A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division.

In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

Lineage- and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells.

Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue in which other cells transition from proliferation to postmitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We show that the D-type cyclin, CYCD7;1, is specifically expressed just prior to the symmetric guard cell-forming division, and that it is limiting for this division. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR1-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell type-specific transcription factors that confine its expression to the appropriate developmental window.

Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, yet the components and structure of the STM gene regulatory network (GRN) are largely unknown. Here, we show that transcriptional regulators are overrepresented among STM-regulated genes and, using these as GRN components in Bayesian network analysis, we infer STM GRN associations and reveal regulatory relationships between STM and factors involved in multiple aspects of SAM function. These include hormone regulation, TCP-mediated control of cell differentiation, AIL/PLT-mediated regulation of pluripotency and phyllotaxis, and specification of meristem-organ boundary zones via CUC1. We demonstrate a direct positive transcriptional feedback loop between STM and CUC1, despite their distinct expression patterns in the meristem and organ boundary, respectively. Our further finding that STM activates expression of the CUC1-targeting microRNA miR164c combined with mathematical modelling provides a potential solution for this apparent contradiction, demonstrating that these proposed regulatory interactions coupled with STM mobility could be sufficient to provide a mechanism for CUC1 localisation at the meristem-organ boundary. Our findings highlight the central role for the STM GRN in coordinating SAM functions.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.

Lack of specificity associated with using molecular beacons in loop mediated amplification assays.

BACKGROUND: Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. RESULTS: We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal. CONCLUSIONS: Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.

DEFECTIVE KERNEL 1 promotes and maintains plant epidermal differentiation.

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis.

Differences in bleeding phenotype and provider interventions in postmenarchal adolescents when compared to adult women with bleeding disorders and heavy menstrual bleeding.

INTRODUCTION: Due to lack of patient/health care provider awareness causing delayed diagnosis, the bleeding phenotype and provider interventions in adolescents with heavy menstrual bleeding (HMB) and bleeding disorders (BD) may be different when compared to adults. AIM: The aim of this study was to compare/characterize bleeding phenotype and provider interventions in postmenarchal adolescents < 18 years and premenopausal adults ≥ 18 years with HMB and BD. METHODS: Patient demographics, BD, and provider interventions/therapy details for HMB were compared between both age groups enrolled in the Centers for Disease Control and Prevention (CDC) Female Universal Data Collection (UDC) surveillance project in United States hemophilia treatment centres. Cross-sectional descriptive analyses including frequency distributions, summary statistics, bivariate and logistic regression analyses were performed. RESULTS: Of 269 females (79 adolescents; median age 16 years, interquartile range (IQR) = 2; 190 adults; median age 27 years, IQR = 13) evaluated, BD distribution was similar in both groups. Compared to adolescents, adults more often had family history of bleeding (Adjusted odds ratios [AOR] = 2.6, 1.3-5.6), delay in diagnosis (AOR = 2.5, 1.2-4.9), bleeding with dental procedures (AOR = 2.0, 1.0-4.0), gastrointestinal bleeding (AOR = 4.6, 1.0-21.9), anaemia (AOR = 2.7, 1.4-5.2), utilized desmopressin less often (AOR = 0.4, 0.2-0.8) and underwent gynaecologic procedure/surgery more frequently (AOR = 5.9, 1.3-27.3). CONCLUSION: Bleeding phenotypes of adolescents and adults with HMB and BD were different with more frequent bleeding complications, anaemia, gynaecologic procedures/surgeries, less desmopressin use and more delay in diagnosing BD in adults. Longitudinal studies are needed to determine whether improved patient/provider awareness and education will translate to early diagnosis and timely management of BD/HMB in adolescents that may prevent/reduce future haematologic/gynaecologic complications.

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.

Reasons for cesarean and medically indicated deliveries in pregnancies in women with systemic lupus erythematosus.

Objective To determine reasons for cesarean and medically indicated deliveries in a registry of pregnant women with SLE compared to RA. Methods Pregnant women with SLE or RA were prospectively followed, and pregnancy outcomes were collected, including whether labor was spontaneous or medically indicated and delivery was vaginal or cesarean. Preterm birth was defined as a birth <37 weeks gestation. Differences in reasons for cesarean delivery and indication of delivery between term and preterm births were determined by Fisher's exact test. Results Compared to RA pregnancies, SLE pregnancies had modestly higher rates of preterm birth (24% SLE vs 14% RA), pre-eclampsia (15% SLE vs 7% RA), and cesarean delivery (48% SLE vs 30% RA). The majority of preterm births among women with SLE were indicated (70%), most commonly for pre-eclampsia or the health of the infant or mother. The majority of preterm births among women with RA, however, were spontaneous, primarily due to premature rupture of membranes. Conclusion Pre-eclampsia and maternal SLE activity appear to be the key drivers for the high rate of preterm birth and medically indicated delivery in SLE. This contrasts with RA, where preterm labor is most often due to spontaneous onset of labor.

Metabolic cost of running is greater on a treadmill with a stiffer running platform.

Exercise testing on motorised treadmills provides valuable information about running performance and metabolism; however, the impact of treadmill type on these tests has not been investigated. This study compared the energy demand of running on two laboratory treadmills: an HP Cosmos (C) and a Quinton (Q) model, with the latter having a 4.5 times stiffer running platform. Twelve experienced runners ran identical bouts on these treadmills at a range of four submaximal velocities (reported data is for the velocity that approximated 75-81% VO2max). The stiffer treadmill elicited higher oxygen consumption (C: 46.7 ± 3.8; Q: 50.1 ± 4.3 ml·kg-1 · min-1), energy expenditure (C: 16.0 ± 2.5; Q: 17.7 ± 2.9 kcal · min-1), carbohydrate oxidation (C: 9.6 ± 3.1; Q: 13.0 ± 3.9 kcal · min-1), heart rate (C: 155 ± 16; Q: 163 ± 16 beats · min-1) and rating of perceived exertion (C: 13.8 ± 1.2; Q: 14.7 ± 1.2), but lower fat oxidation (C: 6.4 ± 2.3; Q: 4.6 ± 2.5 kcal · min-1) (all analysis of variance treadmill comparisons P < 0.01). This study confirms that caution is required when comparing performance and metabolic results between different treadmills and suggests that treadmills will vary in their comparability to over-ground running depending on the running platform stiffness.

Dynamic Equilibrium of the Aurora†A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy.

The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure-based drug discovery.

Gluconeogenesis during endurance exercise in cyclists habituated to a long-term low carbohydrate high-fat diet.

KEY POINTS: Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis). We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization. ABSTRACT: Endogenous glucose production (EGP) occurs via hepatic glycogenolysis (GLY) and gluconeogenesis (GNG) and plays an important role in maintaining euglycaemia. Rates of GLY and GNG increase during exercise in athletes following a mixed macronutrient diet; however, these processes have not been investigated in athletes following a low carbohydrate high fat (LCHF) diet. Therefore, we studied seven well-trained male cyclists that were habituated to either a LCHF (7% carbohydrate, 72% fat, 21% protein) or a mixed diet (51% carbohydrate, 33% fat, 16% protein) for longer than 8 months. After an overnight fast, participants performed a 2 h laboratory ride at 72% of maximal oxygen consumption. Glucose kinetics were measured at rest and during the final 30 min of exercise by infusion of [6,6-(2) H2 ]-glucose and the ingestion of (2) H2 O tracers. Rates of EGP and GLY both at rest and during exercise were significantly lower in the LCHF group than the mixed diet group (Exercise EGP: LCHF, 6.0 ± 0.9 mg kg(-1)  min(-1) , Mixed, 7.8 ± 1.1 mg kg(-1)  min(-1) , P < 0.01; Exercise GLY: LCHF, 3.2 ± 0.7 mg kg(-1)  min(-1) , Mixed, 5.3 ± 0.9 mg kg(-1)  min(-1) , P < 0.01). Conversely, no difference was detected in rates of GNG between groups at rest or during exercise (Exercise: LCHF, 2.8 ± 0.4 mg kg(-1)  min(-1) , Mixed, 2.5 ± 0.3 mg kg(-1)  min(-1) , P = 0.15). We conclude that athletes on a LCHF diet do not compensate for reduced glucose availability via higher rates of glucose synthesis compared to athletes on a mixed diet. Instead, GNG remains relatively stable, whereas glucose oxidation and GLY are influenced by dietary factors.

Activation of CYCD7;1 in the central cell and early endosperm overcomes cell-cycle arrest in the Arabidopsis female gametophyte, and promotes early endosperm and embryo development.

In angiosperms, double fertilization of the egg and central cell of the megagametophyte leads to the development of the embryo and endosperm, respectively. Control of cell cycle progression in the megagametophyte is essential for successful fertilization and development. Central cell-targeted expression of the D-type cyclin CYCD7;1 (end CYCD7;1) using the imprinted FWA promoter overcomes cycle arrest of the central cell in the Arabidopsis female gametophyte in the unfertilized ovule, leading to multinucleate central cells at high frequency. Unlike FERTILIZATION-INDEPENDENT SEED (fis) mutants, but similar to lethal RETINOBLASTOMA-RELATED (rbr) mutants, no seed coat development is triggered. Unlike the case with loss of rbr, post-fertilization end CYCD7;1 in the endosperm enhances the number of nuclei during syncytial endosperm development and induces the partial abortion of developing seeds, associated with the enhanced size of the surviving seeds. The frequency of lethality was less than the frequency of multinucleate central cells, indicating that these aspects are not causally linked. These larger seeds contain larger embryos composed of more cells of wild-type size, surrounded by a seed coat composed of more cells. Seedlings arising from these larger seeds displayed faster seedling establishment and early growth. Similarly, two different embryo-lethal mutants also conferred enlarged seed size in surviving siblings, consistent with seed size increase being a general response to sibling lethality, although the cellular mechanisms were found to be distinct. Our data suggest that tight control of CYCD activity in the central cell and in the developing endosperm is required for optimal seed formation.

State of the art: von Willebrand disease.

The State of the Art in von Willebrand disease (VWD) has been impacted not only by discoveries in the field of haemostasis, but also by changes in practice in other fields. The development of bleeding assessment tools has led to the clarification of bleeding symptoms and phenotype in VWD. New discoveries in the biology and genetics of von Willebrand factor (VWF) are challenging our existing diagnostics and classification(s). An improved understanding of reproductive physiology and the pathology of VWD along with changing obstetric, gynaecologic and haemostatic therapies necessitate an evolving response to the care of women with VWD. The survival of patients with autoimmune disease, malignancies and congenital heart disease along with increasing use of circulatory support devices and extracorporeal membrane oxygenation is increasing the prevalence of acquired von Willebrand syndrome. In each of these challenges, there are opportunities to improve the care of our patients with VWD.

Von Willebrand factor for menorrhagia: a survey and literature review.

BACKGROUND: von Willebrand disease (VWD) is the most common congenital bleeding disorder. In women, menorrhagia is the most common bleeding symptom, and is disabling with iron deficiency anaemia, high health cost and poor quality of life. Current hormonal and non-hormonal therapies are limited by ineffectiveness and intolerance. Few data exist regarding von Willebrand factor (VWF), typically prescribed when other treatments fail. The lack of effective therapy for menorrhagia remains the greatest unmet healthcare need in women with VWD. Better therapies are needed to treat women with menorrhagia. METHODS: We conducted a survey of US haemophilia treatment centres (HTCs) and a literature review using medical subject heading (MeSH) search terms 'von Willebrand factor,' 'menorrhagia' and 'von Willebrand disease' to assess the use of VWF in menorrhagia. Analysis was by descriptive statistics. RESULTS: Of 83 surveys distributed to HTC MDs, 20 (24.1%) provided sufficient data for analysis. Of 1321 women with VWD seen during 2011-2014, 816 (61.8%) had menorrhagia, for which combined oral contraceptives, tranexamic acid and desmopressin were the most common first-line therapies for menorrhagia, whereas VWF was third-line therapy reported in 13 women (1.6%). Together with data from 88 women from six published studies, VWF safely reduced menorrhagia in 101 women at a dose of 33-100 IU kg(-1) on day 1-6 of menstrual cycle. CONCLUSIONS: This represents the largest VWD menorrhagia treatment experience to date. VWF safely and effectively reduces menorrhagia in women with VWD. A prospective clinical trial is planned to confirm these findings.

AINTEGUMENTA and the D-type cyclin CYCD3;1 independently contribute to petal size control in Arabidopsis: evidence for organ size compensation being an emergent rather than a determined property.

Plant lateral aerial organ (LAO) growth is determined by the number and size of cells comprising the organ. Genetic alteration of one parameter is often accompanied by changes in the other, such that the overall effect on final LAO size is minimized, suggested to be caused by an active organ level 'compensation mechanism'. For example, the aintegumenta (ant) mutant exhibits reduced cell number but increased cell size in LAOs. The ANT transcription factor regulates the duration of the cell division phase of LAO growth, and its ectopic expression is correlated with increased levels of the cell cycle regulator CYCD3;1. This has previously led to the suggestion that ANT regulates CYCD3;1. It is shown here that while ANT is required for normal cell proliferation in petals, CYCD3;1 is not, suggesting that ANT does not regulate CYCD3;1 during petal growth. Moreover CYCD3;1 expression was similar in wild-type and ant-9 flowers. In contrast to the compensatory changes between cell size and number in ant mutants, cycd3;1 mutants show increased petal cell size unaccompanied by changes in cell number, leading to larger organs. However, loss of CYCD3;1 in the ant-9 mutant background leads to a phenotype consistent with compensation mechanisms. These apparently arbitrary examples of compensation are reconciled through a model of LAO growth in which distinct phases of division and cell expansion occupy differing lengths of a defined overall growth window. This leads to the proposal that many observations of 'compensation mechanisms' might alternatively be more simply explained as emergent properties of LAO development.

Systems analysis of shoot apical meristem growth and development: integrating hormonal and mechanical signaling.

The shoot apical meristem (SAM) is a small population of stem cells that continuously generates organs and tissues. This review covers our current understanding of organ initiation by the SAM in Arabidopsis thaliana. Meristem function and maintenance involves two major hormones, cytokinins and auxins. Cytokinins appear to play a major role in meristem maintenance and in controlling meristematic properties, such as cell proliferation. Self-organizing transport processes, which are still only partially understood, lead to the patterned accumulation of auxin at particular positions, where organs will grow out. A major downstream target of auxin-mediated growth regulation is the cell wall, which is a determinant for both growth rates and growth distribution, but feedbacks with metabolism and the synthetic capacity of the cytoplasm are crucial as well. Recent work has also pointed at a potential role of mechanical signals in growth coordination, but the precise mechanisms at work remain to be elucidated.

Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis.

The aim of this study was to elucidate the fall in von Willebrand factor (VWF) and factor VIII activity (FVIII) after childbirth in women with and without von Willebrand disease (VWD). VWF:RCo, VWF:Ag, and FVIII were obtained in the third trimester of pregnancy, on admission for childbirth, and 10 times postpartum. Specimens were processed within 4 h and analysed centrally. Means were calculated at each time point. Forty women (40 pregnancies) without VWD and 32 women (35 pregnancies) with VWD were enrolled. 15/32 with VWD were treated (30% of those with type 1 and all of those with type 2) in 17 pregnancies. Treatments prior to delivery consisted of desmopressin (2/17), VWF concentrate (15/17) and after delivery VWF concentrate (16/17). Duration of treatment was 0-21 days (median 6). VWF levels peaked at 250% of baseline--4 h postpartum in women with VWD and 12 h postpartum in women without VWD. Thereafter, VWF levels fell rapidly, approached baseline at 1 week and reached baseline at 3 weeks. Except immediately postpartum, when the levels among treated cases were higher, levels among women with VWD appeared to parallel, but were lower than those among women without VWD. Levels were lowest among those who received treatment. VWF levels fall rapidly after childbirth. Except immediately postpartum, current treatment strategies do not raise VWF levels to the levels of women without VWD or even to the levels of women with milder, untreated VWD. Consequently, women with VWD may be at risk of postpartum haemorrhage despite treatment.