Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3-MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3-MYB transcription factors (GdMYBSG6-1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3-MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data.

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes; however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated Pacific Biosciences (PacBio) long-read data of the genomes of three relatives of the model plant Arabidopsis thaliana and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all, of these misjoints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres were fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences.

Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2.

The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.

EARLY FLOWERING4 recruitment of EARLY FLOWERING3 in the nucleus sustains the Arabidopsis circadian clock.

The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms.

A reduced-function allele reveals that EARLY FLOWERING3 repressive action on the circadian clock is modulated by phytochrome signals in Arabidopsis.

Arabidopsis thaliana EARLY FLOWERING3 (ELF3) is essential for the generation of circadian rhythms. ELF3 has been proposed to restrict light signals to the oscillator through phytochrome photoreceptors, but that has not been explicitly shown. Furthermore, the genetic action of ELF3 within the clock had remained elusive. Here, we report a functional characterization of ELF3 through the analysis of the elf3-12 allele, which encodes an amino acid replacement in a conserved domain. Circadian oscillations persisted, and unlike elf3 null alleles, elf3-12 resulted in a short circadian period only under ambient light. The period shortening effect of elf3-12 was enhanced by the overexpression of phytochromes phyA and phyB. We found that elf3-12 was only modestly perturbed in resetting of the oscillator and in gating light-regulated gene expression. Furthermore, elf3-12 essentially displayed wild-type development. We identified targets of ELF3 transcriptional repression in the oscillator, highlighting the action at the morning gene PSEUDO-RESPONSE REGULATOR9. Taken together, we identified two separable roles for ELF3, one affecting the circadian network and the other affecting light input to the oscillator. This is consistent with a dual function of ELF3 as both an integrator of phytochrome signals and a repressor component of the core oscillator.

A protocol for resuscitation of severe burn patients guided by transpulmonary thermodilution and lactate levels: a 3-year prospective cohort study.

INTRODUCTION: The use of urinary output and vital signs to guide initial burn resuscitation may lead to suboptimal resuscitation. Invasive hemodynamic monitoring may result in over-resuscitation. This study aimed to evaluate the results of a goal-directed burn resuscitation protocol that used standard measures of mean arterial pressure (MAP) and urine output, plus transpulmonary thermodilution (TPTD) and lactate levels to adjust fluid therapy to achieve a minimum level of preload to allow for sufficient vital organ perfusion. METHODS: We conducted a three-year prospective cohort study of 132 consecutive critically burned patients. These patients underwent resuscitation guided by MAP (>65 mmHg), urinary output (0.5 to 1 ml/kg), TPTD and lactate levels. Fluid therapy was adjusted to achieve a cardiac index (CI) >2.5 L/minute/m² and an intrathoracic blood volume index (ITBVI) >600 ml/m2, and to optimize lactate levels. Statistical analysis was performed using mixed models. We also used Pearson or Spearman methods and the Mann-Whitney U-test. RESULTS: A total of 98 men and 34 women (mean age, 48 ± 18 years) was studied. The mean total body surface area (TBSA) burned was 35% ± 22%. During the early resuscitation phase, lactate levels were elevated (2.58 ± 2.05 mmol/L) and TPTD showed initial hypovolemia by the CI (2.68 ± 1.06 L/minute/m²) and the ITBVI (709 ± 254 mL/m²). At 24 to 32 hours, the CI and lactic levels were normalized, although the ITBVI remained below the normal range (744 ± 276 ml/m²). The mean fluid rate required to achieve protocol targets in the first 8 hours was 4.05 ml/kg/TBSA burned, which slightly increased in the next 16 hours. Patients with a urine output greater than or less than 0.5 ml/kg/hour did not show differences in heart rate, mean arterial pressure, CI, ITBVI or lactate levels. CONCLUSIONS: Initial hypovolemia may be detected by TPTD monitoring during the early resuscitation phase. This hypovolemia might not be reflected by blood pressure and hourly urine output. An adequate CI and tissue perfusion can be achieved with below-normal levels of preload. Early resuscitation guided by lactate levels and below-normal preload volume targets appears safe and avoids unnecessary fluid input.

Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency.

Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucine-zipper (bZIP) transcription factor gene family, bZIP19 and bZIP23, that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt- and Irt-related protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils.

Ventilation practices in burn patients-an international prospective observational cohort study.

BACKGROUND: It is unknown whether lung-protective ventilation is applied in burn patients and whether they benefit from it. This study aimed to determine ventilation practices in burn intensive care units (ICUs) and investigate the association between lung-protective ventilation and the number of ventilator-free days and alive at day 28 (VFD-28). METHODS: This is an international prospective observational cohort study including adult burn patients requiring mechanical ventilation. Low tidal volume (V T) was defined as V T ≤ 8 mL/kg predicted body weight (PBW). Levels of positive end-expiratory pressure (PEEP) and maximum airway pressures were collected. The association between V T and VFD-28 was analyzed using a competing risk model. Ventilation settings were presented for all patients, focusing on the first day of ventilation. We also compared ventilation settings between patients with and without inhalation trauma. RESULTS: A total of 160 patients from 28 ICUs in 16 countries were included. Low V T was used in 74% of patients, median V T size was 7.3 [interquartile range (IQR) 6.2-8.3] mL/kg PBW and did not differ between patients with and without inhalation trauma (p = 0.58). Median VFD-28 was 17 (IQR 0-26), without a difference between ventilation with low or high V T (p = 0.98). All patients were ventilated with PEEP levels ≥5 cmH2O; 80% of patients had maximum airway pressures <30 cmH2O. CONCLUSION: In this international cohort study we found that lung-protective ventilation is used in the majority of burn patients, irrespective of the presence of inhalation trauma. Use of low V T was not associated with a reduction in VFD-28. TRIAL REGISTRATION: Clinicaltrials.gov NCT02312869. Date of registration: 9 December 2014.

Asymmetric Transcription Factor Partitioning During Yeast Cell Division Requires the FACT Chromatin Remodeler and Cell Cycle Progression.

The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effects of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved facilitates chromatin transcription (FACT) chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2, and for localization of the RAM network components.

Impact of acetate- or citrate-acidified bicarbonate dialysate on ex vivo aorta wall calcification.

Vascular calcification is highly prevalent in patients with chronic hemodialysis. Increased acetatemia during hemodialysis sessions using acetate-acidified bicarbonate has also been associated with several abnormalities, By contrast, these abnormalities were not induced by citrate-acidified bicarbonate dialysis. Moreover, citrate is biocompatible alternative to acetate in dialysis fluid. However, the effects of citrate on vascular calcification during hemodialysis had not been studied in detail. This study analyzed herein the effects of acetate- or citrate-acidified bicarbonate dialysis on vascular calcification. Citrate has been shown to inhibit calcification in urine in hemodialysis patients. Therefore, our hypothesis is that citrate-acidified bicarbonate dialysis could reduce vascular calcification. Blood samples before and after hemodialysis from patients on acetate- or citrate-acidified bicarbonate dialysis were collected in heparin-containing tubes (n = 35 and n = 25 respectively). To explore the effect of pre- and post-dialysis plasmatic bicarbonate and citrate on vascular calcification, rats aortic rings cultured ex vivo in Minimum Essential Medium containing 0.1% FBS and 45-calcium as radiotracer were used (n = 24). After 7 days of incubation aortic rings were dried, weighed and radioactivity was measured via liquid scintillation counting. Bicarbonate levels increase calcium accumulation in rat aortic wall in a dose-response manner (pH = 7.4). Moreover, citrate prevents calcium accumulation, with a mean inhibitor concentration (IC50) value of 733 µmol/L. During acetate-acidified bicarbonate dialysis, bicarbonate and citrate levels in plasma increase (22.29 ± 3.59 versus 28.63 ± 3.56 mmol/L; p < 0.001) and decrease (133.3 ± 53.6 versus 87.49 ± 32.3 µmol/L, p < 0.001), respectively. These changes in pos-hemodialysis plasma significantly (p < 0.001) alter calcium accumulation in the aortic wall (38.9% higher). Moreover, citrate-acidified bicarbonate dialysis increases post-hemodialysis citrate levels 5-fold (145 ± 79.8 versus 771.6 ± 184.3 µmol/L), reducing calcium accumulation in the aortic wall. Citrate-acidified bicarbonate dialysis reduces plasmatic calcium and pH variations during dialysis session (Δ[Ca2+] = -0.019 ± 0.089; ΔpH = 0.098 ± 0.043) respect to acetate-acidified bicarbonate dialysis (Δ[Ca2+] = 0.115 ± 0.118; ΔpH = 0.171 ± 0.078). To our knowledge, our study is the first to show that citrate protects against calcium accumulation in rat aortic walls ex vivo. Therefore, citrate-acidified bicarbonate dialysis may be an alternative approach to reduce calcification in hemodialysis patients without additional cost.

Prospective randomised multicentre study to demonstrate the benefits of haemodialysis without acetate (with citrate): ABC-treat Study. Acute effect of citrate. / Estudio prospectivo aleatorizado multicéntrico para demostrar los beneficios de la hemodiálisis sin acetato (con citrato): Estudio ABC-treat. Efecto agudo del citrato.

INTRODUCTION: Dialysis fluid (DF), an essential element in hemodialysis (HD), is manufactured in situ by mixing three components: treated water, bicarbonate concentrate and acid concentrate. To avoid the precipitation of calcium and magnesium carbonate that is produced in DF by the addition of bicarbonate, it is necessary to add an acid. There are 2 acid concentrates that contain acetate (ADF) or citrate (CDF) as a stabilizer. OBJECTIVE: To compare the acute effect of HD with CDF vs. ADF on the metabolism of calcium, phosphorus and magnesium, acid base balance, coagulation, inflammation and hemodynamic stability. METHODS: Prospective, multicenter, randomized and crossed study, of 32 weeks duration, in patients in three-week HD, AK-200-Ultra-S or Artis monitor, 16 weeks with ADF SoftPac®, prepared with 3mmol/L of acetate, and 16 weeks with CDF SelectBag Citrate®, with 1mmol/L of citrate. Patients older than 18 years were included in HD for a minimum of 3 months by arteriovenous fistula. Epidemiological, dialysis, pre and postdialysis biochemistry, episodes of arterial hypotension, and coagulation scores were collected monthly during the 8 months of the study. Pre and post-dialysis analysis were extracted: venous blood gas, calcium (Ca), ionic calcium (Cai), phosphorus (P), magnesium (Mg) and parathyroid hormone (PTH) among others. ClinicalTrials.gov NCT03319680. RESULTS: We included 56 patients, 47 (84%) men and 9 (16%) women, mean age: 65.3 (16.4) years, technique HD/HDF: 20 (35.7%)/36 (64.3%). We found differences (p<0.05) when using the DF with citrate (C) versus acetate (A) in the postdialysis values of bicarbonate [C: 26.9 (1.9) vs. A: 28.5 (3) mmol/L], Cai [C: 1.1 (0.05) vs. A: 1.2 (0.08) mmol/L], Mg [C: 1.8 (0.1) vs A: 1, 9 (0.2) mg/dL] and PTH [C: 255 (172) vs. 148 (149) pg/mL]. We did not find any differences in any of the parameters measured before dialysis. Of the 4,416 sessions performed, 2,208 in each group, 311 sessions (14.1%) with ADF and 238 (10.8%) with CDF (p<0.01), were complicated by arterial hypotension. The decrease in maximum blood volume measured by Hemoscan® biosensor was also lower [-3.4 (7.7) vs -5.1 (8.2)] although without statistical significance. CONCLUSION: Dialysis with citrate acutely produces less postdialysis alkalemia and significantly modifies Ca, Mg and PTH. CDF has a positive impact on hemodynamic tolerance.

Prevalence, clinical consequences and management of acute faecal incontinence with diarrhoea in the ICU: The FIRST™ Observational Study.

There are limited data on the incidence and management of acute faecal incontinence with diarrhoea in the ICU. The FIRST™ Observational Study was undertaken to obtain data on clinical practices used in the ICU for the management of acute faecal incontinence with diarrhoea in Germany, UK, Spain and Italy. ICU-hospitalised patients ≥18 years of age experiencing a second episode of acute faecal incontinence with diarrhoea in 24 h were recruited, and management practices of acute faecal incontinence with diarrhoea were recorded for up to 15 days. A total of 372 patients had complete data sets; the mean duration of study was 6.8 days. At baseline, 40% of patients experienced mild or moderate-to-severe skin excoriation, which increased to 63% in patients with acute faecal incontinence with diarrhoea lasting >15 days. At baseline, 27% of patients presented with a pressure ulcer, which increased to 37%, 45% and 49% at days 5, 10 and 15, respectively. Traditional methods (pads, sheets and tubes) were more commonly used compared to faecal management systems during days 1-4 (76% vs. 47% faecal management system), while the use of a faecal management system increased to 56% at days 5-9 and 61% at days 10-15. At baseline, only 26% of nurses were satisfied with traditional management methods compared to 69% with faecal management systems. For patients still experiencing acute faecal incontinence with diarrhoea after 15 days, 82% of nurses using a faecal management systems to manage acute faecal incontinence with diarrhoea were satisfied or very satisfied, compared to 37% using traditional methods. These results highlight that acute faecal incontinence with diarrhoea remains an important healthcare challenge in ICUs in Europe; skin breakdown and pressure ulcers remain common complications in patients with acute faecal incontinence with diarrhoea in the ICU.

Traumatismo por proyectil de arteria poplítea / Trauma by projectile in popliteal artery

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Natural variation reveals that intracellular distribution of ELF3 protein is associated with function in the circadian clock.

Natural selection of variants within the Arabidopsis thaliana circadian clock can be attributed to adaptation to varying environments. To define a basis for such variation, we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant inbred line and localized heritable variation. Extensive variation led us to identify EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal nucleotide polymorphism caused a short-period phenotype under light and severely dampened rhythm generation in darkness, and entrainment alterations resulted. We found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability to accumulate in darkness was compromised. Evidence was provided that the ELF3-Sha allele originated in Central Asia. Collectively, we showed that ELF3 protein plays a vital role in defining its light-repressor action in the circadian clock and that its functional abilities are largely dependent on its cellular localization.

Time for a nuclear meeting: protein trafficking and chromatin dynamics intersect in the plant circadian system.

Circadian clocks mediate adaptation to the 24-h world. In Arabidopsis, most circadian-clock components act in the nucleus as transcriptional regulators and generate rhythmic oscillations of transcript accumulation. In this review, we focus on post-transcriptional events that modulate the activity of circadian-clock components, such as phosphorylation, ubiquitination and proteasome-mediated degradation, changes in cellular localization, and protein-protein interactions. These processes have been found to be essential for circadian function, not only in plants, but also in other circadian systems. Moreover, light and clock signaling networks are highly interconnected. In the nucleus, light and clock components work together to generate transcriptional rhythms, leading to a general control of the timing of plant physiological processes.