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Adenosine triphosphate (ATP) synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. From yeast to vertebrates, cryoelectron tomograms of these membranes have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. The rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. In this study, we present a quantitative molecular-simulation analysis that strongly suggests that the dimers of ATP synthases organize into rows spontaneously, driven by a long-range attractive force that arises from the relief of the overall elastic strain of the membrane. The strain is caused by the V-like shape of the dimers, unique among membrane protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of direct protein-protein interactions or a specific lipid composition of the membrane. We further hypothesize that, once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations by causing macroscopic membrane ridges that ultimately become the edges of cristae. In this way, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline key experiments that would be required to verify or refute this hypothesis., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Background: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models., Results: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models., Conclusions: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization., (© 2018 Plant & Food Research New Phytologist © 2018 New Phytologist Trust.)

We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III 2 was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I 1 III 2 assembly in bovine heart and the yeast Yarrowia lipolytica , but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III 2 , but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

There are no approved drugs for the treatment of heart failure with preserved ejection fraction (HFpEF), which is characterized by left ventricular (LV) diastolic dysfunction. We demonstrate that ITF2357 (givinostat), a clinical-stage inhibitor of histone deacetylase (HDAC) catalytic activity, is efficacious in two distinct murine models of diastolic dysfunction with preserved EF. ITF2357 blocked LV diastolic dysfunction due to hypertension in Dahl salt-sensitive (DSS) rats and suppressed aging-induced diastolic dysfunction in normotensive mice. HDAC inhibitor-mediated efficacy was not due to lowering blood pressure or inhibiting cellular and molecular events commonly associated with diastolic dysfunction, including cardiac fibrosis, cardiac hypertrophy, or changes in cardiac titin and myosin isoform expression. Instead, ex vivo studies revealed impairment of cardiac myofibril relaxation as a previously unrecognized, myocyte-autonomous mechanism for diastolic dysfunction, which can be ameliorated by HDAC inhibition. Translating these findings to humans, cardiac myofibrils from patients with diastolic dysfunction and preserved EF also exhibited compromised relaxation. These data suggest that agents such as HDAC inhibitors, which potentiate cardiac myofibril relaxation, hold promise for the treatment of HFpEF in humans., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Black raspberry ( Rubus occidentalis L.) is a niche fruit crop valued for its flavor and potential health benefits. The improvement of fruit and cane characteristics via molecular breeding technologies has been hindered by the lack of a high-quality reference genome. The recently released draft genome for black raspberry (ORUS 4115-3) lacks assembly of scaffolds to chromosome scale. We used high-throughput chromatin conformation capture (Hi-C) and Proximity-Guided Assembly (PGA) to cluster and order 9650 out of 11,936 contigs of this draft genome assembly into seven pseudo-chromosomes. The seven pseudo-chromosomes cover ~97.2% of the total contig length (~223.8 Mb). Locating existing genetic markers on the physical map resolved multiple discrepancies in marker order on the genetic map. Centromeric regions were inferred from recombination frequencies of genetic markers, alignment of 303 bp centromeric sequence with the PGA, and heat map showing the physical contact matrix over the entire genome. We demonstrate a high degree of synteny between each of the seven chromosomes of black raspberry and a high-quality reference genome for strawberry ( Fragaria vesca L.) assembled using only PacBio long-read sequences. We conclude that PGA is a cost-effective and rapid method of generating chromosome-scale assemblies from Illumina short-read sequencing data., Competing Interests: The authors declare that they have no conflict of interest.

Background: GH insensitivity (GHI) is characterised by short stature, IGF-1 deficiency and normal/elevated serum GH. IGF-1 insensitivity results in pre- and post-natal growth failure with normal/high IGF-1 levels. The prevalence of genetic defects is unknown., Objective: To identify the underlying genetic diagnoses in a paediatric cohort with GH or IGF-1 insensitivity using candidate gene (CGS) and whole-exome sequencing (WES) and assess factors associated with the discovery of a genetic defect., Methods: We undertook a prospective study of 132 patients with short stature and suspected GH or IGF-1 insensitivity referred to our centre for genetic analysis. 107 (96 GHI, 88 probands; 11 IGF-1 insensitivity, 9 probands) underwent CGS. WES was performed in those with no defined genetic aetiology following CGS., Results: A genetic diagnosis was discovered 38/107 (36%) patients (32% probands) by CGS. WES revealed 11 patients with genetic variants in genes known to cause short stature. A further 2 patients had hypomethylation in the H19/IGF2 region or mUPD7 consistent with Silver-Russell Syndrome (total with genetic diagnosis 51/107, 48% or 41/97, 42% probands). WES also identified homozygous putative variants in FANCA and PHKB in 2 patients. Low height SDS and consanguinity were highly predictive for identifying a genetic defect., Conclusions: Comprehensive genetic testing confirms the genetic heterogeneity of GH/IGF-1 insensitivity and successfully identified the genetic aetiology in a significant proportion of cases. WES is rapid and may isolate genetic variants that have been missed by traditional clinically driven genetic testing. This emphasises the benefits of specialist diagnostic centres., (© 2017 European Society of Endocrinology.)

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies.

The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F 1 F O -ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F 1 F O -ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F 1 F O -ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F 1 F O -ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F 1 F O -ATP synthase. We propose a model in which part of the F 1 F O -ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interest associated with this manuscript.

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson's disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson's disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson's disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson's disease.

Growing access to diverse 'real-world' data sources is enabling new approaches to close persistent evidence gaps about the optimal use of medical products in real-world practice. Here, we argue that contrary to widespread impressions, existing FDA regulations embody sufficient flexibility to accommodate the emerging tools and methods needed to achieve this goal.

Background: Tumor epithelial cells (TEpCs) and spindle-shaped stromal cells, not associated with the vasculature, of patients with early breast cancer express osteoprotegerin (OPG), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand, stromal cell derived factor-1, interleukin-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2) and their receptors at significantly higher levels compared with non-neoplastic breast tissues. We evaluated the clinicopathological significance of these ligands and receptors in TEpC and spindle-shaped stromal cells, not associated with the vasculature, to determine their impact on prognosis of patients with early-stage breast cancer., Methods: We conducted immunohistochemical analyses of protein expression in primary tumors of patients with early breast cancer and analyzed their association with standard prognostic parameters and clinical outcomes, including local relapse, metastatic recurrence, disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS)., Results: Elevated levels of TRAIL-R3 and chemokine (C-C motif) receptor 2 (CCR-2) in TEpCs and OPG and CCL-2 in stromal cells were significantly associated with a higher risk of metastasis (p = 0.032, p = 0.003, p = 0.038, and p = 0.049; respectively). Moreover, high expression of TRAIL-R3 and CCR-2 in TEpCs was associated with shorter DFS, MFS, and OS. High TRAIL-R3 expression in TEpCs was an independent prognostic factor for DFS and OS, and high CCR-2 expression in these cells was an independent prognostic factor for MFS., Conclusions: High levels of TRAIL-R3 and CCR-2 expression in TEpCs identified patients with early breast cancer with poor outcomes.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F 1 head, in which the catalytic (αβ) 3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal α C fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the α C fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis., Competing Interests: The authors declare no conflict of interest.

In evaluating the insurance hypothesis as an explanation for obesity, we propose one missing piece of the puzzle. Our suggested explanation for why individuals report food insecurity is that an individual may have an impaired episodic ability to plan for the future.

Bulb color is an important consumer trait for onion ( Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic ( Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum maju s of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

Spindle-shaped stromal cells, like carcinoma-associated fibroblasts and mesenchymal stem cells, influence tumor behavior and can serve as parameters in the clinical diagnosis, therapy, and prognosis of early breast cancer. Therefore, the aim of this study is to explore the clinicopathological significance of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) receptors (Rs) 2 and 4 (TRAIL-R2 and R4), and interleukin-6 R (IL-6R) in spindle-shaped stromal cells, not associated with the vasculature, as prognostic determinants of early breast cancer patients. Receptors are able to trigger the migratory activity, among other functions, of these stromal cells. We conducted immunohistochemical analysis for the expression of these receptors in spindle-shaped stromal cells, not associated with the vasculature, of primary tumors from early invasive breast cancer patients, and analyzed their association with clinicopathological characteristics. Here, we demonstrate that the elevated levels of TRAIL-R2, TRAIL-R4, and IL-6R in these stromal cells were significantly associated with a higher risk of metastatic occurrence (p = 0.034, 0.026, and 0.006; respectively). Moreover, high expression of TRAIL-R4 was associated with shorter disease-free survival and metastasis-free survival (p = 0.013 and 0.019; respectively). Also, high expression of IL-6R was associated with shorter disease-free survival, metastasis-free survival, and overall survival (p = 0.003, 0.001, and 0.003; respectively). Multivariate analysis showed that IL-6R expression was an independent prognostic factor for disease-free survival and metastasis-free survival (p = 0.035). This study is the first to demonstrate that high levels of IL-6R expression in spindle-shaped stromal cells, not associated with the vasculature, could be used to identify early breast cancer patients with poor outcomes.

Vegetative shoots of a naturalized population of purple-leaved plectranthus (Plectranthus ciliatus, Lamiaceae) were found to contain four main anthocyanins: peonidin 3-(6″-caffeoyl-β-glucopyranoside)-5-β-glucopyranoside, peonidin 3-(6″-caffeoyl-β-glucopyranoside)-5-(6‴-malonyl-β-glucopyranoside), peonidin 3-(6″-E-p-coumaroyl-β-glucopyranoside)-5-(6‴-malonyl-β-glucopyranoside), and peonidin 3-(6″-E-p-coumaroyl-β-glucopyranoside)-5-β-glucopyranoside. The first three of these pigments have not been reported previously from any plant. They all follow the typical anthocyanin pattern of Lamiaceae, with universal occurrence of anthocyanidin 3,5-diglucosides and aromatic acylation with p-coumaric and sometimes caffeic acids; however, they differ by being based on peonidin. The four anthocyanins were present in the leaves (22.2 mg g(-1) DW), and in the xylem and interfascicular parenchyma of the stem. They were exceptionally abundant, among the highest reported for any plant organ, in epidermal hairs on some of the stem internodes (101 mg g(-1) DW). Anthocyanin content in these hairs increased more than three-fold from the youngest to the fourth-youngest internodes. In situ absorbances (λmax ≈ 545 nm) were bathochromic in comparison to absorbances of the isolated anthocyanins in their flavylium form in acidified aqueous solutions (λmax = 525 nm), suggesting that the anthocyanins occur both in quinoidal and flavylium forms in constant proportions in the anthocyanic hair cells. The most distinctive observation with respect to relative proportions of individual anthocyanins was found in de-haired internodes, for which anthocyanin caffeoyl-derivatives decreased, and anthocyanin coumaroyl-derivatives increased, from the youngest to the fourth-youngest internode., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chronic cardiac hypertrophy is maladaptive and contributes to the pathogenesis of heart failure. The objective of this study was to identify small molecule inhibitors of pathological cardiomyocyte hypertrophy. High content screening was performed with primary neonatal rat ventricular myocytes (NRVMs) cultured on 96-well plates and treated with a library of 3241 distinct small molecules. Non-toxic hit compounds that blocked hypertrophy in response to phenylephrine (PE) and phorbol myristate acetate (PMA) were identified based on their ability to reduce cell size and inhibit expression of atrial natriuretic factor (ANF), which is a biomarker of pathological cardiac hypertrophy. Many of the hit compounds are existing drugs that have not previously been evaluated for benefit in the setting of cardiovascular disease. One such compound, the anti-malarial drug artesunate, blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). These findings demonstrate that phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery. Using annotated libraries of compounds with known selectivity profiles, this screening methodology also facilitates chemical biological dissection of signaling networks that control pathological growth of the heart., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

To develop more ecologically valid models of the neurobiology of obesity, it is critical to determine how the neural processes involved in food-related decision-making translate into real-world eating behaviors. We examined the relationship between goal-directed valuations of food images in the MRI scanner and food consumption at a subsequent ad libitum buffet meal. We observed that 23 lean and 40 overweight human participants showed similar patterns of value-based neural responses to health and taste attributes of foods. In both groups, these value-based responses in the ventromedial PFC were predictive of subsequent consumption at the buffet. However, overweight participants consumed a greater proportion of unhealthy foods. This was not predicted by in-scanner choices or neural response. Moreover, in overweight participants alone, impulsivity scores predicted greater consumption of unhealthy foods. Overall, our findings suggest that, while the hypothetical valuation of the health of foods is predictive of eating behavior in both lean and overweight people, it is only the real-world food choices that clearly distinguish them.

Copper is a biometal essential for normal brain development and function, thus copper deficiency or excess results in central nervous system disease. Well-characterized disorders of disrupted copper homoeostasis with neuronal degeneration include Menkes disease and Wilson's disease but a large body of evidence also implicates disrupted copper pathways in other neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Huntington's disease and prion diseases. In this short review we critically evaluate the data regarding changes in systemic and brain copper levels in Parkinson's disease, where alterations in brain copper are associated with regional neuronal cell death and disease pathology. We review copper regulating mechanisms in the human brain and the effects of dysfunction within these systems. We then examine the evidence for a role for copper in pathogenic processes in Parkinson's disease and consider reports of diverse copper-modulating strategies in in vitro and in vivo models of this disorder. Copper-modulating therapies are currently advancing through clinical trials for Alzheimer's and Huntington's disease and may also hold promise as disease modifying agents in Parkinson's disease., (© 2016 Authors; published by Portland Press Limited.)

Unlabelled: Angiogenesis is a key process for metastatic progression. While it has been established that the evaluation of breast tumoral microvessel density by CD105 marker is a potential prognostic parameter, its evaluation by CD146 marker has been poorly studied., Aim: The purpose of this study was to compare the prognostic value of intra-tumoral microvessel density assayed by CD105 and CD146 in early breast cancer patients., Methods: 42 women with breast infiltrative ductal carcinoma (I and II-stages) were retrospectively reviewed. Intra-tumoral microvessel density was immunohistochemically examined using antibodies anti-CD105 and CD146 in paraffin-embedded tissues, and their association with classical prognostic-markers, metastatic recurrence, metastasis-free survival and overall survival was analyzed., Results: High microvessel density assessed by CD146 was significantly associated with a higher risk of developing metastasis (p=0.0310) and a shorter metastasis-free survival (p=0.0197). In contrast, when we used the CD105-antibody, we did not find any significant association. Finally, CD146 showed to be an independent predictive indicator for metastasis-free survival (p=0.0055)., Conclusion: Our data suggest that the intra-tumoral microvessel density evaluated by CD146 may be a more suitable predictor of metastatic development than that evaluated by CD105 in early breast cancer., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Rotary ATPases are energy-converting nanomachines found in the membranes of all living organisms. The mechanism by which proton translocation through the membrane drives ATP synthesis, or how ATP hydrolysis generates a transmembrane proton gradient, has been unresolved for decades because the structure of a critical subunit in the membrane was unknown. Electron cryomicroscopy (cryoEM) studies of two rotary ATPases have now revealed a hairpin of long, horizontal, membrane-intrinsic α-helices in the a-subunit next to the c-ring rotor. The horizontal helices create a pair of aqueous half-channels in the membrane that provide access to the proton-binding sites in the rotor ring. These recent findings help to explain the highly conserved mechanism of ion translocation by rotary ATPases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Key Message: The Md - MYB10 R6 gene from apple is capable of self-regulating in heterologous host species and enhancing anthocyanin pigmentation, but the activity of MYB10 is dependent on endogenous protein partners. Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10 R6 , are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10 R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10 R6 transgene (MYB10-R6 pro :MYB10:MYB10 term ) activated anthocyanin synthesis when transiently expressed in Antirrhinum rosea (dorsea) petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10 R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of 'Mitchell' petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10 R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10 R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

Redox-active metals in the brain mediate numerous biochemical processes and are also implicated in a number of neurodegenerative diseases. A number of different approaches are available for quantitatively measuring the spatial distribution of biometals at an image resolution approaching the subcellular level. Measured biometal levels obtained using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS; spatial resolution 15 μm × 15 μm) were within the range of those obtained using X-ray fluorescence microscopy (XFM; spatial resolution 2 μm × 7 μm) and regional changes in metal concentration across discrete brain regions were replicated to the same degree. Both techniques are well suited to profiling changes in regional biometal distribution between healthy and diseased brain tissues, but absolute quantitation of metal levels varied significantly between methods, depending on the metal of interest. Where all possible variables affect metal levels, independent of a treatment/phenotype are controlled, either method is suitable for examining differences between experimental groups, though, as with any method for imaging post mortem brain tissue, care should be taken when interpreting the total metal levels with regard to physiological concentrations.

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

The mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F(1)F(o) ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F(1)F(o) ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F(1)F(o) ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae.

Several studies have confirmed that the breast tumor microenvironment drives cancer progression and metastatic development. The aim of our research was to investigate the prognostic significance of the breast tumor microenvironment in untreated early breast cancer patients. Therefore, we analyzed the association of the expression of α-SMA, FSP, CD105 and CD146 in CD34-negative spindle-shaped stromal cells, not associated with the vasculature, in primary breast tumors with classical prognostic marker levels, metastatic recurrence, local relapse, disease-free survival, metastasis-free survival and the overall survival of patients. In the same way, we evaluated the association of the amount of intra-tumor stroma, fibroblasts, collagen deposition, lymphocytic infiltration and myxoid changes in these samples with the clinical-pathological data previously described. This study is the first to demonstrate the high CD105 expression in this stromal cell type as a possible independent marker of unfavorable prognosis in early breast cancer patients. Our study suggests that this new finding can be useful prognostic marker in the clinical-pathological routine.

The anthocyanin biosynthetic pathway is regulated by a transcription factor complex consisting of an R2R3 MYB, a bHLH, and a WD40. Although R2R3 MYBs belonging to the anthocyanin-activating class have been identified in many plants, and their role well elucidated, the subgroups of bHLH implicated in anthocyanin regulation seem to be more complex. It is not clear whether these potential bHLH partners are biologically interchangeable with redundant functions, or even if heterodimers are involved. In this study, AcMYB110, an R2R3 MYB isolated from kiwifruit (Actinidia sp.) showing a strong activation of the anthocyanin pathway in tobacco (Nicotiana tabacum) was used to examine the function of interacting endogenous bHLH partners. Constitutive expression of AcMYB110 in tobacco leaves revealed different roles for two bHLHs, NtAN1 and NtJAF13. A hierarchical mechanism is shown to control the regulation of transcription factors and consequently of the anthocyanin biosynthetic pathway. Here, a model is proposed for the regulation of the anthocyanin pathway in Solanaceous plants in which AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Despite advances in the study of breast cancer (BC), it remains the second leading cause of mortality among women. BC is a heterogeneous system, mainly composed of tumor epithelial cells (TEpCs) and stromal cells (SCs); the interaction through the ligands and their receptors (Rs) plays a major role in BC progression. The aim of the present study was to evaluate the association between ligands, such as osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand (RANKL), stromal cell-derived factor (SDF)-1, interleukin (IL)-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2), and their Rs in TEpC and spindle-shaped SCs not closely associated with the vasculature., Patients and Methods: We studied the expression of all those factors in 63 primary tumors of untreated patients with BC with infiltrative ductal carcinoma (I/II stage) and 10 non-neoplastic tissues. The percentage of positive cells and the staining intensity were analyzed by immunohistochemistry. Mann-Whitney test and Spearman's rank correlation coefficient were used (P ≤ .05)., Results: We found a significant association between the expression of RANKL, IL-6, SDF-1, and CCL-2 in TEpC and the receptor activator of nuclear factor kappa B (RANK), IL-6R, C-X-C chemokine R type 4, and chemokine (C-C motif) R-2 (CCR-2) in spindle-shaped SC. The expression of TRAIL, RANKL, and CCL-2 in spindle-shaped SC also was associated with the expression of TRAIL-receptor 1, TRAIL-receptor 4, RANK, and CCR-2 in TEpC., Conclusions: Because the described ligands and Rs are implicated in BC progression, our results suggest that these factors could be involved in the crosstalk between TEpC and SC in the early stages of BC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Patients with pulmonary arterial hypertension (PAH) are treated with vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, soluble guanylyl cyclase activators, and prostacyclin. Despite recent advances in pharmacotherapy for individuals with PAH, morbidity and mortality rates in this patient population remain unacceptably high. Here, we tested the hypothesis that combination therapy with two PAH drugs that target distinct biochemical pathways will provide superior efficacy relative to monotherapy in the rat SU5416 plus hypoxia (SU-Hx) model of severe angioproliferative PAH, which closely mimics the human condition., Methods: Male Sprague Dawley rats were injected with a single dose of SU5416, which is a VEGF receptor antagonist, and exposed to hypobaric hypoxia for three weeks. Rats were subsequently housed at Denver altitude and treated daily with the PDE-5 inhibitor, tadalafil (TAD), the type A endothelin receptor (ETA) antagonist, ambrisentan (AMB), or a combination of TAD and AMB for four additional weeks., Results: Monotherapy with TAD or AMB led to modest reductions in pulmonary arterial pressure (PAP) and right ventricular (RV) hypertrophy. In contrast, echocardiography and invasive hemodynamic measurements revealed that combined TAD/AMB nearly completely reversed pulmonary hemodynamic impairment, RV hypertrophy, and RV functional deficit in SU-Hx rats. Efficacy of TAD/AMB was associated with dramatic reductions in pulmonary vascular remodeling, including suppression of endothelial cell plexiform lesions, which are common in human PAH., Conclusions: Combined therapy with two vasodilators that are approved for the treatment of human PAH provides unprecedented efficacy in the rat SU-Hx preclinical model of severe, angioproliferative PAH.

Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor [TF; ROSEA1 (ROS1)] that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related basic helix-loop-helix TF transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high-light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1 × 35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment in the petal throat region, and the anthers changed from yellow to purple pigmentation. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1 × 35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants.

Main Conclusion: This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3' hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3'H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure., (Copyright © 2014 the American Physiological Society.)

Background: Cardiovascular diseases such as atherosclerosis and vascular calcification are a major cause of death in patients with chronic kidney disease (CKD). Recently, the long-awaited results of the Study of Heart and Renal Protection trial were reported. This large randomized clinical trial found that an extensive cholesterol-lowering therapy through the combination of simvastatin and ezetimibe significantly reduced cardiovascular diseases in a wide range of patients with CKD. However, the mechanism by which this cholesterol-lowering therapy reduces CKD-dependent vascular diseases remains elusive. The objective of the present study was to determine the contribution of the oxysterol-induced pro-apoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) on the pathogenesis of CKD-dependent cardiovascular diseases through endoplasmic reticulum stress signaling., Methods and Results: CKD increased levels of serum oxysterols such as 7-ketocholesterol in human patients and ApoE(-/-) mice. Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction. This therapy also reduced aortic endoplasmic reticulum stress induced by CKD. The short hairpin RNA-mediated knockdown of CHOP and activating transcription factor-4 in vascular smooth muscle cells attenuated oxysterol-induced mineralization, osteogenic differentiation, and endoplasmic reticulum stress. In addition, CHOP deficiency protected ApoE(-/-) mice from CKD-dependent vascular calcification, cardiac dysfunction, and vascular cell death., Conclusions: These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress. CHOP plays a crucial role in the pathogenesis of CKD-dependent vascular calcification., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss. These data suggest that regions affected by α-synuclein pathology may display enhanced vulnerability and cell loss if Cu-dependent protective mechanisms are compromised. Additional investigation of copper pathology in PD may identify novel targets for the development of protective therapies for this disorder., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Plant cell cultures can offer continuous production systems for high-value food and health ingredients, independent of geographical or environmental variations and constraints. Yet despite many improvements in culture technologies, cell line selection, and bioreactor design, there are few commercial successes. This is principally due to the culture yield and market price of food products not being sufficient to cover the plant cell culture production costs. A better understanding of the underpinning biological mechanisms that control the target metabolite biosynthetic pathways may allow the metabolic engineering of cell lines to provide for economically competitive product yields. However, uncertainty around the regulatory and public acceptance of products derived from engineered cell cultures presents a barrier to the uptake of the technology by food product companies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots.

The combination of bridging bis-bidentate redox-active ligands derived from 3,3-bis-2-hydroxy-1,4-naphthoquinone (bhnqH2), ancillary ligands based on tris(2-pyridylmethyl)amine (tpa) and cobalt salts has afforded a new family of dinuclear cobalt complexes. Compounds of the complexes [Co2(bhnq)(tpa)2](2+) (1), [Co2(bhnq)(Metpa)2](2+) (2), [Co2(bhnq)(Me2tpa)2](2+) (3) [Co2(bhnq)(Me3tpa)2](2+) (4), [Co2(bhnq)(tpa)2](4+) (5), [Co2(bhMenaph)(tpa)2](2+) (6) and [Co2(bhPronaph)(tpa)2](2+) (7) (Mentpa involves n = 0, 1, 2 and 3 methyl groups at the 6-position of the tpa pyridine rings; bhMenaphH4 = bis-3,4-dihydroxy-4-methoxynaphthalene-1-one; bhPronaphH4 = bis-3,4-dihydroxy-4-(2-oxopropyl)naphthalen-1(4H)-one) have been characterised by single crystal X-ray diffraction. While complexes 1-4 possess divalent cobalt centres, trivalent cobalt is evident in complexes 5-7. The bis-bidentate redox-active bridging ligand remains in the diamagnetic quinone bhnq(2-) redox state in complexes 1-5. Metal-catalysed reaction with methoxide or acetone enolate ions gives rise to the derivatised bridging ligands present in 6 and 7. The electronic properties of compounds of 1-7 have been explored in the solid state by infrared spectroscopy and variable temperature magnetic measurements and in solution by electronic absorption spectroscopy and cyclic voltammetry.

Background: Adolescents and Young Adults (AYAs) with cancer are being disadvantaged within the present health care system. Some of the factors identified as leading to this disadvantage include medical issues specific to AYAs with cancer, delay in diagnosis, fragmented services, lack of access to clinical trials and psychosocial life stage issues., Objective: A major study investigated the experience that accompanies the stages of diagnosis, treatment, dying and death of an AYA from the perspective of family members. This paper discusses the major theme of the reality of hospitalisation., Methods: Narrative inquiry was the methodology for this study. The participants were a self-selected purposeful sample of 26 family members. Open-ended interviews were conducted to obtain a story of the experience of having an AYA family member live with and die of cancer. A meta-narrative of the family member's experience was developed by NVivo8., Results: In amongst the mass of data this study produced, a major theme to emerge was the experience 'of the reality of hospitalisation'. Within this theme issues regarding: The place of treatment; the hospital experience; not fitting in; and, confronting illness and death were revealed., Conclusion: While on the whole the cancer was treated with state of the art medicine, the experiences of the hospitalisation repeated consistently throughout this narrative reveals a failure to meet the higher order needs specific to adolescents and young adults and their families As a result this cohort were exposed to a landscape which did not facilitate a therapeutic experience, as well as would be expected for children and older adults.

The diversity of pigmentation patterns observed in plants occurs due to the spatial distribution and accumulation of colored compounds, which may also be associated with structural changes to the tissue. Anthocyanins are flavonoids that provide red/purple/blue coloration to plants, often forming complex patterns such as spots, stripes, and vein-associated pigmentation, particularly in flowers. These patterns are determined by the activity of MYB-bHLH-WDR (MBW) transcription factor complexes, which activate the anthocyanin biosynthesis genes, resulting in anthocyanin pigment accumulation. Recently, we established that the MBW complex controlling anthocyanin synthesis acts within a gene regulation network that is conserved within at least the Eudicots. This network involves hierarchy, reinforcement, and feedback mechanisms that allow for stringent and responsive regulation of the anthocyanin biosynthesis genes. The gene network and mobile nature of the WDR and R3-MYB proteins provide exciting new opportunities to explore the basis of pigmentation patterning, and to investigate the evolutionary history of the MBW components in land plants.

Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era, originating from charophycean green algae. The successful adaptation to land of these prototypes of amphibious plants - when they emerged from an aquatic environment onto the land - was achieved largely by massive formation of "phenolic UV light screens". In the course of evolution, plants have developed the ability to produce an enormous number of phenolic secondary metabolites, which are not required in the primary processes of growth and development but are of vital importance for their interaction with the environment, for their reproductive strategy and for their defense mechanisms. From a biosynthetic point of view, beside methylation catalyzed by O-methyltransferases, acylation and glycosylation of secondary metabolites, including phenylpropanoids and various derived phenolic compounds, are fundamental chemical modifications. Such modified metabolites have altered polarity, volatility, chemical stability in cells but also in solution, ability for interaction with other compounds (co-pigmentation) and biological activity. The control of the production of plant phenolics involves a matrix of potentially overlapping regulatory signals. These include developmental signals, such as during lignification of new growth or the production of anthocyanins during fruit and flower development, and environmental signals for protection against abiotic and biotic stresses. For some of the key compounds, such as the flavonoids, there is now an excellent understanding of the nature of those signals and how the signal transduction pathway connects through to the activation of the phenolic biosynthetic genes. Within the plant environment, different microorganisms can coexist that can establish various interactions with the host plant and that are often the basis for the synthesis of specific phenolic metabolites in response to these interactions. In the rhizosphere, increasing evidence suggests that root specific chemicals (exudates) might initiate and manipulate biological and physical interactions between roots and soil organisms. These interactions include signal traffic between roots of competing plants, roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil properties to the roots. Plant phenolics can also modulate essential physiological processes such as transcriptional regulation and signal transduction. Some interesting effects of plant phenolics are also the ones associated with the growth hormone auxin. An additional role for flavonoids in functional pollen development has been observed. Finally, anthocyanins represent a class of flavonoids that provide the orange, red and blue/purple colors to many plant tissues. According to the coevolution theory, red is a signal of the status of the tree to insects that migrate to (or move among) the trees in autumn., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Abstract Background: Adolescents and Young Adults (AYA) with cancer are being disadvantaged within the present health care system. Some of the factors identified as leading to this disadvantage include medical issues specific to AYAs with cancer, delay in diagnosis, fragmented services, lack of access to clinical trials and psychosocial life stage issues Objective: A major study investigated the experience that accompanies the stages of diagnosis, treatment, dying and death of an AYA from the perspective of family members. This paper discusses the major theme of the reality of hospitalisation Methods: Narrative inquiry was the methodology for this study. The participants were a self-selected purposeful sample of 26 family members. Open-ended interviews were conducted to obtain a story of the experience of having an AYA family member live with and die of cancer. A meta-narrative of the family member's experience was developed by NVivo8. Results: In amongst the mass of data this study produced, a major theme to emerge was the experience 'of the reality of hospitalisation.' Within this theme issues regarding: the place of treatment; the hospital experience; not fitting in; and, confronting illness and death were revealed. Conclusion: While on the whole the cancer was treated with state of the art medicine, the experiences of the hospitalisation repeated consistently throughout this narrative reveals a failure to meet the higher order needs specific to adolelscents and young adults and their families As a result this cohort were exposed to a landscape which did not facilitate a therapeutic experience, as well as would be expected for children and older adults.