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Supplemental material, sj-docx-1-hpp-10.1177_15248399231175843 for Remote Evidence-Based Health Promotion Programs During COVID: A National Evaluation of Reach and Implementation for Older Adult Health Equity by Lesley Steinman, Erica Chavez Santos, Kelly Chadwick, Caitlin Mayotte, Selisha (Snowy) Johnson, Marlana Kohn, James Kelley, Paige Denison, Clarissa Montes, Lesha Spencer-Brown and Kate Lorig in Health Promotion Practice

In Chapter 1, I use a novel equity lending dataset to show that equity lending fees are highly predictable out-of-sample. I find that the two strongest predictors of future stock loan fees are the past loan fee and the stock's rational expected return. Expected returns strongly and negatively predict future equity lending fees, as a decline in expected returns predicts both higher loan demand and lower loan supply. In comparing two expected return measures, I find that a rational expected return has stronger predictive power of future equity lending activity than an extrapolative expected return, implying that equity lending market participants are primarily rational, forward-looking agents. Past loan fee predictability has implications for future loan demand, suggesting that short sellers are drawn to short stocks with predictable loan fees in order to mitigate the risk of unexpected loan fee increases. In Chapter 2 (co-authored with Christian Lundblad and Adam Reed), we document the existence of commonality in equity lending fees, and we show that the common component of loan fees explains a high degree of their variation. Stocks with high loan fees tend to exhibit high sensitivity to the common component. The common component is highly correlated with several asset pricing factors, suggesting loan fee commonality is associated with consequential states. Loan fee commonality is an important limit to arbitrage, as evidenced by a strong relationship between loan fee commonality and both low future returns and decreased price efficiency. Loan demand is the primary driver of the observed commonality. In Chapter 3 (co-authored with Ric Colacito, Max Croce, and Federico Gavazzoni), we study returns to several international carry trade strategies. The slope carry takes a long (short) position in the long-term bonds of countries with steeper (flatter) yield curves. The traditional carry takes a long (short) position in countries with high (low) short-term rates. We document that: (i) the slope carry return is slightly negative (strongly positive) in the pre (post) 2008 period, whereas it is concealed over longer samples; (ii) the traditional carry return is lower post-2008; and (iii) expected global growth and inflation declined post-2008. We connect these findings through an equilibrium model in which countries feature heterogeneous exposure to news shocks about global output and global inflation. In Chapter 4 (co-authored with Andrei Goncalves), we construct a Stochastic Discount Factor (SDF) that prices bond, equity, and real estate portfolios sorted on cash flow duration. Using this SDF and the dynamics of cash flow yields in these three asset classes, we estimate the bond, equity, and real estate term structures monthly from 1974 to 2019. We find that while (nominally) safe and risky cash flows have risk premia term structures that are upward sloping on average and move together over time, the term structure dynamics are fundamentally different after we remove the safe component of the risky cash flows. Specifically, equity and real estate maturity-matched risk premia, on average, increase over short maturities but decline over long maturities. Moreover, their term structures comove positively with each other but negatively with the bond term structure.

Objectives Radiation therapy (RT) and intrathecal drug delivery systems (IDDS) are often used concurrently to optimize pain management in patients with cancer. Concern remains among clinicians regarding the potential for IDDS malfunction in the setting of RT. Here we assessed the frequency of IDDS malfunction in a large cohort of patients treated with RT. Materials and methods Cancer patients with IDDS and subsequent RT at our institution from 2011 to 2019 were eligible for this study. Patients were excluded in the rare event that their IDDS was managed by an outside clinic and follow-up documentation was unavailable. Eighty-eight patients aged 22-88 years old (43% female, 57% male) representing 106 separate courses of RT were retrospectively identified. Patients received varying levels of radiation for treatment of cancer and cumulative dose to the IDDS was calculated. IDDS interrogation was subsequently performed by a pain specialist. Malfunction was recorded as deviation from the expected drug volume and/or device errors reported upon interrogation as defined by the manufacturer. Results Total measured RT dose to the IDDS ranged from 0 to 18.0 Gy (median = 0.2 Gy) with median dose of 0.04 Gy/fraction (range, 0-3.2 Gy/fraction). Ten pumps received a total dose >2 Gy and three received ≥5 Gy. Eighty-two percentage of patients had follow-up with a pain specialist for IDDS interrogation and all patients underwent follow-up with a healthcare provider following RT. There were zero incidences of IDDS malfunction related to RT. No patient had clinical evidence of radiation related pump malfunction at subsequent encounters. Conclusions We found no evidence that RT in patients with IDDS led to device failure or dysfunction. While radiation oncologists and pain specialists should coordinate patient care, it does not appear that RT dose impacts the function of the IDDS to warrant significant clinical concern.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand–receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. Implications: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Introduction: The current treatment paradigm of abdominal aortic aneurysms (AAA) focuses on observing patients until their disease reaches certain thresholds for intervention, with no preceding treatment available. There is an opportunity to develop drug or cell-based therapies to prevent further aneurysmal growth and decrease the risk of a highly morbid rupture. A previously validated acute AAA swine model was treated directly with gelfoam containing human adipose-derived stem cells (h-ADSCs). Methods: Male Yorkshire swine (n=16) received periadventitial injections (type 1 collagenase and porcine pancreatic elastase) into a 4 cm segment of infrarenal aorta. Experimental group (n=7) received gel foam soaked with 10 million h-ADSCs and control group (n=9) media-soaked gel foam. Aortic diameters (AD) were monitored at post-operative day (POD) 7 and 14 using ultrasound with euthanasia on POD 21. Suprarenal (control) and infrarenal aortas (experimental) were harvested for PCR, mechanical tensiometry testing, histopathological analyses as well as immunohistochemistry (IHC). Each animal served as their own control. Groups were compared with paired t-tests and Mann Whitney U tests. Results: Twelve animals survived until POD 21. Stem cell treated AAAs, unlike controls, demonstrated no growth (-1.6 ± 8.8% vs. 34.8 ± 13.7%, p Conclusion: Stem cell therapy prevents the development of aortic aneurysms in a swine model. Stem cell treated swine with induced AAAs show more normal gross features, histologic, and biochemical properties compared to untreated controls with induced AAAs. Further investigation is merited as a possible translatable therapy for aneurysmal disease.

Purpose and objectives Effective community-based programs to manage arthritis exist, but many adults with arthritis are unaware that these programs are available in their communities. An electronic health record (EHR) referral intervention was designed to strengthen health care and community-based partnerships and increase participation in these arthritis programs. The intervention was developed in response to a national effort that aimed to enhance the health, wellness, and quality of life for people with arthritis by increasing the awareness and availability of, and participation in arthritis-appropriate evidence-based interventions. Intervention approach The National Recreation and Park Association recruited 4 park and recreation agencies and their health care partners to implement an EHR-based retrospective and point-of-care referral intervention. Eligible for referral were adults aged 45 or older with an arthritis condition who were seen by a physician within the past 18 months, and were living within the park and recreation service area. After health care organizations identified eligible adults, they either mailed communication packages describing the availability and benefits of the intervention and conducted phone calls to encourage arthritis-appropriate intervention participation or counseled and referred patients during an office visit. Evaluation methods The pilot was assessed by using semi-structured interviews with key intervention staff members and the Consolidated Framework for Implementation Research. Results Our approach resulted in referrals for 3,660 people, 1,063 (29%) of whom participated in an intervention. Analysis of key informant interviews also highlighted the specific contextual factors, facilitators, and barriers that influenced the adaptation and overall implementation of the referral intervention. Implications for public health Our pilot demonstrates that successful coordination between health care organizations and community-based organizations can promote awareness of and participation in community-based programs. An understanding of the contextual factors and lessons learned can be used to inform processes that can lead to more effective and sustainable health care and community-based partnerships.

The operation of smart power grids is supported by interconnected sensors and devices that provide real-time monitoring. Smart grids provide more secure and dependable electricity than traditional grids because of constant monitoring. This paper reviews the technology used in smart grids, specifically sensors and communication technologies. It also talks about the challenges that smart grids face, such as providing power to the network's sensors, islanding, cyber-security, and issues that occur when using distributed generators. Current solutions for sensing changes and communication of sensed data in smart grid networks are also discussed.

Cerebral organoids provide unparalleled access to human brain development in vitro. However, variability induced by current culture methodologies precludes using organoids as robust disease models. To address this, we developed an automated Organoid Culture and Assay (ORCA) system to support longitudinal unbiased phenotyping of organoids at scale across multiple patient lines. We then characterized organoid variability using novel machine learning methods and found that the contribution of donor, clone, and batch is significant and remarkably consistent over gene expression, morphology, and cell-type composition. Next, we performed multi-factorial protocol optimization, producing a directed forebrain protocol compatible with 96-well culture that exhibits low variability while preserving tissue complexity. Finally, we used ORCA to study tuberous sclerosis, a disease with known genetics but poorly representative animal models. For the first time, we report highly reproducible early morphological and molecular signatures of disease in heterozygous TSC+/− forebrain organoids, demonstrating the benefit of a scaled organoid system for phenotype discovery in human disease models.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis. We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.

International audience; Genome size evolution, and its relationships with pollen grain size, has been investigated in the sweet potato (Ipomoea batatas), an economically important crop, and closely related diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, 10 sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined by flow cytometry. Pollen grains have been studied at scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged over 3.12-3.29 pg, mean monoploid genome size being 0.539 pg (527 Mbp) much as for the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was respectively 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida, I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% of AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, genome size and ploidy levels were correlated with pollen size. Our results allow us proposing alternative or complementary hypotheses to the one currently proposed for the formation of hexaploid Ipomoea batatas. This article is protected by copyright. All rights reserved.

TheVenturiagenus comprises fungal species that are pathogens onRosaceaehost plants, includingV. inaequalisandV. asperataon apple,V. aucupariaeon sorbus andV. pirinaon pear. Although the genetic structure ofV. inaequalispopulations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87Venturiastrains that represent each species and each population withinV. inaequalis. We present a PacBio genome assembly for theV. inaequalisEU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies ofV. inaequalisandV. aucupariaecontain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly ofV. inaequalispresents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research ofVenturiapathogens. Especially, it will help understanding the evolutionary history ofVenturiaspecies that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.

Background Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. Results This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. Conclusions The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses. Electronic supplementary material The online version of this article (10.1186/s12870-018-1567-z) contains supplementary material, which is available to authorized users.

The genome size of eight species from different wild Algerian populations of diploid and one tetraploid Medicago L., initially analysed for chromosome numbers and seed storage proteins, has been assessed by flow cytometry. Genome size ranged from 2C = 0.94 pg in Medicago orbicularis (L.) Bartal. to 2C = 1.80 pg in Medicago laciniata (L.) Miller. The competence for direct somatic embryo formation in liquid medium was studied for 4 of these species having distinct genome sizes: M. orbicularis (2C = 0.94 pg); M. truncatula Gaertn. (2C = 1.08 pg); M. scutellata (L.) Miller (2C = 1.11 pg); M. arabica (L.) Huds. (2C = 1.22 pg). M. orbicularis, with the smallest genome size, formed somatic embryos most quickly, with a high frequency of reactive explants and with numerous somatic embryos per explant. It was followed by M. truncatula, M. scutellata and M. arabica, which in fact represents the order of increasing genome size.

With the present work, we aim to provide a better understanding of chromosome evolutionary trends among southern Brazilian species of Iridoideae. Chromosome numbers and genome sizes were determined for 21 and 22 species belonging to eight genera of Tigridieae and two genera of Trimezieae, respectively. The chromosome numbers of nine species belonging to five genera are reported here for the first time. Analyses of meiotic behaviour, tetrad normality and pollen viability in 14 species revealed regular meiosis and high meiotic indexes and pollen viability (> 90%). The chromosome data obtained here and compiled from the literature were plotted onto a phylogenetic framework to identify major events of chromosome rearrangements across the phylogenetic tree of Iridoideae. Following this approach, we propose that the ancestral base chromosome number for Iridoideae is x = 8 and that polyploidy and dysploidy events have occurred throughout evolution. Despite the variation in chromosome numbers observed in Tigridieae and Trimezieae, for these two tribes our data provide support for an ancestral base number of x = 7, largely conserved in Tigridieae, but a polyploidy event may have occurred prior to the diversification of Trimezieae, giving rise to a base number of x2 = 14 (detected by maximum-parsimony using haploid number and maximum likelihood). In Tigridieae, polyploid cytotypes were commonly observed (2x, 4x, 6x and 8x), whereas in Trimezieae, dysploidy seems to have been the most important event. This feature is reflected in the genome size, which varied greatly among species of Iridoideae, 4.2-fold in Tigridieae and 1.5-fold in Trimezieae. Although no clear difference was observed among the genome sizes of Tigridieae and Trimezieae, an important distinction was observed between these two tribes and Sisyrinchieae, with the latter possessing the smallest genome sizes in Iridoideae. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 177, 27–49.

The National Recreation and Park Association (NRPA) is one of four national organizations to receive funding from CDC to disseminate arthritis-appropriate evidence-based interventions (AAEBIs). NRPA has partnered with Fit & Strong!, a CDC-approved AAEBI and National Council on Aging falls prevention program, to disseminate through local park and recreation agencies across the country. To meet the needs of this dissemination effort, Fit & Strong! adapted its in-person instructor training into an online curriculum which became available March 2018. After initial pilot-testing, Fit & Strong! created a five-module training curriculum using Moodle teaching software. The training curriculum uses slides, graphics, videos, and interactive questions to assess mastery. After completing the training, fidelity checks are performed on all instructors by having them video record a class session and share it with the Fit & Strong! team for review. After evaluating the videos, the team meets with the instructors by phone to debrief and advise on any needed improvements. Through this partnership, Fit & Strong! trained an initial cohort of 39 instructors across 18 states within a month’s time. Training evaluation results have been positive. To date, over 900 participants have been reached. A second cohort of approximately 40 new instructors will be trained in Spring 2019. This presentation will review the processes of creating and executing the online training, potential cost and time-saving benefits to CBOs, instructor feedback, and the ease with which instructors can complete the training and offer the program anywhere across the country.

Four taxa of Algerian Astragalus L. were studied for their chromosome number, karyotype features, and genome size. Fluorochrome banding was done for detection of GC-rich DNA regions, fluorescence in situ hybridization (FISH) for physical mapping of 35S and 5S rRNA genes, and flow cytometry for nuclear DNA content. All the taxa present the same chromosome number (2n = 2x = 16) and single 35S and 5S rDNA loci, but their distributions on chromosomes are different. The GC heterochromatin pattern was different among studied taxa and an unusually high number of chromomycin-positive bands were observed in A. pseudotrigonus Batt. & Trab. The genome size differed between species, ranging from 2C = 1.39 pg in A. cruciatus Link. to 2C = 2.71 pg in A. armatus subsp. tragacanthoides (Desf.) Maire. No difference in nuclear DNA amount was detected between the 2 subspecies of A. armatus. Although Astragalus is a large genus comprising some 3000 species, such morphometric and molecular cytogenetic karyotype analyses, with genome sizes, are particularly scarce therein. Therefore, published genome sizes have also been compiled into one table.

The geographic position of Lebanon in the Mediterranean basin at the transition of two major landmasses, Eurasia and Africa, has contributed to its high plant diversity and makes its flora particularly interesting to study. This paper contributes to the plant DNA C-value database of native Lebanese taxa. These data should reinforce biodiversity evaluation, systematic and evolution studies involving processes of speciation such as polyploidisation. C-values have been estimated by flow cytometry using propidium iodide as intercalary fluorochrome stain. Each sample comprised at least five individuals. Where possible, several populations were measured for each species. This study presents C-values for 225 taxa belonging to 55 families and 141 genera. C-values are novel for 193 taxa including 126 plants endemic to the Eastern Mediterranean region. These are the first values for 50 genera. In this panel, genome size ranged from 1C = 0.28 pg in Hypericum thymifolium to 54.69 pg in Fritillaria alfredae. The life ...

Fleshy fruit species such as tomato are important because of their nutritional and economic value. Several stages of fruit development such as ovary formation, fruit set, and fruit maturation have already been the subject of many developmental studies. However, fruit growth per se has been much less addressed. Fruit growth like all plant organs depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will compose the fruit; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by the means of endoreduplication, i.e. genome amplification in the absence of mitosis, appears to be of great importance in fleshy fruits. In tomato fruit, endoreduplication is associated with DNA-dependent cell expansion: cell size can reach spectacular levels such as hundreds of times its initial size (e.g. >0.5 mm in diameter), with as much as a 256-fold increase in nuclear DNA content. Using tomato fruit development as a model, recent investigations combining the use of flow cytometry, cellular imaging and molecular analyses have provided new data in favor of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication acts as a morphogenetic factor supporting cell growth during tomato fruit development. In the context of plant breeding, deciphering the mechanisms controlling fruit growth, in particular those connecting the process of nuclear endoreduplication with modulation of gene expression, the regulation of cell size and final fruit size and composition, is necessary to understand better the establishment of fleshy fruit quality traits.

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast ( Saccharomyces cerevisiae ), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana , which is devoid of CL. In contrast to yeast cls , where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

Arabidopsis thaliana L. (Brassicaceae) and its close relative Thellungiella salsuginea (Pallas) O.E. Schulz have been widely used as genetic models by researchers in their quest of understanding salt tolerance mechanisms in plants. Despite the fact that significant knowledge has been gained, both of these plants present some limitations mainly in relation to their response to salinity. Indeed, Arabidopsis is a glycophyte, whereas Thellungiella is a facultative halophyte. Among the Brassicaceae, Cakile maritima Scop. is an annual succulent obligate halophyte with a small size genome (1C = 719 Mb) and short life cycle. With these attributes, C. maritima presents a potential as a genetic model system to address salt stress adaptations at the molecular level in the quest to identify salt stress tolerance mechanisms. Beside their potential as promising model species, halophytes might also be valued for their potential as cash crops themselves. The present paper aims to highlight the main results gained on C. maritima using multidisciplinary approaches in complement to those obtained on plant model species of the Brassicaceae family.

Habitats on ultramafic substrate present a hostile environment for plant development. We aimed to determine whether any particular range of genome size is favoured in such habitats. Genome sizes of natural serpentinophyte populations were estimated using propidium iodide cytometry and compared with published data by phylogeny paired t-tests with plants from other substrata. The panel included 308 taxa belonging to 213 genera, with new values for 28 genera and 93 species. Using Leitch’s criteria, 56 % taxa belong to the group very small genomes (1C ≤ 1.4 pg), 22 % to small (1.4–3.5 pg), 19 % to intermediary (3.5–14 pg), 3 % to large (14–35 pg) and 0.31 % to very large (1C ≥ 35 pg). The majority of species were either indifferent for substrate (56 %) or facultative serpentinophytes (33 %). Most obligate serpentinophytes possessed very small genomes, and none exceeded 5 pg (1C). On average, plants growing on serpentine exhibited lower Cx-values than the same taxa growing on other soil types. About 4 % of species were annuals and 88 % perennials. Hemicryptophytes were dominant. Presence of at least two ploidy levels was recorded for 10 species. Water stress, high temperatures and presence of heavy metals in serpentine habitats impose a high selective pressure and favour perennial species with very small genomes.

International audience; In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at post-embryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used 3-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the post-embryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies, but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. This article is protected by copyright. All rights reserved.

Belonging to the genus Cenchrus with 16–22 species, Cenchrus ciliaris L. (syn. Pennisetum ciliare (L.) Link, buffelgrass) is a perennial, common in warmer regions of both hemispheres, growing as a C4 grass in a wide range of habitats. In the present study we determined chromosome number and nuclear DNA content (2C DNA) for 28 natural populations collected from northern to southern Tunisia. Three ploidy levels were found: one tetraploid population (2n = 4x = 36), three pentaploid (2n = 5x = 45), and 24 hexaploid populations (2n = 6x = 54). The hexaploid chromosome number has already been reported for Tunisian populations of C. ciliaris but tetraploid and pentaploid (2n = 45) are new for this area. The tetraploid population was found in the semi-arid north; pentaploids were mostly on the northern side of the arid region, while the hexaploids were located mainly in the arid southern Tunisian and Saharan region. 2C DNA values, assessed using flow cytometry, correlated with chromosome counts. Nuclear DNA content ranged from 2C = 3.03 to 4.61 pg, revealing three ploidy levels corresponding to 4x, 5x, 6x, and mean 2C DNA amounts were of 3.03, 3.7 and 4.48 pg, respectively. Each cytotype produced viable pollen. Flow cytometric seed screening neither proved nor disproved apomixis. The most frequent hexaploid populations seem best adapted to arid conditions in southern Tunisia. The monoploid value, 1Cx, was constant. The existence of pentaploid cytotype suggests hybridization ability between tetraploids and hexaploids. It appears that polyploidization is the major evolutionary mechanism in the speciation of C. ciliaris.

International audience; Bananas (Musa spp.), including dessert and cooking types, are giant perennial monocotyledonous herbs of the order Zingiberales, a sister group to the well-studied Poales, which include cereals. Bananas are vital for food security in many tropical and subtropical countries and the most popular fruit in industrialized countries. The Musa domestication process started some 7,000 years ago in Southeast Asia. It involved hybridizations between diverse species and subspecies, fostered by human migrations, and selection of diploid and triploid seedless, parthenocarpic hybrids thereafter widely dispersed by vegetative propagation. Half of the current production relies on somaclones derived from a single triploid genotype (Cavendish). Pests and diseases have gradually become adapted, representing an imminent danger for global banana production. Here we describe the draft sequence of the 523-megabase genome of a Musa acuminata doubled-haploid genotype, providing a crucial stepping-stone for genetic improvement of banana. We detected three rounds of whole-genome duplications in the Musa lineage, independently of those previously described in the Poales lineage and the one we detected in the Arecales lineage. This first monocotyledon high-continuity whole-genome sequence reported outside Poales represents an essential bridge for comparative genome analysis in plants. As such, it clarifies commelinid-monocotyledon phylogenetic relationships, reveals Poaceae-specific features and has led to the discovery of conserved non-coding sequences predating monocotyledon-eudicotyledon divergence.

International audience; Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.

International audience; BACKGROUND AND AIMS: Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined. METHODS: Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species. KEY RESULTS AND CONCLUSIONS: Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.

International audience; Thirty-three populations belonging to the three Retama species, Retama monosperma, Retama raetam and Retama sphaerocarpa, were collected to study species differentiation using flow cytometry for 2C DNA assessment and molecular cytogenetics for karyotype organisation. All were 2n=48. Genome size ranged from 1.76 to 1.97 pg and revealed significant intraspecific variation correlated to the geographic distribution of the populations. The number and position of the two ribosomal gene families 5S and 45S were determined by fluorescent in situ hybridization, revealing chromosome reorganisation between species. In R. raetam and R. monosperma, the minor 5S loci co-localised with 45S on the satellite chromosome pair. Fluorochrome banding identified GC- and AT-rich DNA regions. In R. monosperma a unique chromomycin positive GC-rich band was observed associated with the secondary constriction. In contrast, an original pattern showing two chromomycin positive bands localised at each side of the extended rDNA locus was observed in R. sphaerocarpa and R. raetam. The polymorphism revealed in our cytogenetic data allowed us to separate the group of R. raetam and R. monosperma from R. sphaerocarpa

Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.

Background information. The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype). Results. We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.

The genus Ramonda includes three preglacial paleoendemic species surviving as the rare resurrection angiosperms of the Northern hemisphere in refugia habitats in the Balkan (Ramonda nathaliae and Ramonda serbica) and Iberian Peninsulas (Ramonda myconi). This study focuses on: assessing genome size and base composition, determining chromosome number and ploidy level in several populations, evaluating inter- and intra-specific variations in DNA content and chromosome number as well as looking for the possible hybridization in the sympatric zones of Balkan species. R. nathaliae and R. myconi are diploid species (2n = 2x = 48) while R. serbica is hexaploid (2n = 6x = 144). The mean 2C DNA values ranged from 2.30 pg for R. nathaliae to 2.59 pg for R. myconi compared to 7.91 pg for R. serbica. The base composition for R. nathaliae was 42.1% GC, for R. myconi 39.9% and for R. serbica 41.2%. In one population of R. serbica the DNA content ranged from 2C = 7.65 to 11.82 pg, revealing different ploidy levels among its individuals. In sympatric populations genome size was intermediary (∼5 pg) between the diploid and hexaploid classes which indicates the hybridization ability between R. serbica and R. nathaliae. It appears that polyploidization is the major evolutionary mechanism in the genus Ramonda.

Original and simple procedures for glassy carbon electrode modification with polyoxometalates (POMs), phosphotungstate [H7P8W48O184]33-, and Co(II)-containing silicotungstates [Co6(H2O)30{Co9Cl2(OH)3(H2O)9(β-SiW8O31)3}]5- and [{Co3(B-β-SiW9O33(OH))(B-β-SiW8O29OH)2}2]22- give stable and very active surfaces for the hydrogen-evolution reaction (HER). For this purpose, the selected POMs fixed on Vulcan XC-72 were adsorbed on the electrode surface or were directly entrapped in polyvinylpyridine films on the electrode. Cyclic voltammetry and confocal microscopy results converge to indicate that the activation is related to the proton and electron reservoir-like behaviors of these molecular oxides and not to any electrode surface area increase. However, the Tafel parameters of the HER process, which are different from one POM to the next, are in the range of those of the best metallic electrodes.

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.

Background: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in critical limb ischemia patients. Fibroblast growth factors (FGF) play a major role in angiogenesis and wound healing. The purpose of this study was to evaluate the effects of FGF and VEGF on human ASCs proliferation, migration and potential endothelial differentiation regards to their use as endothelial cell (EC) substitutes. Methods: ASCs were isolated from clinical lipoaspirates and cultured in M199 medium with FBS (10%), FGF2 (10ng/ml), VEGF (50ng/ml) or combinations of FGF2 and VEGF. Cell proliferation rates, viability, and migration were measured by growth curves, MTT and scratch assays. For cell attachment determinations, ASCs were seeded onto scaffold (small intestinal submucosa) for 5d. Endothelial differentiation capabilities of ASC were confirmed by expression of EC specific markers using qPCR, immunofluorescence staining and cord formation on Matrigel. PD173074, a selective inhibitor of FGF receptor, was used to confirm the importance of FGF signaling. Results: Increased proliferation rates were observed in ASCs treated with FGF2 when compared to ASCs cultured with VEGF and control groups (3.2 fold, p Conclusions: Our results indicate that: 1) FGF is an ASC promoter for proliferation, migration, attachment and endothelial differentiation; 2) FGF and VEGF have a co-stimulatory effect on ASC endotheliogenesis; and 3) FGF signaling mediates ASC EC differentiation. These results suggest that ASCs with enhanced FGF2 signaling may potentially be used for tissue engineering and cell-based therapies in critical limb ischemia patients.

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcAp2S). Genetic algorithm-based computational docking and filtering suggested that GlcAp2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcAp2S and 2-N-sulfonated glucosamine residues, labeled as HS2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcAp2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-O-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences.

Intraspecific variation in nuclear DNA content in 19 populations of Armeria maritima was investigated. Significant differences in DNA amount among ecogeographic groups and among populations were observed. These differences are related to geographic origin, with Californian populations having 7 % more DNA than their European counterparts. Within Europe, the four ecogeographic groups tested are not distinct, whatever their geographic origin (maritime versus continental) and their ecological characteristics (heavy-metal polluted versus non-toxic soils). However, significant differences in DNA amount appeared among population clusters identified from biochemical classification of European populations based on flavonoid compounds and isozyme markers. Variation in genome size among populations was found to be significantly correlated to variation in pollen size. Weak interpopulational variation has been detected for the nucleotidic base ratio, but was not correlated to variation in DNA amount.

Flow-cytometric analysis was carried out to determine the effect of low temperature on the changes in proportion of 2C:4C complement of DNA in root cells from three different zones (meristem apex, elongation and differentiation) in seedlings of Medicago sativa cv. Sitel. 2C:4C complement of DNA in cells were estimated after the treatments of the roots at control temperature of24°C, elevated temperatures of26°C, 30°C, 37°C and low temperatures—10°C and 5°C (in three different periods of duration 15, 38, 45 days). The observed results showed that at control temperature percentage of nuclei 2C:4C in meristem apex is 65:35, in elongation zone 50:50 and in differentiation zone is 40:60. The elevated temperatures (26°C, 30°C, 37°C) influenced this proportion very slightly while treatments of roots with low temperature especially in a period of 45 days changed 2C:4C complement of DNA drastically. It was found that root treated for 45 days at temperature of 10°C the 2C:4C in meristem apex is 78:22, elonga...

Four wild Ribes species (Ribes alpinum L., Ribes petraeum Wulf., Ribes rubrum L., and Ribes uva- crispa L.; all 2n = 2x = 16) were surveyed for their chromosome and genome organisation. Their genome size was assessed using flow cytometry. Ribes alpinum had 5.3% more nuclear DNA than did the three other species, whose average was 2C = 1.91 pg with 40.4% GC. In addition, GC- and AT-rich heterochromatin and rDNA (18S–5.8S–26S and 5S) patterns were studied using fluorochrome banding and double-target fluorescence in situ hybridization (FISH), respectively. Only GC-rich heterochromatin was detected, co-localizing with 18S–26S rDNA. Fluorochrome banding and FISH patterns revealed marked differences between species. Ribes alpinum and R. uva-crispa differed from R. rubrum and R. petraeum by the number of 18S–26S sites and the localization of 5S rDNA. Ribes alpinum and R. uva-crispa were differentiated by the number of 5S sites. Ribes rubrum and R. petraeum also differed by the number of 5S sites and by the size of the GC-rich band on the satellite chromosome pair. These results should contribute to a better understanding of phylogenetic relationships among these species.Key words: Ribes, flow cytometry, fluorochrome banding, FISH, rDNA, NORs.

This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on beta-COP-like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian beta-COP proteins. In mammalian cells, beta-COP proteins are part of a complex named the coatomer, which probably recruits some specific areas of the endomembrane system. Immunofluorescence analyses by confocal laser scanning microscopy showed that beta-COP-like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (gamma-COP, beta'-COP and Arf1 proteins) were immunolocalized on the same membraneous structures as beta-COP. Moreover, beta-COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of beta-COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on trafficking events in plant cells. They also report for the first time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant-specific way.

The genome sizes of eight species from different wild population of diploid Medicago with origin from Algeria have been assessed by flow cytometry. This parameter vary from 2C=0.94 pg in Medicago orbicularis (the smallest genome) to 2C=1.80 pg in Medicago laciniata (the largest one). The relationship between genome size and competence for direct somatic embryo formation in liquid medium for four of the tested species (M. orbicularis Orbi DZ19 C 2C=0.94; M. truncatula Tru DZ 2B 2C=1.08; M.scutelata Scu 274 2C=1.11; M. arabica Ara 46 Ina 2C=1.22) was studied. It was found that Medicago orbicularis with the smallest genome size formed somatic embryos for a shortest period of time, with a high percent of reacting explants and number of somatic embryos per explant. It was followed by M. truncatula, M. scutelata and M. arabica.

The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.

Summary Imaging or quantifying protein synthesis in cellulo through a well-resolved analysis of the cell cycle (also defining G1 subcompartments) is a methodological challenge. Click chemistry is the method of choice to reveal the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) and track proliferating nuclei undergoing DNA synthesis. However, the click reaction quenches fluorescent proteins. Our challenge was to reconcile these two tools. A robust protocol based on a high-resolution cytometric cell cycle analysis in tobacco (Nicotiana tabacum) BY2 cells expressing fluorescent Golgi markers has been established. This was broadly applicable to tissues, cell clusters, and other eukaryotic material, and compatible with Scale clearing. EdU was then used with the photoconvertible protein sialyl transferase (ST)-Kaede as a Golgi marker in a photoconversion pulse-chase cytometric configuration resolving, in addition, subcompartments of G1. Quantitative restoration of protein fluorescence was achieved by introducing acidic EDTA washes to strip the copper from these proteins which were then imaged at neutral pH. The rate of synthesis of this Golgi membrane marker was low during early G1, but in the second half of G1 (30% of cycle duration) much of the synthesis occurred. Marker synthesis then persisted during S and G2. These insights into Golgi biology are discussed in terms of the cell's ability to adapt exocytosis to cell growth needs.

Background and Aims Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities. Methods Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined. Key Results Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance. Conclusions Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.

Genome size has been studied for the first time in the Colchicum genus. Values obtained by flow cytometry were quite stable and specific to each taxon: C. autumnale L., 2C=5.89±0.22 pg, equivalent to 5.7×10 9 bp (2 n=4 x=36); C. alpinum DC., 2C=8.06±0.24 pg, equivalent to 7.8×10 9 bp (2 n=6 x=56); C. lusitanum Brot., 2C=10.7±0.67 pg, equivalent to 10.3×10 9 bp (2 n= approx. 10 x=90–92 and 94–96); C. multiflorum Brot., 2C=16.5±0.69 pg, equivalent to 15.9×10 9 bp, (2 n= approx. 16 x=140–148); C. corsicum Baker, 2C=21.3±0.99 pg, equivalent to 20.6×10 9 bp (2 n=22 x= approx. 198±2). These values are well below those published for Liliaceae stricto sensu. In Colchicum species from the western Mediterranean area, genome size was highly correlated with ploidy level ( R 2=0.99, P

Heparan sulfate (HS) polysaccharide chains have been shown to orchestrate distinct biological functions in several systems. Study of HS structure-function relations is, however, hampered due to the lack of availability of HS in sufficient quantities as well as the molecular heterogeneity of naturally occurring HS. Enzymatic synthesis of HS is an attractive alternative to the use of naturally occurring HS, as it reduces molecular heterogeneity, or a long and daunting chemical synthesis of HS. Heparosan, produced by E. coli K5 bacteria, has a structure similar to the unmodified HS backbone structure and can be used as a precursor in the enzymatic synthesis of HS-like polysaccharides. Here, we describe an enzymatic approach to synthesize several specifically sulfated HS polysaccharides for biological studies using the heparosan backbone and a combination of recombinant biosynthetic enzymes such as C5-epimerase and sulfotransferases.