Asymmetric supercapacitors based on ultrathin MSs and graphene exhibit a very high energy density up to 97.2 Wh kg−1 with no more than 3% capacitance loss after 10000 cycles in aqueous electrolyte. Most interestingly, we show that the energy storage device can have an aesthetic property. For instance, a “Chinese panda” supercapacitor is capable of lighting up a red light emitting diode. This work has another, quite different aspect that a supercapacitor is no longer a cold industry product, but could have the meaning of art.Flexible asymmetric supercapacitors with excellent electrochemical performance and aesthetic property are realized by using ultrathin two-dimensional (2D) MnO Flexible asymmetric supercapacitors using the ultrathin 2D MSs and graphene in aqueous Ca(NO3)2-SiO2 gel electrolyte realized excellent electrochemical performance . Most interestingly, these supercapacitors can be designed to show desirably aesthetic property, for example letters, words and patterns. It provides a versatile tool to design and fabricate the supercapacitor with a certain shape through a simple process and almost all electrode materials are available. For instance an asymmetric supercapacitor with a vivid “Chinese panda” pattern has a voltage of 2.0 V capable of lighting up a red light emitting diode (LED). Our work has another, quite different aspect that a battery or supercapacitor is no longer a cold industry product, but could have the meaning of art.Herein, for the first time, flexible asymmetric supercapacitors with excellent electrochemical performance and aesthetic property were fabricated by using two kinds of ultrathin 2D nanosheet materials (MSs and graphene) through a novel strategy . The MSs4 can be dissolved mainly within the aqueous region, which indicates a binary liquid lamellar structure consisting of surfactant AOT and KMnO4 aqueous solution. Our previous work found that KMnO4 can be reduced by the hydrophilic head of AOT2 ultrathin lamellae. At first, the AOT and water are mixed to form a binary lamellar structure. Then KMnO4 is dissolved within the aqueous region (Ws). Finally MnO2 will be automatically synthesized and the growth of MnO2 will be restricted in the aqueous region by extra AOT to form MSs.We firstly use the soft template to synthesize the ultrathin MSs. A binary system consisting of surfactant (sodium bis(2-ethylhexyl)sulfosuccinate, AOT) and water can form unique structure automatically. The hydrophobic long chain of surfactant (S) tends to intertwine from each other to force hydrophilic heads toward the water phase in a binary phase. The common phase of a binary phase is the lamellar structure (referred as G phase) as shown in 6] octahedron known as the basic structure unit of manganese dioxide. Atomic force microscopy (AFM) study on the thickness of MS is shown in 2 nanosheets with a high magnification inserted are shown in 2 nanosheets. X-ray diffraction (XRD) pattern of the amorphous MSs with a few of broad peaks is shown in −1 and 1630 cm−1 represent stretching and bending vibrations of water molecules or hydroxyl groups in the tunnel−1 to 400 cm−1 corresponds to the characteristic Mn-O stretching vibrations in manganese oxides. In the Raman spectral window, the band of 580 cm−1 is attributed to the vibration of Mn-O in MnO229302 adsorption/desorption analysis  method, which is close to the theoretical value of 220 m2 g−1 for a MnO2 sheet with 2 nm in thickness and 50 nm in diameter.As shown in analysis . The sur2 g−1 with nitrogen absorption/desorption analysis (−1. The intensity ratio (ID/IG) of D and G bands is 0.9 in GO and increases to 1.1 in GA, which indicates some defects are introduced in GA due to the hydrothermal reduction of GOThe formation of graphene with continuous architecture (GA) is derived by some non-covalent bonding such as hydrogen bonding, coordination, electrostatic interaction and Π-Π stacking interactions during the hydrothermal processanalysis . TEM imaanalysis , the curanalysis . Both GO2 flexible electrodes can be designed as various pictures, words or letters, and patterns designed by us as shown in T) of electrode can be calculated by α is opacity of active material film on printing stock ranging from 0 to1, k is rate of coverage that can be calculated by using a screen printing unit, 0T is the transparency of blank unprinted sample characterized by ultraviolet and visible spectrophotometer (UV-vis). For example, toward the printed electrode with the amplified 0.5 × 0.5 (mm2) square lattice patterns in T0 is 85% 2 aqueous solution as electrolyte. The Ca(NO3)2 electrolyte is used because the capacitance of MnO2 in aqueous electrolyte is much higher by replacing univalent cation with bivalent cation such as Ca2+ ion34−1 in −1 at a scan rate of 2 mV s−1 and with about 76% of capacity retention rate at 50 mV s−1 (−1 at a current density of 0.1 ampere per gram (A g−1)  induces a very high capacitance because of high utilization of MSs, direct contact of sheet surface to the electrolyte and fast ion diffusion. Besides, the good contact between active materials and high conductive ITO film also facilitates the transfer of electrons. The performances of GA electrodes at a potential window of −1 ~ 0 V are presented in The electrochemical performance of the flexible MS and GA electrodes is investigated in a three-electrode system by using 2 molar per liter (M) Ca2-SiO2 composite gel electrolyte. The aqueous Ca(NO3)2-SiO2 composite gel electrolyte with high transparency and safety was synthesized by a sol-gel method (See methods in 0T is 72% characterized by UV-vis from a supercapacitor assembled by Ca(NO3)2-SiO2 electrolyte sandwiched with ITO-PET, where k is about 0.071  composites, our flexible supercapcitor based on ultrathin MSs and GA also shows relatively high energy density16These screen-printed supercapacitors have been measured by the electrochemical tests. The CV measurement has performed on the MS/GA supercapacitor before and after being bended 180° at a scan rate of 20 mV s 5 A g−1 . The RagWe have also performed the electrochemical tests on the symmetric MS/MS supercapacitor. This MS/MS supercapacitor is also optical see-through and flexible. However, the operating voltage of the symmetric MS/MS supercapacitor is only half of the operating voltage of the asymmetric MS/GA supercapacitor. The capacitance and Ragone plots of the symmetric MS/MS supercapacitor are presented in Long cycle life tests have been performed on the symmetric MS/MS and asymmetric MS/GA supercapacitors . These s48495051−1 with a long cycle stability and good rate performance. Asymmetric screen-printed supercapacitors based on ultrathin 2D MSs and graphene realize good flexibility and excellent electrochemical performance, for example, a higher energy density (97.2 Wh kg−1) than traditional MnO2 based supercapacitors. Interestingly, a desirably visual and/or aesthetic property  of the supercapacitor can be realized and almost all electrode materials are available through this new strategy. This work has another and quite different aspect that the battery or supercapacitor is no longer a cold industry product, but could have the meaning of art. For example a “Chinese panda” supercapacitor is capable of lighting a LED light. We believed that it may open a new era of the flexible energy storage devices in future to meet the specific demand of the consumer electronics.In summary, flexible asymmetric supercapacitors with excellent electrochemical performance and aesthetic property are fabricated based on ultrathin 2D nanosheets through a novel strategy. Ultrathin MSs with thickness of ca. 2 nm show a high capacitance of 774 F g4 aqueous solution was prepared by adding potassium permanganate  in deionized water. Next 0.25 L of 0.1 M KMnO4 solution was added in 0.5 L AOT/isooctane solution, followed by stirring 5 h to obtain a dark brown precipitate. Then MS powders were prepared by washing the precipitates with ethanol and distilled water several times, and drying at 80°C for 12 h.MSs were prepared by using a soft template method. Firstly, a 0.1 M sodium bis(2-ethylhexyl)sulfosuccinate aqueous solution was prepared by dissolving AOT in water and a 0.1 M KMnOGraphite oxide (GO) powders were initially prepared by a modified Hummers method. 160 mg GO powders was added in 80 mL deionized water followed by ultrasonication for 2 h to obtain a homogeneous graphene oxide aqueous dispersion. Then it was sealed in a 100 mL Teflon-lined autoclave and maintained at 180°C for 24 h. The autoclave was naturally cooled to room temperature and a cylindrical graphene body is self-assembled. Finally, a vacuum freeze-drying procedure was used to remove water from graphene body obtaining GA.α radiation), Fourier transformation infrared spectroscopy , and Raman spectroscopy . Nitrogen adsorption and desorption isotherms for the porosities were measured on Micromeritics ASAP 2020 surface area analyzer. Surface area was calculated by the Brunauer-Ennett-Teller (BET) method. Electrochemical tests were performed with an Im6e (Zahner) electrochemical station. All electrodes are tested by using a typical three-electrode assembly, in which a piece of platinum gauze and saturated calomel electrode were assembled as the counter and reference electrode. A mild 2 M Ca(NO3)2 aqueous solution was used as electrolyte. The capacitance values calculated from CV plots and charge-discharge curves are by following: m is the mass of active materials excluding binder and conductive additive, ΔU is the potential window, Q is charge, I is the applied current, and Δt is discharging time.Morphologies were characterized by high-resolution transmission electron microscopy , atomic force microscopy , and field emission scanning electron microscopy . The structure of samples were studied by X-ray diffraction  and power density (P) of supercapacitor devices are calculated from charge-discharge curves according to following equations: S.S. and C.X. wrote the manuscript, performed the experiments and prepared figures 1–4. C.Y. provided the screen printing method. Y.C. and J.L. synthesized the graphene. S.S., C.X. and F.K. conceived the concept and revised the manuscript. All authors reviewed the manuscript.Supplementary InformationMovie


Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage. Loss of replication fork integrity is a primary source of genetic instability. In eukaryotes DNA synthesis is rapidly followed by its assembly into chromatin, and these two processes are tightly connected. Defective chromatin assembly mutants accumulate DNA damage and are sensitive to genotoxic agents, even though the mechanisms responsible for this genetic instability remain unclear because chromatin assembly also plays essential roles in transcription, silencing, DNA repair, and checkpoint signaling. A good example is the acetylation of histone H3 at lysine 56, which promotes histone deposition by the chromatin assembly factors CAF1 and Rtt106. In this case, the absence of this modification also causes a loss of structural and/or coding information at chromatin. Here we show that defective replication-coupled chromatin assembly leads to an accumulation of recombinogenic DNA damage by affecting the integrity of advancing, but not stalled, replication forks. Therefore, we propose that H3K56ac/CAF1/Rtt106-dependent chromatin assembly provides a mechanism for the stabilization of replication forks. Besides, H3K56 acetylation promotes replicative DNA damage repair/tolerance through a function that is independent of CAF1/Rtt106 and likely subsequent to its deposition at chromatin, revealing this modification as a key regulator of genome integrity. Saccharomyces cerevisiae) that lead to specific responses such as the maintenance of replication fork stability, inhibition of late replication origins, DNA repair modulation and cell cycle arrest Problems in DNA replication are a direct cause of genetic instability and are associated with early tumor development 2 tetramer to which an H2A/H2B dimer binds on each side In eukaryotes DNA is packaged into a highly specialized and dynamic nucleoprotein structure called chromatin, which is actually the substrate for cell machineries that deal with DNA. The repetitive unit of chromatin, the nucleosome, is formed by ∼146 base pairs of DNA wrapped 1.65 times around an octamer of histones. Nucleosome assembly of the replicated DNA is conducted by histone chaperones and chromatin assembly factors that first deposit two heterodimers of histones H3 and H4 to form a core (H3/H4)Newly synthesized histones H3 and H4 are acetylated before being deposited at the fork, and this modification is required for nucleosome assembly asf1Δ, rtt109Δ and H3K56R mutants increases the frequency of HR and gross chromosomal rearrangements (GCRs) A number of results have clearly shown over the last few years that defective chromatin assembly causes genetic instability. In plants and human cells, the absence of CAF1 causes inhibition of DNA synthesis, accumulation of DNA damage and activation of the S-phase checkpoint We have recently shown that defective chromatin assembly by partial depletion of H4 is rapidly followed by the collapse of replication forks, which are efficiently rescued via HR, suggesting that correct nucleosome deposition is required for replication fork stability asf1Δ, rtt109Δ and H3K56R mutants increases the frequency of genetic recombination and budded cells with foci of the recombination protein Rad52 fused to the yellow-fluorescence protein (Rad52-YFP)  . As prev mutants .hir1Δ did not affect recombination.Histone H3K56 acetylation marks nucleosomes incorporated into chromatin via both RC and replication independent mechanisms asf1Δ, rtt109Δ and H3K56R could be associated with defective histone deposition but also with a loss of structural and/or coding information because of the absence of H3K56ac at chromatin. To distinguish between these possibilities we analyzed the role of CAF1 and Rtt106 in preventing the accumulation of recombinogenic DNA damage; CAF1 and Rtt106 have redundant chromatin assembly functions as shown by the fact that cac1Δ rtt106Δ, but not cac1Δ and rtt106Δ, is defective in histone deposition cac1Δ rtt106Δcac1Δ and rtt106Δ were not affected in HR, the double mutant cac1Δ rtt106Δ increased the frequency both of genetic recombination and budded cells with Rad52-YFP foci as compared to the wild type  in wild type and mutants by 2D-gel electrophoresis. For this, cells were synchronized in G1 with α-factor and released into S phase, and DNA samples were analyzed at different times to follow the progression of replication forks from the early replication origin ARS305 , taking the total amount of wild-type RIs as 100, was reduced to ∼50% in  rtt109Δ . In agre was 33% . An incr rtt109Δ , which mcac1Δ, rtt106Δ and cac1Δ rtt106Δ mutants synchronized in G1 and released into S phase was analyzed. As shown in cac1Δ and rtt106Δ accumulated wild-type levels of RIs, the double mutant cac1Δ rtt106Δ displayed a ∼50% reduction in the amount of RIs at the origin, indicating that CAF1- and Rtt106-mediated chromatin assembly pathways have redundant roles in preventing the loss of replication forks. Besides, the levels of RIs in cac1Δ rtt106Δ were the same as in asf1Δ and rtt109Δ (∼50%), suggesting that the major role of H3K56 acetylation in replication fork stability is through its function in chromatin assembly. Consistently, the reduction in RIs in the triple mutant asf1Δ cac1Δ rtt106Δ was neither synergistic nor additive as compared to asf1Δ , taking rtt109Δ  and this rtt109Δ . Also, a rtt106Δ , indicatrad52Δ was not affected by HU . Strikis arrest ; consistasf1Δ and rtt109Δ, rtt101Δ was not sensitive to HU  or the DNA alkylating agent methyl methane sulfonate (MMS) . Again,  rtt109Δ . Our com rtt109Δ ; thus, tcac1Δ rtt106Δ to resume DNA replication upon the treatment of G1 released cells with a high concentration (0.033%) of MMS. cac1Δ rtt106Δ cells resumed and completed DNA replication but remained partially arrested in mitosis  accumulate recombinogenic DNA damage as determined by genetic recombination, cells with Rad52 foci and molecular analysis of sister-chromatid exchange hir1Δ exhibited wild-type levels of recombination. Alternatively, and in agreement with a model in which spontaneous genetic instability stems from defective DNA damage repair/tolerance, hyper-recombination might result from defective repair/tolerance and channelling to HR of spontaneous DNA lesions. In this case, DNA damage induction with genotoxic agents to which these mutants are sensitive should further increase their levels of recombination. In contrast, Asf1, Rtt109 and the Rtt101 complex are required for HR induced by MMS and CPT asf1ΔH3K56 acetylation is a histone modification required for chromatin assembly. Notably, mutants defective in H3K56 acetylation  or delayed just 10–20 minutes  the time required for DNA duplication despite the loss of RIs. Replication fork rescue by HR cannot account for completion of DNA replication because asf1Δ rad52Δ cells are also capable of completing DNA duplication , a result that is not unexpected because of the redundancy of DNA nucleases in DNA damage repair asf1Δ rad52Δ) is not affected by the presence of HU. Second, RC-chromatin assembly mutants  are proficient in stalled fork stability and restart upon an acute treatment with HU as determined by FACS analysis, checkpoint recovery and cell viability. Therefore, our results point to defects in the stability of advancing forks as the cause of the genetic instability in RC-nucleosome assembly mutants, further supporting the idea that defective histone deposition uncouples DNA synthesis and nucleosome assembly. Notably, asf1Δ cells treated with HU also exhibited an accumulation of Polα at the fork and an uncoupling of the MCM helicase DNA synthesis and histone deposition are physically and genetically connected to ensure the exact supply of histones at the fork H3K56Q, which mimics constitutive acetylation, suppresses asf1Δ sensitivity to HU and CPT cac1Δ rtt106Δ is only sensitive to high concentrations of MMS and CPT and is not sensitive to chronic treatment with HU, suggesting that the function of H3K56ac in the replicative DNA damage response can be separated from its role in CAF1/Rtt106-mediated chromatin assembly. This points to a role subsequent to its deposition into chromatin. In agreement with this idea, it has recently been shown that a change of lysine 56 to glutamic acid in H3 generates a histone proficient in binding to CAF1 and Rtt106 but sensitive to replicative DNA damage H3K56 acetylation – and by extent Asf1 and Rtt109 – is required for promoting resistance to replicative DNA damage rtt101Δ, suggesting an additional function for this histone modification in response to replicative DNA damage. This role could be to open the chromatin and facilitate the access of repair proteins to DNA. Other possibility is that H3K56 acetylation promotes checkpoint deactivation via CAF1/Rtt106-chromatin assembly upon the repair of the replicative DNA damage, as previously demonstrated for DSB repair cac1Δ rtt106Δ becomes temporally arrested at mitosis by sustained phosphorylation of Rad53 upon DNA damage release, even though this defect might also be a consequence of an incomplete accumulation of H3K56ac behind the fork of the double mutant.Besides, our comparative analysis shows that H3K56 acetylation mutants are slightly more sensitive to DNA damage than Our results in yeast anticipate a similar role for chromatin assembly in the stability of advancing replication forks through the more demanding chromatin structure of mammalian genomes. It will thereby be well worth the effort to address replication fork integrity in human cells defective in RC-chromatin assembly, which are known to arrest in the S phase and accumulate DNA damage H3K56R mutants that are isogenic to MSY421. pRS316-SU RAD52-YFP, respectively. Yeast cells were grown in supplemented minimal medium (SMM), except for nocodazole (NCD) synchronization that were grown in YPD medium asf1Δ rad52Δ, cac1Δ rtt106Δ and asf1Δ cac1Δ rtt106Δ) or 0.25 µg/ml (rest of strains). Then, cells were washed three times and released into the S phase at different times in fresh medium with or without 0.2 M HU and 50 µg/ml pronase. Cell cycle progression was followed by DNA content analysis (data not shown). To prevent cells from re-entering a new cell cycle in Yeast strains used in this study are listed in + recombinants generated by recombination between inverted repeat sequences was determined in cells transformed with plasmid pRS316-SU by fluctuation tests as the median value of six independent colonies The frequency of LeuThe proportion of budded cells with Rad52-YFP foci was performed as described previously DNA content analysis was performed by fluorescence-activated cell sorting (FACS) as reported previously 32P-labelled probes . All signals were quantified using a Fuji FLA5100 and ImageGauge as analysis program. Quantification of the RIs was normalized to the total amount of DNA, including linear monomers (n), to the size of the restriction fragment, and to the percentage of cells synchronized in G1; thus, the total amount of RIs at each specific region and time point was calculated as [ΣRIs/Σ(RIs+n×g)]×f, where f is the ratio between the size of the DNA fragment containing the origin and the size of the specific DNA fragment, and g is the proportion of cells in G1 after α-factor synchronization.Each replication kinetic was conducted in parallel with the mutants and the wild type. Cell cultures were arrested with sodium azide  and cooled down in ice. Total DNA was isolated either in agarose plugs or with the G2/CTAB protocol as previously reported ARS305 and a non-replicated control region (located at ∼7 kb from the late replicating origin ARS609) determined by qPCR . The efficiency of replication initiation was calculated as the ratio between the amount of DNA in HU-arrested cells and the amount of DNA in G1-arrested cells at the origin normalized to the same ratio at the control DNA region.Total DNA from mid-log phase cells synchronized in G1 and released into S phase in the presence of 0.2 M HU for 50 minutes was extracted and the amount of DNA at the origin in situ kinase assay, respectively. Rad53 was detected either with rabbit polyclonal antibody JDI47 Yeast protein extracts were prepared from mid-log-phase cultures using the TCA protocol as described Figure S1asf1Δ and cac1Δ rtt106Δ causes a loss of RIs at ARS315. Analysis of RIs at the EcoRI fragment encompassing the ARS315 origin of cells synchronized in G1 and released into S phase. A representative kinetics with its quantification, as well as the average and standard deviation of RIs at the ARS315 during the kinetics of 3 independent experiments, are shown.Defective chromatin assembly in (TIF)Click here for additional data file.Figure S2ARS305 with DNA collected and restricted in agarose plugs. DNA from wild type and asf1Δ cells released into S phase upon G1 synchronization was extracted and restricted with EcoRV and HindIII in agarose plugs and analyzed by 2D-gel electrophoresis. Quantification of RIs, taken the total amount of wild-type RIs over the region as 100, is shown.Analysis of RIs at the (TIF)Click here for additional data file.Figure S3ARS305 and four adjacent regions of cells synchronized in G1 and released into the S phase in the presence of 0.2 M HU for 1 hour. Quantification of RIs, taken the total amount of wild-type RIs over the region as 100, is shown. A schematic representation of the telomere-proximal region replicated from the early origin ARS305 with the restriction fragments analyzed by 2D-gel electrophoresis is also shown on top.Analysis of stalled RIs at the (TIF)Click here for additional data file.


Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver∶brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor  Epidemiological studies, large-scale clinical cohorts, and experimental animal models have shown that hormonal, metabolic and nutritional disturbances during critical periods of early development can significantly impact the propensity to develop adverse health outcomes in later life . We previously reported that these animals display significant “catch up” growth during the first few weeks of postnatal life p<0.05). As the liver is known to possess strong regenerative capabilities throughout life, we hypothesised that an increase in liver size would parallel this overall catch up growth, and to address this we investigated liver-specific effects of MHF diet in offspring at an early post-weaning time-point (i.e. P27) subsequent to catch-up growth. No differences in cell cycle dynamics were observed between CONT and MHF offspring in P27 livers, with the majority of cells appearing in a quiescent (G0/G1) phase irrespective of maternal diet (Most cells (including hepatocytes) require DNA replication to occur prior to mitosis; therefore fluorescently labelling the DNA of all cells within a population and then measuring fluorescence emitted by each individual cell  provides an accurate estimation of cell cycle dynamics. To determine whether differences exist in cell cycle dynamics within the liver of rats born to MHF versus CONT offspring during the first week of postnatal life (i.e. a period of active proliferation), we fluorescently labelled the DNA of dissociated liver cells from P2 rats, and measured DNA content by flow cytometric analysis. Interestingly, MHF diet was associated with a significant inhibition of transition from Gffspring . These dnal diet . Interesnal diet , suggestnal diet . OverallCdkn1a, which encodes the cell cycle inhibitory protein p21CIP1/WAF1We next set out to investigate the mechanism regulating altered hepatic cell cycle dynamics in P2 neonates as a consequence of MHF diet. Many genes involved in the regulation of cell cycle progression are well characterised and we suspected that altered regulation of these genes might be an important contributing factor. As such, using a validated quantitative RT-PCR array (SABiosciences) in P2 neonatal livers, we undertook differential expression analysis of 84 cell cycle-related genes. Of these, 7 genes were significantly differentially expressed between the groups p<0.05; , the majCdkn1a mRNA levels were significantly up-regulated in MHF neonatal livers, we hypothesised that alterations in DNA methylation might play a role in regulating the expression of this gene. We therefore performed quantitative DNA methylation analysis using the SEQUENOM MassArray platform. Nineteen T-cleavage restriction fragments from two PCR amplicons spanning a combined ∼600 bp region across the Cdkn1a CpG island (CGI) (UCSC coordinates chr20:7384350–7384590 NCBI build 39) allowed us to analyse 28 CpG dinucleotides, 12 of which at single CpG dinucleotide resolution  that independently displayed significant hypomethylation relative to controls  diet during pregnancy and lactation leads to changes in phenotypic outcomes resulting in obesity in later life irrespective of postnatal diet. In the present study, we report novel observations that a MHF diet during pregnancy and lactation leads to changes in epigenetic gene regulation in the neonatal liver that result in compromised regulation of cell cycle progression, changes in 1 cell cycle arrest in late gestation fetal rats The final three days of gestation in the fetal rat are marked by a tripling of liver mass and the replacement of haematopoietic cells by hepatocytes, which is accounted for by a high rate of hepatocyte proliferation 1 to S-phase transition is expected to confer, we hypothesised that such differences would be reversed as a consequence of postnatal liver rejuvenation. However, this proved not to be the case, and MHF livers remained significantly smaller than controls by P27 despite somatic “catch up” growth . It is possible that such disproportionality between liver and body weight is in itself one of the factors that contributes to the metabolic complications that arise in the offspring of MHF-fed mothers et al. showed that a MHF diet during pregnancy and lactation resulted in “developmental priming” of non-alcoholic fatty liver disease (NAFLD) whereby maternal fat intake led to disease progression in offspring via impaired hepatic mitochondrial metabolism and up-regulated hepatic lipogenesis ob/ob mouse and hepatocyte expression of p21 is increased in liver biopsies from patients with alcoholic liver disease who exhibit impaired hepatocyte proliferation In contrast to many other differentiated cell types, hepatocytes are known to retain a strong propensity to replicate and rejuvenate into adulthood  in neonatal livers. We therefore investigated epigenetic regulation of Cdkn1a expression in the liver of offspring, specifically focussing on DNA methylation. DNA methylation is the best-characterised mechanism of epigenetic regulation, and in most cases, high levels of DNA methylation at CpG Islands (CGI's) have been strongly correlated with transcriptional repression Cdkn1a mRNA expression in MHF offspring at P2 is associated with subtle but significantly lower levels of DNA methylation. Interestingly, these effects were seen across the entire CGI, as well as at specific CpG dinucleotides. These observations are suggestive of a mechanism by which transcriptional activity of Cdkn1a becomes transiently increased in P2 MHF offspring as a consequence of relatively lower DNA methylation levels. Interestingly, no differences in DNA methylation at the Cdkn1a CGI were observed at P27, suggesting that this epigenetic modification is dynamically regulated during early postnatal development in the rat. It will be important for future studies to determine whether differences in other epigenetic modifications such as trimethylated histone 3 lysine 27 (H3K27me3) persist at later developmental time-points. For example, previous work in cancer has shown that the polycomb modification H3K27me3 and DNA methylation are mutually exclusive and that switching between these two states can occur; thus representing a means by which epigenetic states may change during disease and development Epigenetic mechanisms are known to play an important role in the regulation of gene expression and developmental processes Cdkn1a CGI region, using TRANSFAC software. This led to the identification of numerous transcription factor binding sites associated with the Cdkn1a CGI (see TP53) was previously shown to play an essential role in transcriptional activation Cdkn1a ETF binding site plays a critical role in the modulation of transcriptional activity, and cell proliferation, in rodent hepatocytes.High levels of DNA methylation presumably bring about their transcriptionally repressive effects by causing local chromatin compaction and thus preventing transcriptional activators from binding to gene promoter and enhancer regions. Indeed, such a phenomenon has previously been reported in the brains of rodents exposed to poor maternal care during early postnatal life  CGI see . Of partet al. has demonstrated significant differences in cell-type specific epigenetic regulation within a single tissue The liver is a dynamic organ comprised of many cell types, each of which possesses distinct epigenotypes. These different epigenotypes ultimately control cell functionality, and thus phenotype. In the present study we have shown that a maternal obesogenic diet is associated with changes in gene expression and DNA methylation patterns in male offspring related to cell cycle control which are paralleled by a reduced maturation of hepatocytes during a critical period of liver development. We recognise that these data are limited however, due to the presence of a mixed cell population via use of whole liver homogenates. Recent work by Grigoriu All animal work was approved by the Animal Ethics Committee at the University of Auckland .ad libitum until postnatal day 27 (P27). Hepatic tissue samples were collected from males at 2 developmental ages, postnatal day P2 and P27 . Livers were weighed and a sample of liver homogenate collected in Type IV collagenase (Sigma-Aldrich) for flow cytometry and another sample from the same lobe snap frozen in liquid nitrogen for molecular analyses.A rat model of maternal high fat nutrition was used as described previously 6 cells/ml. Cells were co-stained with LIVE/DEAD Fixable Green (Invitrogen) and Vybrant DyeCycle Orange (Invitrogen) dyes according to the manufacturer's instructions, and fluorescence data were collected for 20,000 viable cells using the FACSAria II flow cytometry system (Becton Dickinson). Proportions of cells in the various stages of the cell cycle were determined using FCS Express Multicycle software.A single suspension of liver-derived cells was prepared using an enzymatic dissociation procedure. Freshly excised liver was minced using a scalpel blade and approximately 200 mg was transferred to 10 ml of ice-cold HBSS solution containing 1.25 mg/ml Type IV collagenase . Suspensions were incubated at 37°C for 30 minutes with occasional gentle mixing by inversion, followed by triturating by gentle pipetting, and then filtering first through 100 µm and then 40 µm cell strainers (Becton Dickinson). Cells were pelleted by centrifugation at 200× g for 5 minutes at 4°C, washed twice in PBS, and resuspended in PBS to a final concentration of 1×10Snap frozen liver (30 mg) was homogenised using a TissueLyser (Qiagen), and total RNA and genomic DNA were co-extracted using the AllPrep DNA/RNA Mini Kit (Qiagen), according to the manufacturers recommended protocol. Extracted nucleic acids were resuspended in sterile RNase/DNase free water (Ambion), and concentrations and purity of DNA and RNA were assessed using a NanoDrop 1000 spectrophotometer  using NanoDrop software (version 3.1.2).2 First Strand Kit (SABiosciences). The differential expression (CTRL versus HF) of 84 genes key to cell cycle regulation were analysed using the Rat Cell Cycle RT2 Profiler PCR array (SABiosciences). Quantitative PCR was performed according to the manufacturer's recommended protocol, using the SYBR Green detector on a 7900HT Sequence Detection System (Applied Biosystems). Relative mRNA expression levels were determined by the ΔΔ-Ct method t) of a panel of housekeeping genes (included on the array) as the normalisation factor .Total RNA (1 µg) was used to generate cDNA using the RTCdkn1a gene under the following PCR conditions: 2 mM MgCl2, 200 µM dNTP mix, 0.2 Units Taq polymerase, 400 nM forward (F) and reverse (R) primers, and 10 ng sodium bisulphite treated DNA template. Primer sequences were:The EZ DNA Methylation Kit (Zymo) was used to prepare sodium bisulphite treated genomic DNA, predominantly according to the manufacturer's instructions, but with the inclusion of an “Alternative Cycling Protocol” . Sodium bisulphite treated DNA was used as template to amplify two discrete regions within the CpG Island (CGI) of the rat 1:aggaagagagGGGTTTAGGTAGATTTTGGGTAGTT; R1:cagtaatacgactcactatagggagaaggctACCTATTCCACACAAAAACAAAAT; F2:aggaagagagGAGAAAAAGGTTTGGATTATGTGAT; R2:cagtaatacgactcactatagggagaaggctAAACCCAAAACTACCCAAAATCTAC . A GeneAMP PCR System 9700 (Applied Biosystems) was used to carry out the PCR under the following cycling conditions: step 1: 95°C 4 min; step 2: 95°C, 20 sec; 60°C, 30 sec; 72°C, 60 sec – 45 cycles; step 3: 72°C, min; 4°C hold. The proportion of DNA methylation at defined CpG units was then determined using the MassCLEAVE for MassARRAY procedure (SEQUENOM) according to the manufacturers recommended protocols, followed by analysis using EpiTyper 1.0 software (SEQUENOM).Fp<0.05.Data were analyzed using JMP 7  for Windows. All data were first tested for normality and equal variances. Data are presented as mean ± SEM unless otherwise stated. In all cases, “litters” were considered to be individual biological replicates. At least four litters per treatment group were used, and significant differences between treatment groups were determined using Student's t-test. Correlations between gene expression and DNA methylation were analysed using the Pearson test. Level of significance was set at Figure S1Cdkn1a CpG island in CTRL and MHF offspring at postnatal day 2  and postnatal day 27 . CTRL, control offspring; MHF, maternal high fat offspring. * p<0.05.Absolute DNA methylation levels at 19 individual CpG units across the (TIF)Click here for additional data file.Figure S2Cdkn1a CpG island.Identification of numerous transcription factor binding sites associated with the (TIF)Click here for additional data file.


Women known to carry a BRCA1/2 mutation have to make complex decisions regarding the management of their increased cancer risk, such as whether or not to undergo risk-reducing surgery and how to communicate this risk information to family members. These women may face a unique set of supportive care needs, although unlike the situation for breast and other cancer patients, few supportive care services for mutation carriers exist outside the Familial Cancer Centres. The aim of this presentation is to describe the unmet support needs of women with a known BRCA 1/2 mutation volunteering for a randomised control trial (RCT) testing the effectiveness of a telephone based peer support program.Participants were recruited through Familial Cancer Centres in Victoria, New South Wales and South Australia. Women between the ages of 18-70, who tested positive for a BRCA1/2 mutation within the last 4 years were eligible for participation in the study. Participants were recruited as part of an ongoing RCT. Women completed a baseline survey after enrolling in the study and prior to randomisation. The baseline survey included a 16-item scale assessing unmet support needs, such as dealing with fears about developing cancer, wanting reassurance that feelings are normal, and talking to others in a similar situation. Women were asked to indicate their level of need in the past month on each item using a 5-point likert scale.216 participants have completed the baseline survey to date (response rate of 46%). The average age of participants was 47.2 years (SD:13.6) and 44.0% (n=95) had a previous diagnosis of breast or ovarian cancer. The mean time since notification of carrier status was 2.5 years (SD=2.1).The average number of moderate to very high needs among all participants was 4.7 (S.D 4.6). Twenty six percent (n=52) of participants had no moderate to very high needs, while 23.1% (n=46) had a moderate to very high need on eight or more items. The most commonly endorsed moderate to very high needs were: “dealing with uncertainty about the future” (42%), “dealing with the impact a faulty gene has had on your family” (40%), and “dealing with fears about developing cancer” (39%). The least endorsed items were: “dealing with insurance issues” (61% indicated no need), “dealing with feelings of isolation” (53% no need) and “obtaining more information about your risk for breast cancer” (53%). The average number of moderate to very high unmet needs was not related to previous history of cancer (P=0.94), having children (p=0.87) or time since receiving BRCA1/2 mutation status results (P=0.36).Many women with a known BRCA 1/2 mutation have unmet supportive care needs that are not currently being addressed. Level of unmet need is not related to time since receiving test results, history of cancer or having children. The RCT trial will assess whether a telephone-based peer support program meets these unmet needs.


Maximum-likelihood and Bayesian methods were used to explore the degree of phylogenetic mixing of subtype C sequences from different cities and to reconstruct the geographical pattern of viral spread in this country region. Phylogeographic analyses supported the monophyletic origin of the HIV-1 subtype C clade circulating in southern Brazil and placed the root of that clade in Curitiba (Paraná state). This analysis further suggested that Florianópolis (Santa Catarina state) is an important staging post in the subtype C dissemination displaying high viral migration rates from and to the other cities, while viral flux between Curitiba and Porto Alegre (Rio Grande do Sul state) is very low. We found a positive correlation (r2 = 0.64) between routine travel and viral migration rates among localities. Despite the intense viral movement, phylogenetic intermixing of subtype C sequences from different Brazilian cities is lower than expected by chance. Notably, a high proportion (67%) of subtype C sequences from Porto Alegre branched within a single local monophyletic sub-cluster. These results suggest that the HIV-1 subtype C epidemic in southern Brazil has been shaped by both frequent viral migration among states and in situ dissemination of local clades.The HIV-1 subtype C has spread efficiently in the southern states of Brazil . Phylogeographic studies indicate that the subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population at some time point between 1960 and 1980, but little is known about the spatial dynamics of viral spread. A total of 135 Brazilian HIV-1 subtype C  Through June 2011, about 610,000 cases of AIDS have been cumulatively reported in Brazil since the first identification of AIDS in the early 1980s Phylogeographic studies indicate that the HIV-1 subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population probably originated from East Africa pol datasets that included a very small number of sequences (n = 14) per state, which may bias the results. In the present study, we reevaluated the spatial pattern of HIV-1 subtype C dissemination in southern Brazilian states, based on the analysis of a larger subtype C pol data set containing sequences collected at the capital cities of Rio Grande do Sul (n = 55), Santa Catarina (n = 41), and Paraná (n = 39).Although several studies have explored the origin and time-scale of the HIV-1 subtype C epidemic in Brazil, there is little information about the spatial dynamic of dispersion of this clade in the country. The only phylogeographic study points toward Paraná or Rio Grande do Sul states as the possible entrance points of HIV-1 subtype C and further indicate an asymmetrical net viral flow following a north to south axis et aln = 55) and Curitiba (n = 21) described elsewhere New protease and partial reverse transcriptase (PR/RT) subtype C Brazilian sequences were obtained from 41 HIV-infected antiretroviral therapy-naïve patients from Florianópolis  and 18 HIV-infected treated-patients from Curitiba . Patients from Florianópolis were followed up at the “Hospital Homero de Miranda Gomes", had their samples taken from 2008 to 2009 and were described in more detail by Gräf pol gene sequences of African origin was obtained from the Los Alamos HIV Sequence Database (www.hiv.lanl.gov). The reference set includes subtype C sequences representative of the East , Southern , Central , and West  African regions, sampled over a time interval of 18 years (1986–2004). Among those subtype C references we included some sequences from Burundi, Kenya, and Ethiopia that were described as the ones most closely related to the Brazilian subtype C clade A reference set of 31 subtype C aLRT) Sequences were aligned using CLUSTAL X program 6 generations with a burn-in of 3–5×106 generations. Adequate chain mixing was checked by calculating the effective sample size (ESS) using program TRACER v1.4 The hypothesis of restricted phylogenetic mixing of HIV-1 subtype C sequences obtained from different cities in Southern Brazil was tested using program BaTS n = 3) and a discrete phylogeographic model was used in which all three possible reversible exchange rates between locations were equally likely (flat prior) −3 substitutions/site/year) equal to the mean rate previously estimated for the Brazilian subtype C clade at pol gene 4 nucleotide substitution model, an uncorrelated Lognormal relaxed molecular clock model BayesTraits program Ancestral reconstruction of the locations at the interior nodes of a time-scale phylogenetic tree was obtained using the Bayesian statistical framework implemented in BEAST 1.6.2 pol (PR/RT) sequences from Porto Alegre (n = 55), Florianópolis (n = 41), and Curitiba (n = 39) were analyzed together with some subtype C reference sequences of African origin, including those sequences from East Africa that were described as the ones most closely related to the Brazilian subtype C lineage. ML and Bayesian phylogenetic analyses revealed that all Brazilian subtype C sequences formed a highly supported  monophyletic group , here called BR-PA, that was almost exclusively composed by sequences isolated in that state , Porto Alegre/Curitiba (PA/CU), and Florianópolis/Curitiba (FL/CU). The null hypothesis of panmixis  was rejected by both the AI and PS statistics for all comparisons , demonstPP≥0.96) and that the ancestor of the BR-PA clade probably originated in Florianópolis (PP = 1).Having established the existence of statistically significant geographic subdivision as well as evidence of frequent migrations of Brazilian subtype C strains, we applied a Bayesian phylogeographic approach in order to investigate how this structure was established and to estimate the net viral flow among southern states. The overall topology of the Bayesian MCC tree obtained with BEAST corroborates the monophyletic nature of the Brazilian subtype C clade and the existence of the BR-PA sub-clade circulating in Porto Alegre . Ancestrr2 = 0.64), albeit not completely  followed by Florianópolis to Porto Alegre (956.2), Porto Alegre to Florianópolis (143.1), Florianópolis to Curitiba (86.4), Curitiba to Porto Alegre (40.1), and Porto Alegre to Curitiba (0.2) . As a pompletely . InteresPP≥0.96). Differences in the Brazilian and/or African subtype C sequences used in each study may have a great influence in the exact position of the root of the Brazilian subtype C clade. Because we only included sequences retrieved from the southern state capitals, it will be important to test whether the support of Curitiba (Paraná) as the entrance point of subtype C is maintained or not after addition of new sequences sampled at other Brazilian cities.This study supports the notion that the HIV-1 subtype C epidemic affecting the south Brazilian region resulted from a single founder event, consistent with previous findings et alAfter its introduction into Brazil, the subtype C clade was rapidly disseminated through the south region Florianópolis is the most important resort locality in the region and a partial correlation of routine travel patterns and HIV-1 movement was found in our study. The most intense routine travel and the highest viral migration rates were from Paraná (Curitiba) to Santa Catarina (Florianópolis). At the other extreme, Paraná and Rio Grande do Sul displayed the less frequent routine travel and also the lowest viral migration rates within the region. People mobility, however, may only explain part of the subtype C dispersal dynamics between southern Brazilian states. This seems to be particularly evident for Rio Grande do Sul that receives much more subtype C lineages and sends much less than would be expected according to routine travel among the states. No relationship between the estimated subtype C migration rates and the size of the AIDS epidemic nor the subtype C prevalence was observed. Porto Alegre has the larger number of HIV-1 positive patients , but exhet alin situ dissemination of a single local lineage, here called BR-PA, which was probably introduced from Santa Catarina. Of note, previous studies have shown a high prevalence (10–35%) of the Circulating Recombinant Form 31_BC in Porto Alegre While the previous work of Veras In conclusion, the results presented here suggest that the subtype C epidemic spreading in the southern region of Brazil was initiated by the introduction of a single founder strain probably through Paraná. This subtype C variant was rapidly disseminated to the other southern states and during this process, Florianópolis (Santa Catarina) acted as an important staging post between Curitiba (Paraná) and Porto Alegre (Rio Grande do Sul), both receiving and sending viral lineages to the neighboring cities. Meanwhile, the direct viral migration flow between Curitiba and Porto Alegre seems to be much lower. The current subtype C diversity in the south Brazilian region has also been shaped by the local dispersion of a limited set of founding strains, particularly in the southern-most capital. These data provide a more comprehensive understanding of the spatial dynamics of the subtype C epidemic spreading in southern Brazil and may be helpful to guide HIV prevention strategies in this region of the country.


The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ40WT is significantly impaired to acquire the fibrillar architecture of Aβ40Arc. A detailed characterisation demonstrated that Aβ40WT has a restricted ability to dock and isomerise with high binding affinity onto Aβ40Arc fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided “dock and lock” mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1–40 Glu22Gly (Aβ This mutation is associated with an aggressive form of AD Alzheimer's disease (AD) is the most common form of dementia today and results in both individual suffering and major economical costs for society Amyloid polymerisation is a complicated process where monomers are incorporated into the amyloid fibril form in several steps. The polymerisation process has been studied in detail and it is widely accepted that the process involves a dock and lock mechanism Within this work we can show that the architecture of Aβ fibrils is determined by constraints imposed by the monomer conformation during docking and isomerisation. This finding has general implications and suggests that although Aβ is considered to be unstructured it is restrained to adopt certain conformations which impair its ability to acquire certain fibrillar structures. Interestingly, our results moreover show that the kinetic rate of fibril formation determines the fibrillar architecture rather than the thermodynamically lowest energy state.www.alexotech.com). Due to the aggregation properties of Aβ-peptides, an appropriate solubilisation scheme was essential. Prior to use, lyophilised peptides were solubilised in 10 mM NaOH at pH 12, followed by 30 s sonication in a water bath and 5 min centrifugation at 20 000 g to remove residual oligomeric species. This treatment efficiently monomerised the peptides and facilitated dilution in phosphate buffered saline  to the selected concentrations. All samples were verified to have pH 7.4 or a pD corresponding to 7.0 by direct pH meter reading. Fibrillar forms were acquired by incubating 100 µM Aβ at 37°C under agitation for 48 hours. μ.All peptides were obtained from Alexotech AB, Umeå, Sweden  14N-AβWT40 fibrils as seeds. Agitation was omitted during cross-seeding experiments.The fibrillar forms of 2O solution  followed by 24 h incubation at 37°C. The second fraction was used without further treatment as a fully protonated reference sample to identify and exclude amide exchange resulting from the experimental procedure (i.e. highly exposed amides within the monomeric state). At the end of the incubation period and immediately prior to NMR analysis, Aβ assemblies were recovered by short centrifugations (20 000 g) and rapidly converted into NMR-detectable monomers using an optimised solution of hexafluoroisopropanol as described previously To probe solvent accessibility, aggregate/fibrillar solutions of each peptide type were split into two fractions and recovered by short centrifugation at 20 000 g. Hydrogen-deuterium (H/D) exchange was carried out on one of the fractions by diluting the pellets 30 times using a D15N-HSQC experiments, typically starting 6–8 minutes after fibril dissolution. All experiments were performed at 15°C on a 600 MHz Bruker AVANCE spectrometer, equipped with a 5 mm triple-resonance, pulsed-field z-gradient cryoprobe. The acquisition time for each 15N-HSQC experiment was 10 min using four transients per increment and 128 (t1)×1024 (t2) complex data points. Prior to each 15N-HSQC experiment, a 1D proton NMR spectrum was acquired to quantitatively monitor the dissolution of fibrils into monomers. Protection ratios and experimental errors were determined as described previously Hydrogen exchange was subsequently monitored by recording a series of heteronuclear 2D N-(3-dimethylaminopropyl)-N′-ethylcarbodimidehydrochloride) 0.4 M and NHS (N-hydroxysuccinimide) 0,1 M for 7 min.The different Aβ variants, including both monomeric and fibrillar, forms were immobilized at 10 µM peptide concentration to a final density corresponding to 2000–5 000 RU to a CM5 chip or to the dextran free chip C1  using standard amine-coupling chemistry at pH 5. Briefly, to activate the chip a 50/50 mixture of EDC  of monomers at equilibrium. Therefore, the dissociation constant can be used in combination with SPR data obtained with known concentrations of peptides in the running buffer to determine the affinity between the monomers and fibrillar ends Fibrillar samples were analysed directly on the surface of C1 chips  that do not have a dextran surface. Analysis was performed using a Nanoscope IIIa multimode AFM  in tapping mode™ in air. A silicon probe was oscillated at around 280 kHz and images were collected at an optimised scan rate corresponding to 1–2 Hz.40Arc and Aβ40WT, their corresponding fibrillar form were analysed through quenched H/D exchange NMR experiments. 40Arc and Aβ40WT, respectively. The result clearly shows that no significant difference can be identified between the two forms, only a slightly lower protection is observed for the four C-terminal residues of Aβ40WT. 40Arc when seeded with Aβ40WT fibrils and further strengthen the notion that no obvious difference can be detected on the backbone structure, apart from a moderate reduction of the solvent protection at the mutation site of Aβ40Arc.To evaluate whether there are any structural differences between the fibrillar cores of Aβ40WT fibrils recruited free Aβ40WT monomers by a fibrillar polymerisation process . As a consequence, two different fibrillar architectures can be anticipated where Aβ40Arc monomers can more easily polymerise onto either fibrillar form than Aβ40WT monomers.Surface plasmon resonance (SPR) enables the changes in bound mass to a surface to be monitored and therefore provides a convenient tool to follow fibril formation. Immobilised Aβ process . Bindingmonomers . To furton phase . In conton rates . The effImmobilised monomeric Aβ on the chip surface could not recruit monomeric variants from the solution, probed in an identical manner as described above. This important control verified the specificity of the system and also highlights the dependency of a specific structure for an efficient peptide assembly (data not shown).40WT and Aβ40Arc fibril morphologies and verify preservation of fibril integrity during immobilisation on the SPR chip. Sonicated fibrils of Aβ40WT or Aβ40Arc were immobilised followed by a continuous polymerisation. The surface of the C1 chips were directly analysed using AFM  was employed to compare Aβsing AFM . The pre40WT and Aβ40WT fibrils was determined to be 200 nM  would favour an increase in the fraction of high affinity bound peptides. As a consequence, the time-dependent isomerisation event would be enhanced at lower monomer concentrations where the time between each new docking event would be longer, thereby increasing the maturation time. However, our results showed that the impaired ability of Aβ40WT monomers into the fibrillar form of Aβ40Arc is significantly impaired as result of a reduced ability to dock and isomerise relative to Aβ40Arc monomers. Upon docking, the fibril structure is determined by a balance between intra-peptide and peptide-fibril interactions. At this point, it is not possible to determine if the impaired incorporation of Aβ40WT to adopt into Aβ40Arc fibrils is due to structural limitations of monomeric Aβ40WT in solution, interactions of the monomer with the fibril end or a combination of the two effects. Nonetheless, Aβ40WT monomer binding to Aβ40Arc is of low affinity and cannot compete efficiently with the Aβ40Arc monomer binding. Therefore, the properties of Aβ fibrils are controlled to a great extent by the properties and constraints of the precursor molecules.From our results, we can conclude that incorporation of Aβ40Arc monomers have a significantly stronger affinity for Aβ40WT fibrils than Aβ40Arc fibrils. Since Aβ40Arc monomers can adopt the conformation of both Aβ40WT and Aβ40Arc fibrils, it is not thermodynamic stability that determines fibrillar architecture but rather the rate of formation. Due to the high kinetic barriers, a subsequent re-arrangement of the fibril into a thermodynamic more stable form is prevented.From a general point of view, the initial formation of a nucleus and the architecture of the resulting amyloid fibrils are controlled by both thermodynamic and kinetic factors. As the aggregates increase in size, the energetic barriers between different states increases and kinetic barriers essentially block interconversion between different fibrillar forms. On this basis we hypothesise that the predominant fibrillar structure would be the structure with the fastest kinetics of formation even though more thermodynamically stable states might exist. Interestingly, this hypothesis is supported by the results shown here as the measurements of affinity between the monomers and the fibrillar ends are directly related to the thermodynamic stability of the fibril 40WT and Aβ40Arc are similar and suggest a similar structure overall. However, through cross-seeding experiments, striking differences regarding aggregation rates was seen, indicating structural differences. Our results suggest that Aβ40WT docking and subsequent isomerisation into Aβ40Arc fibrils is restricted. This finding highlights the importance of structural constraints at an early point in the process of incorporating free monomers into Aβ fibrils. As a consequence, structural constraints of the monomer, possibly already in solution, determine the rate of fibril assembly. We further showed that formation of the predominant Aβ fibrillar architecture is controlled by kinetics rather than thermodynamics where the most thermodynamically stable form is not necessarily the predominant structure within a sample.Development of AD is the consequence of an imbalance between aggregate formation and degradation. The rate of peptide assembly as well as the stability of the formed aggregates is consequently of high importance. We can within this work show that the solvent protection patterns of the fibrillar forms of Aβ


Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus  Although often inconspicuous, Eumycota fungi constitute one of the dominant life forms of our planet. They inhabit nearly all biotopes and their total biomass is huge Recent developments in our understanding of Eumycota phylogeny Although basidiomycetes and ascomycetes follow the same general pattern of dikaryophase maintenance, they also exhibit some differences. Foremost is the stage at which dikaryophase occurs. In most filamentous ascomycetes, the dikaryon forms following the fusion of male and female gametes and is located inside the fruiting body. Only in Taphrinomycotina, a basal sub-phylum of ascomycetes, have dikaryotic vegetative mycelia been described Three Amino acid Length Extension), which contain three extra amino acids between helix 1 and helix 2, and non-TALE. Interestingly, HD factors often function as homo- or hetero-dimers. In animals, they are present as dozens of paralogs, which include notably the hox genes, which participate in the definition of the body plan Clamp connections are under the control of the mating-type in Basidiomycetes, which encodes either pheromones and receptors or homeoproteins . The first HD protein characterized in filamentous ascomycetes was PAH1 from P. anserina genome using the sequence of pah1 and HD transcription factors from N. crassa and M. griseapah1 (P. anserina homeobox 1) and six were novel genes , the only M. grisea transcription factor without a P. anserina ortholog in the study by Kim et al. M. grisea, N. crassa and P. anserina, and shows that the HD proteins can be subdivided into two distinct classes, TALE and non-TALE e.g., N. crassa, Sordaria macrospora, Chaetomium globosum, M. grisea, Aspergillus nidulans and A. fumigatus). Transcriptomic analysis showed that all seven of the homeobox genes were expressed in P. anserina , some triple mutants, and some other multiple mutants . These experiments were also performed at 18°C and 35°C to test for possible cold- or thermo-sensitivity. The ΔP. anserina male gametes. The Δpah1 mutant is known to produce more spermatia than the wild-type strain pah1 mutant pah1 mutants, none of the mutations affected sexual events occurring before fertilization.We then tested the efficiency of sexual reproduction in the mutant strains, including the formation of sexual cells and the differentiation of fertilized fruiting bodies. Although none of the HD transcription factors were involved in crozier formation, alterations in the sexual cycle were detected in some mutants. The homeogene deletion mutants were tested for impaired formation of spermatia, the mat+/mat− heterokaryons that were homozygous for a homeobox gene deletion. Wild-type mat+/mat− heterokaryotic culture inoculated in the center of an M2 plate differentiates perithecia mostly in a ring area 1 cm thick and located about 1 cm away from the inoculation point  and ascospore autonomous. The pigmentation defect had a variable expressivity, and Δpah5 ascospores could be white, light green or dark green.In addition to the beak defect, there was also a defect in ascospore pigmentation. It was investigated in ♂ Δ progeny . Geneticpah5 ascospores also had a low germination rate. To quantify the germination rate, asci were recovered after their ejection from the perithecia on a layer of germination medium solidified by 1.2% agar. The layer was then observed under the light microscope after incubation for 6 h at 27°C. In these conditions, WT ascospores germinated with almost 100% efficiency, while the germination rate of Δpah5 ascospores was reduced to 42% (60/144 ascospores). Furthermore, Δpah5 ascospores had an altered mode of germination . The Δpah1Δpah2Δpah7 triple mutant had a similar percentage (95.5%) to the Δpah1Δpah2 double mutant. Thus, Δpah3 appeared to antagonize the cooperative effect of Δpah1 and Δpah2, which contrasts with the synergistic interaction it displayed with Δpah2 in the Δpah2Δpah3 double mutant. The percentage of perithecia without beaks was not further modified (90.5%) when a fourth mutation, Δpah4, was added (Δpah1Δpah2Δpah3Δpah4 quadruple mutant). Similarly, the addition of the Δpah4 mutation did not modify the Δpah2Δpah3 interaction (44% perithecia without beaks in both the Δpah2Δpah3Δpah4 triple mutant and the Δpah2Δpah3 double mutant).To further explore the genetic interactions between the homeobox gene mutations, we generated by genetic crosses three triple mutants, Δvaluated . A slighpah1Δpah2Δpah3Δpah4Δpah5 quintuple and the Δpah1Δpah2Δpah3Δpah4Δpah5Δpah6 sextuple mutants differentiated only tiny fruiting bodies without ascospores, indicating that defects in the perithecial wall and beaks increased as more deletions were associated with the Δpah5 deletion. As for Δpah5, this defect could be fully rescued by Δmat hyphae, which enabled normal development of the fruiting body in the quintuple and sextuple mutants , it is written that “Sexually reproducing Ascomycota also form dikaryons, although they are not as long-lived as those of Basidiomycota. The clade that includes Ascomycota and Basidiomycota has been called the Dikaryomycotina, reflecting this presumably homologous similarity… Croziers may be homologous to clamp connections”.Since their discovery, there has been debate about whether clamps and croziers are analogous or homologous. Analogy is the opinion of, for example, Buller P. anserina. In basidiomycetes, the initial stages, including nuclear pairing, initial clamp cell formation, conjugate mitosis and first septum formation, are regulated by homeodomain transcription factors located at one of the two mating-type loci in tetrapolar Basidiomycetes . The later stages of peg formation and cell fusion are controlled by the other mating-type locus , which encodes pheromones and receptors. In the dimorphic pathogenic fungus Ustilago maydis, the formation of clamp-like structures is dependent on the homeodomain transcription factors encoded by the b mating-type locus, as in the higher basidiomycetes, while it is independent of the pheromone–receptor system encoded by the a mating-type locus P. anserina strain had no impact on crozier formation. Ascomycetes homeobox genes may be orthologous or paralogous to those present in basidiomycetes. If the homeobox genes present in ascomycetes are paralogous to those of basidiomycetes, this means that key genes involved in the control of clamp formation are absent from ascomycetes. Alternatively, if ascomycetes homeobox genes are orthologous to those of basidiomycetes, then these genes are not involved in crozier formation. This result suggests that the molecular mechanisms controlling the formation of crozier in ascomycetes and clamp in basidiomycetes are different, suggesting that clamp and crozier are analogous rather than homologous structures. This has previously been proposed based on the many differences that exist in their formation, such as the fusion of the apex for crozier and of a lateral peg for clamp, and in the different stages of the lifecycle at which they take place Unlike basidiomycetes, for which the homeobox genes present at one of the mating-type loci are mandatory for clamp formation, homeodomain transcription factors appear dispensable for crozier formation in the filamentous ascomycete Cryptococolax abnorme, another Pucciniomycotina belonging to the Cryptococolomycetes, has hyphae with clamp Colacosiphon filiformis, lack clamps Cystobasidiopsis nirenbergiaeRecent advances in understanding the phylogeny of fungi provide additional evidence for analogy . Among tclampless1, has previously been identified as being involved in clamp formation based on studies in Coprinopsis cinereaCryptococcus neoformansUstilago maydisclampless1 gene is not conserved outside the basidiomycetes cro1, encodes a protein belonging to the family of UCS proteins, which are assumed to interact with various myosins cro1 mutants of Podospora anserina have a defect in the establishment and/or maintenance of a true cellular state: the actin belts and septa formation between the daughter nuclei after division are misplaced or absent, leading to a giant, multinucleated crozier prm1, which is involved in cell fusion P. anserinaAlthough they play a major role in the biology of fungi, the presence and formation of clamp and crozier are neglected phylogenetic and molecular topics. Apart from the role of the mating-types, to our knowledge, only one gene, P. anserina used in this study were derived from the “S” (big S) wild-type strain, which is the strain used for sequencing of the P. anserina genome http://podospora.igmors.u-psud.fr. Details of the culture conditions and experimental procedures to work with this fungus can be accessed at the same internet address.All strains of pah2 to pah5), plasmids containing homeobox deletion cassettes conferring resistance to hygromycin B were constructed according to the AscI/MluI method Pah6 and pah7 deletion cassettes were constructed according to the N. crassa strategy for high-throughput gene deletion pah6 and pah7, a phleomycin resistance cassette was used. Each deletion plasmid was digested to generate the deletion cassette prior to transformation of Δmus-51 protoplasts P. anserina. This was done to eliminate potential untransformed nuclei and to segregate out the Δmus-51 mutation. Several mat+ and mat− strains containing the deletion but lacking Δmus-51 were selected from each progeny and subjected to Southern blot analysis for validation  of the hygromycin phenotype, i.e., asci with two HygR (resp. PhleR) and two HygS (resp. PhleS) ascospores. Sensitive ascospore-derived cultures from these asci carried the wild-type alleles of both genes; hence, resistant cultures had mutations in both genes. A heterokaryotic mat+/mat− resistant culture was self-crossed and mat+ and mat− homokaryotic double mutants were isolated from the progeny. Colonial growth (for Δpah1) and ascospore pigmentation defect (for Δpah5) constituted two easily scorable phenotypes, allowing easy identification of the corresponding mutations. In all crosses involving Δpah5, it was used as the male partner since it exhibited a maternal sexual defect. To generate double mutants for pah1 and pah2, which are closely linked on chromosome 2, 70 asci were analyzed and double mutants were isolated from tetratype asci. Only Δpah5Δpah7 double mutants could not be recovered, which was due to the tight linkage (d<0.1 cM) of these two genes. The genotype of all double mutants was confirmed by PCR assays directed at the specific junctions for each deletion.Double mutants were constructed by crossing single mutants of opposite mating-type in all pairwise combinations. In crosses involving pah2Δpah3Δpah4 was isolated from the Δpah2×Δpah3Δpah4 cross by screening asci exhibiting the 2 hygR/2 hygS segregation described above. Δpah1Δpah2Δpah3 mutants were isolated from Δpah1Δpah2×Δpah3 using a similar strategy. The Δpah1Δpah2Δpah3Δpah4 quadruple mutants were isolated from Δpah1Δpah2×Δpah3Δpah4. Sixty-four spored asci were analyzed to determine their germination phenotype. Eight asci with 2 colonial/2 non-colonial segregation, indicative of Δpah1 FDS, were further tested on hygromycin B medium. One ascus with 2 hygR colonial/2 hygS non-colonial spores was identified. The mat+ or mat− homokaryotic quadruple mutants were obtained in the progeny of one hygR colonial ascospore from this ascus. Presence of the four deletions was confirmed by PCR. The Δpah1Δpah2Δpah3Δpah4Δpah5 quintuple mutants were isolated from Δpah1Δpah2Δpah3Δpah4×Δpah5. More than 100 asci with 2 pigmented/2 unpigmented ascopores (2+pah5/2Δpah5) were analyzed on hygromycin B medium. Two asci with 2 hygR colonial/2 hygS non-colonial segregation pattern were identified. The quintuple mutant was generated from a hygR colonial culture from one of these two asci. The presence of all five deletions was confirmed by PCR analysis. The sextuple mutant Δpah1Δpah2Δpah3Δpah4Δpah5Δpah6 was generated by crossing Δpah6 as female with the quintuple mutant as male. One hundred and sixty asci with 2 pigmented/2 unpigmented ascospores (2+pah5/2Δpah5) were analyzed. Unpigmented ascospores gave rise to colonial mycelium (Δpah5Δpah1) in 18 asci, among which one ascus exhibited 2 hygR phleR/2 hygS phleR segregation. The hygR phleR ascospores were thus homokaryotic for Δpah1Δpah2Δpah3Δpah4Δpah5 and heterokaryotic for Δpah6. mat+ and mat− homokaryotic sextuple mutants were then isolated from the progeny of self-fertilization of one of the mycelia recovered from the hygR phleR ascospores. When Δpah5 was homozygous in a cross, the maturation defect of the perithecia was rescued with the Δmat helper strain . Cells were observed in a three-dimensional deconvolution microscope  equipped with an HCxPL APO 100× oil CS objective, NA = 1.40 (Leica). The images were captured by a 10 MHz Cool SNAPHQ charge-coupled device camera (Roper Instruments), with a z-optical spacing of 0.5 µm. METAMORPH software  was used to acquire z-series. Images were processed using ImageJ software .Cells were fixed in 7.4% paraformaldehyde and processed for fluorescence microscopy as previously described Supporting Information S1pah7 increased after fertilization, which corresponded to the timing at which some of these genes are required for proper development of the fruiting bodies, i.e., for neck differentiation. The expression of most of the genes was decreased at the end of the development period (96 h). The expression level was measured as described by Bidard et al. pah mutants. DNA was isolated from wild-type P. anserina and one or several purified transformants  and digested with appropriate restriction enzymes. The blots were probed either with a sequence containing the relevant CDS and its flanking region  or with the ble sequence . For each pah gene, a restriction map of the wild-type and mutant locus is presented. The sizes of the expected fragments are indicated on the maps and are reported close to the corresponding fragment on the Southern blot. The DNA sequence around pah7 is not well defined because of sequencing errors and the restriction pattern cannot be confidently predicted for the pah7 deletion. To confirm the deletion of pah7, the blot was stripped and further probed with the pah7 CDS to confirm that it was absent in the Δpah7 candidates. Figure S3. Fertility of Δpahx mutants on medium containing wood shavings. Strains of the indicated genotypes were inoculated on medium containing wood shavings as the sole carbon source. The images were taken 10 days later. Even after prolonged incubation for up to three weeks, no perithecia were observed in the Δpah1Δpah2 and Δpah2Δpah5 cultures. The small dots visible on the Δpah2Δpah5 cultures correspond to dark, insoluble product(s) excreted by the hyphae. These products are also excreted by wild-type and all the other pahx mutants. Figure S4. Fertility rescue by Δmat hyphae. Heterokaryons with the following components: pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat+, pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat− andΔmat are able to differentiate fruiting bodies of wild-type appearance, which produce abundant ascospores.Figure S1. Expression of homeobox genes during fruiting body maturation. Differential expression in space and time of the seven homeobox genes was assessed by microarray analysis in a time course of mycelium growth  and perithecium maturation . No difference in expression was detected for the seven genes in one-day, two-day or three-day old mycelia. In contrast, statistically significant differential expression of the homeobox genes was detected during perithecium maturation. Each gene had its own pattern of expression. However, the expression of each homeobox gene except (DOC)Click here for additional data file.