Traits in Boars Used for Artificial Insemination.

The length of sperm tail midpiece, occupied by the mitochondrial sheath (MS), has been correlated with reproductive traits of mice, fish, and birds; however, it is not known whether such a correlation exists in higher order species such as domestic pigs. As the mitochondria provide for sperm motility and generate the fertility-affecting reactive oxygen species (ROS), we hypothesized that MS length correlates with boar semen parameters and artificial insemination (AI) fertility. Sperm samples collected from 57 boars and used for single sire AI were labeled with ProteoStat Aggresome probe (AGG; Enzo Life Sciences) for MS imaging by epifluorescence microscopy and image-based flow cytometry (IBFC). The mean boar MS length was 7.26 ± 0.2 µm, ranging from 6.94 ± 0.18 µm to 7.65 ± 0.31 µm. The absolute longest MS measured was 9.19 µm and the shortest was 5.83 µm. Boars in the high tertile of MS length had significantly higher conception rate (CR; p = 0.05) and sperm parameters. Boars within the high tertile of average number piglets born per litter had significantly shorter MS and more varied MS length than boars in the low tertile (p = 0.04). MS length data correlated with conventional sperm parameters including percent viable and intact acrosomes (p = 0.03), basal:induced oxidation ratio (measure of intracellular ROS levels; p = 0.02) and Comp DNA (chromatin integrity; p = 0.06) along with many flow cytometric AGG parameters in IBFC. Sperm head AGG intensity median absolute deviation had a negative correlation with total born (r = -0.423 p = 0.004). These data reveal a complex relationship between sperm MS length and aggresome abundance to sperm parameters and boar reproductive success in AI service.