Circadian regulation of the transcriptome in a complex polyploid crop.

The circadian clock is a finely balanced timekeeping mechanism that coordinates programmes of gene expression. It is currently unknown how the clock regulates expression of homoeologous genes in polyploids. Here, we generate a high-resolution time-course dataset to investigate the circadian balance between sets of 3 homoeologous genes (triads) from hexaploid bread wheat. We find a large proportion of circadian triads exhibit imbalanced rhythmic expression patterns, with no specific subgenome favoured. In wheat, period lengths of rhythmic transcripts are found to be longer and have a higher level of variance than in other plant species. Expression of transcripts associated with circadian controlled biological processes is largely conserved between wheat and Arabidopsis; however, striking differences are seen in agriculturally critical processes such as starch metabolism. Together, this work highlights the ongoing selection for balance versus diversification in circadian homoeologs and identifies clock-controlled pathways that might provide important targets for future wheat breeding.

Interpreting machine learning models to investigate circadian regulation and facilitate exploration of clock function.

The circadian clock is an important adaptation to life on Earth. Here, we use machine learning to predict complex, temporal, and circadian gene expression patterns in Arabidopsis Most significantly, we classify circadian genes using DNA sequence features generated de novo from public, genomic resources, facilitating downstream application of our methods with no experimental work or prior knowledge needed. We use local model explanation that is transcript specific to rank DNA sequence features, providing a detailed profile of the potential circadian regulatory mechanisms for each transcript. Furthermore, we can discriminate the temporal phase of transcript expression using the local, explanation-derived, and ranked DNA sequence features, revealing hidden subclasses within the circadian class. Model interpretation/explanation provides the backbone of our methodological advances, giving insight into biological processes and experimental design. Next, we use model interpretation to optimize sampling strategies when we predict circadian transcripts using reduced numbers of transcriptomic timepoints. Finally, we predict the circadian time from a single, transcriptomic timepoint, deriving marker transcripts that are most impactful for accurate prediction; this could facilitate the identification of altered clock function from existing datasets.

Circadian and diel regulation of photosynthesis in the bryophyte Marchantia polymorpha.

Circadian rhythms are 24-h biological cycles that align metabolism, physiology, and development with daily environmental fluctuations. Photosynthetic processes are governed by the circadian clock in both flowering plants and some cyanobacteria, but it is unclear how extensively this is conserved throughout the green lineage. We investigated the contribution of circadian regulation to aspects of photosynthesis in Marchantia polymorpha, a liverwort that diverged from flowering plants early in the evolution of land plants. First, we identified in M. polymorpha the circadian regulation of photosynthetic biochemistry, measured using two approaches (delayed fluorescence, pulse amplitude modulation fluorescence). Second, we identified that light-dark cycles synchronize the phase of 24 h cycles of photosynthesis in M. polymorpha, whereas the phases of different thalli desynchronize under free-running conditions. This might also be due to the masking of the underlying circadian rhythms of photosynthesis by light-dark cycles. Finally, we used a pharmacological approach to identify that chloroplast translation might be necessary for clock control of light-harvesting in M. polymorpha. We infer that the circadian regulation of photosynthesis is well-conserved amongst terrestrial plants.

Naturally occurring circadian rhythm variation associated with clock gene loci in Swedish Arabidopsis accessions.

Circadian clocks have evolved to resonate with external day and night cycles. However, these entrainment signals are not consistent everywhere and vary with latitude, climate and seasonality. This leads to divergent selection for clocks which are locally adapted. To investigate the genetic basis for this circadian variation, we used a delayed fluorescence imaging assay to screen 191 naturally occurring Swedish Arabidopsis accessions for their circadian phenotypes. We demonstrate that the period length co-varies with both geography and population sub-structure. Several candidate loci linked to period, phase and relative amplitude error (RAE) were revealed by genome-wide association mapping and candidate genes were investigated using TDNA mutants. We show that natural variation in a single non-synonymous substitution within COR28 is associated with a long-period and late-flowering phenotype similar to that seen in TDNA knock-out mutants. COR28 is a known coordinator of flowering time, freezing tolerance and the circadian clock; all of which may form selective pressure gradients across Sweden. We demonstrate the effect of the COR28-58S SNP in increasing period length through a co-segregation analysis. Finally, we show that period phenotypic tails remain diverged under lower temperatures and follow a distinctive "arrow-shaped" trend indicative of selection for a cold-biased temperature compensation response.

Clinical characteristics and outcomes of infants compared with children diagnosed with rhabdomyosarcoma: Analysis of surveillance, epidemiology and end results data from 2000 to 2016.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, but occurs infrequently in infants (<1 year). Historically, infants with RMS have worse overall survival compared to other pediatric age groups. AIM: This study aims to assess the clinical features and treatment factors associated with survival comparing infants to children aged 1-9 years diagnosed with RMS. METHODS: Children aged <10 years diagnosed with RMS between 2000 and 2016 were identified using the SEER database. Descriptive statistics were used to assess demographic, clinical, and treatment characteristics of infants and children with RMS. Kaplan-Meier estimates and Cox proportional hazards regression were performed to assess for factors associated with survival. RESULTS: Age <1 year was independently associated with an increased risk of mortality. Compared to children aged 1-9 years, fewer infants received standard of care therapy, that is, chemotherapy combined with local control (surgery and/or radiation; 86.8 vs. 75.7%; p = .009). In comparing the frequency of specific treatment modalities (used alone or in combination with other modalities), infants were less likely to receive radiation therapy (34.0 vs. 66.4%; p < .001) and more likely to receive surgery (68.9 vs. 57.5%; p = .02) than children aged 1-9 years. Across age groups, chemotherapy combined with local control was significantly associated with reduced mortality. Alveolar histology, metastatic disease, and Hispanic ethnicity were negatively associated with survival. CONCLUSIONS: Age of <1 year was an independent risk factor for increased mortality from RMS compared to ages 1-9 years. Fewer infants were treated with chemotherapy combined with local control, the therapy associated with best survival in all age groups. Other factors contributing to differences in survival should be further explored.

A high-throughput delayed fluorescence method reveals underlying differences in the control of circadian rhythms in Triticum aestivum and Brassica napus.

BACKGROUND: A robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. A huge number of physiological processes are under circadian control in plants including: responses to biotic and abiotic stresses; flowering time; plant metabolism; and mineral uptake. Understanding how the clock functions in crops such as Triticum aestivum (bread wheat) and Brassica napus (oilseed rape) therefore has great agricultural potential. Delayed fluorescence (DF) imaging has been shown to be applicable to a wide range of plant species and requires no genetic transformation. Although DF has been used to measure period length of both mutants and wild ecotypes of Arabidopsis, this assay has never been systematically optimised for crop plants. The physical size of both B. napus and T. aestivum led us to develop a representative sampling strategy which enables high-throughput imaging of these crops. RESULTS: In this study, we describe the plant-specific optimisation of DF imaging to obtain reliable circadian phenotypes with the robustness and reproducibility to detect diverging periods between cultivars of the same species. We find that the age of plant material, light regime and temperature conditions all significantly effect DF rhythms and describe the optimal conditions for measuring robust rhythms in each species. We also show that sections of leaf can be used to obtain period estimates with improved throughput for larger sample size experiments. CONCLUSIONS: We present an optimized protocol for high-throughput phenotyping of circadian period specific to two economically valuable crop plants. Application of this method revealed significant differences between the periods of several widely grown elite cultivars. This method also identified intriguing differential responses of circadian rhythms in T. aestivum compared to B. napus; specifically the dramatic change to rhythm robustness when plants were imaged under constant light versus constant darkness. This points towards diverging networks underlying circadian control in these two species.

Coordination of robust single cell rhythms in the Arabidopsis circadian clock via spatial waves of gene expression.

The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24-hr rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.

Knowledge and beliefs regarding cervical cancer screening and HPV vaccination among urban and rural women in León, Nicaragua.

BACKGROUND: In Nicaragua, cervical cancer is the leading cause of cancer-related death for women ages 15-44, yet access to the HPV vaccine is limited to those with financial resources to pay for it. Cervical cytology is provided free of charge in public clinics; however, only 10% of women receive Pap smears at the nationally recommended frequency. Previous studies have not investigated how beliefs regarding cervical cancer screening may differ for urban and rural populations in Nicaragua. Furthermore, no investigation has assessed Nicaraguan women's beliefs about a potential HPV immunization campaign. Given beliefs' influence on health behavior, we investigated the structural, sociocultural, and knowledge-based factors influencing women's beliefs regarding cervical cancer screening among urban and rural women in León, Nicaragua, and assessed acceptance of a potential HPV immunization program. METHODS: Our sequential explanatory mixed-methods study consisted of two phases: (1) a close-ended questionnaire, followed by (2) a qualitative, in-depth interview. Our quantitative sample contained 117 urban and 112 rural participants aged 18-49. We assessed beliefs regarding cervical cancer screening using a 22-item scale, with higher scores indicating screening-promoting beliefs in simple linear and multiple linear regressions. Twenty qualitative interviews, exploring the sociocultural dimensions of knowledge and attitudes indicated by our quantitative findings, were conducted with a sample of 13 urban and 7 rural women aged 19-46. RESULTS: The multiple linear regression indicates that greater knowledge of Pap smears, HPV, and cervical cancer is significantly associated with screening-promoting beliefs after adjusting for other relevant factors. There was no significant difference in screening knowledge and beliefs for urban and rural women. Four recurrent themes representing determinants of knowledge, beliefs, and attitudes regarding cervical cancer screening arose from interviews and built on quantitative findings: (1) women's embarrassment due to the intimate nature of the Pap smear and male gender of exam provider discourages screening; (2) women believe Pap smears and cervical cancer are associated with sexual promiscuity, and this association stigmatizes women with the disease; (3) knowledge of cervical cancer prevention is limited to those who regularly attend health centers; and (4) women find screening inconvenient, believing understaffed clinics increase patient wait time, limit time patients spend with clinicians, and delay Pap results. A fifth theme indicates (5) participants' acceptance of a potential HPV immunization program. DISCUSSION: Future interventions should focus on increasing access to information about cervical cancer prevention for women who do not regularly attend health centers. Furthermore, our results suggest that if funding were allocated to make the HPV vaccine accessible in Nicaragua, it would be well received.