CHRNA5 is strongly associated with the level of nicotine consumption in humans and manipulation of the expression or function of Chrna5 similarly alters nicotine consumption in rodents. In both humans and rodents, reduced or complete loss of function of Chrna5 leads to increased nicotine consumption. However, the mechanism through which decreased function of Chrna5 increases nicotine intake is not well-understood. Toward a better understanding of how loss of function of Chrna5 increases nicotine consumption, we have initiated efforts to identify genetic modifiers of Chrna5 deletion-dependent oral nicotine consumption in mice. For this, we introgressed the Chrna5 knockout (KO) mutation onto a panel of C57BL/6J-Chr#A/J/NAJ chromosome substitution strains (CSS) and measured oral nicotine consumption in 18 CSS and C57BL/6 (B6) mice homozygous for the Chrna5 KO allele as well as their Chrna5 wild type littermates. As expected, nicotine consumption was significantly increased in Chrna5 KO mice relative to Chrna5 wildtype mice on a B6 background. Among the CSS homozygous for the Chrna5 KO allele, several exhibited altered nicotine consumption relative to B6 Chrna5 KO mice. Sex-independent modifiers were detected in CSS possessing A/J chromosomes 5 and 11 and a male-specific modifier was found on chromosome 15. In all cases nicotine consumption was reduced in the CSS Chrna5 KO mice relative to B6 Chrna5 KO mice and consumption in the CSS KO mice was indistinguishable from their wild type littermates. Nicotine consumption was also reduced in both Chrna5 KO and wildtype CSS mice possessing A/J chromosome 1 and increased in both KO and wild type chromosome 17 CSS relative to KO and wild type B6 mice. These results demonstrate the presence of several genetic modifiers of nicotine consumption in Chrna5 KO mice as well as identify loci that may affect nicotine consumption independent of Chrna5 genotype. Identification of the genes that underlie the altered nicotine consumption may provide novel insight into the mechanism through which Chrna5 deletion increases nicotine consumption and, more generally, a better appreciation of the neurobiology of nicotine intake.The gene  CHRNA5, the gene that codes for the nAChR α5 subunit, nicotine dependence and other nicotine dependence relevant phenotypes (CHRNA5 (rs16969968) exhibited the strongest association with cigarettes per day of all tested SNPs (P = 1.2 × 10−278) (It has become evident that nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit play a critical role in the risk for nicotine dependence. Human studies repeatedly have found an association between genetic variants in enotypes –7. In faChrna5 in nicotine-related behaviors. For example, a few early studies demonstrated that Chrna5 played a role in sensitivity to the acute effects on nicotine in mice  mice, unlike wildtype controls, did not reduce their responding for i.v. nicotine self-administration as the unit dose increased. At the highest nicotine dose tested, Chrna5 KO mice self-administered five times more nicotine than did their wildtype controls. Similarly, Jackson et al.  often without having a measurable effect on the phenotype itself (Chrna5 KO-dependent nicotine consumption. Chromosome substitution strains (CSS) are a panel of strains that share a common genetic background but differ from one another for a single whole chromosome that comes from a donor strain. A full panel of mouse CSS consists of twenty-two strains, one strain for each of the 19 donor strain autosomes, one each for the donor strain sex chromosomes and one strain harboring the mitochondrial genome from the donor strain  and body weight , respectively. Therefore, the main measure we used to assess nicotine consumption was μg of nicotine consumed per ml of total fluid drank (μg/ml) which should minimize any confound caused by strain-dependent differences in total fluid intake and weight.Because the most common measure used to assess oral nicotine intake is dose and dose is dependent upon the amount of fluid intake as well as the weight of the mice, the CSS panel was assessed for whether there was a main effect of strain on either fluid intake or animal body weight across the study. Results indicated main effects of strain on both fluid consumption [Chrna5 genotype impacts nicotine consumption as previously reported (Chrna5 null mutation as well as their wildtype (WT) littermates were tested in an ascending two-bottle choice test for oral nicotine consumption at nicotine concentrations 100, 200, and 300 μg/ml. Results (Chrna5 genotype on nicotine consumption on a B6 background (P = 0.003) with Chrna5 null mutant mice consuming significantly more nicotine than WT mice. There was no main effect of sex and no genotype by sex interaction. Examination of the entire population of tested CSS mice (P = 5.9 × 10−20) as well as sex (P = 1.6 × 10−10) but no genotype x sex interaction. Consistent with B6 background mice, CSS mice harboring the Chrna5 null mutation, on average, consuming more nicotine than CSS mice that were WT for Chrna5.To confirm that reported , 13, fem Results  confirmeCSS mice  also indChrna5 genotype impacts nicotine intake in the CSS, it was then determined whether individual strain impacted nicotine consumption and/or the effect of Chrna5 genotype on consumption. In order to simplify the phenotype, total μg/ml of nicotine consumed across the experiment  was used for the analysis. For total μg/ml of nicotine consumed, main effects of strain (P = 5.02 × 10−35), genotype (P = 1.88 × 10−25) and sex (P = 2.33 × 10−12) as well as a strain x genotype interaction (P = 0.006) were observed  was compared to B6 mice homozygous for the null mutation (B6Chrna5KO) in a within genotype analysis (P = 1.1 × 10−18) and sex (P = 1 × 10−6). Due to the main effect of sex, the CSSChrna5KO strains were individually assessed for the effect of sex on nicotine intake , CSS5Chrna5KO (q < 0.005), and CSS11Chrna5KO (q < 0.005) consumed less nicotine then did B6Chrna5KO mice  and increased relative to B6Chrna5KO for both sexes in CSS17Chrna5KO mice   . IncreasChrna5 null mutation could be specific to the Chrna5 deletion or could be a main effect of the CSS on nicotine consumption . To establish whether the effect of strain on nicotine consumption was specific to mice possessing the Chrna5 null mutation or a general effect on nicotine intake, nicotine consumption was assessed in all of the CSS strains possessing the WT allele of Chrna5 and compared to B6 WT mice (P = 6.7 × 10−13) and sex (P = 1.1 × 10−7). Again, due to the main effect of sex, individual CSS strains were evaluated for significant sex effects (q < 0.05) consumed less nicotine than did B6 WT mice while CSS17 WT (q < 0.05) drank more nicotine than did B6 WT mice  also exhibited a main effect of sex. However, the lack of a significant genotype by sex interaction for any of these strains indicates that the effect of Chrna5 genotype on nicotine consumption did not appreciably differ between female and male animals. For six strains , there was a main effect for both genotype and sex suggesting the possibility that the effect of Chrna5 genotype on nicotine intake might be sex dependent. Among these six strains, four  exhibited modest sex differences in the effect of Chrna5 genotype on intake. Nonetheless, the lack of a genotype by sex interaction suggests no meaningful difference of the effect of Chrna5 genotype on nicotine consumption between female and male mice of these strains.Lastly, CSS were examined individually using a between Chrna5-dependent nicotine consumption as indicated by either a within genotype  and/or between genotype comparison , one strain (CSS17) impacted nicotine intake independent of Chrna5 genotype and one strain (CSS1) altered nicotine intake in both a Chrna5 genotype dependent and independent manner.In summary , amongstChrna5, the gene that encodes the nicotinic acetylcholine receptor subunit α5, significantly increases nicotine consumption  includes the observation that nicotine intake in CSS5Chrna5KO, CSS11Chrna5KO, and male CSS15Chrna5KO mice significantly differs from the reference B6Chrna5KO strain. In all cases, the CSSChrna5KO animals consume less nicotine than B6Chrna5KO mice. In addition, mice that possess the WT allele of Chrna5 in these three strains do not significantly differ from B6 mice harboring the WT allele indicating that the strain effect is specific to the Chrna5 null allele. Further, the effect of Chrna5 genotype on nicotine consumption in CSS5, CSS11, and male CSS15 mice is abolished . Removing CSS9 from this analysis due to potential genetic differences from our CSS9 vs. the JAX CSS9 leads to a significant positive correlation . Using dose (mg/kg/day) as an alternative measure of nicotine consumption in the CSS WT mice led to significant correlations with locomotor activity with  or without  the inclusion of CSS9. In sum, these correlations are suggestive of a genetic overlap between oral nicotine intake and the acute effects of nicotine on locomotor activity with animals less sensitive to the locomotor depressant effects/more sensitive to the locomotor stimulating effects of nicotine consuming greater amounts of nicotine.In the only other study that measured a nicotine response in the C57BL/6J-Chr#and Gill  assessedand Gill  and dataChrna5 genotype in CSS1 mice remains to be determined.Genetic mapping of oral nicotine consumption in mice also has been reported in one previous study . In thisChrna5 deletion on nicotine consumption and one CSS was identified that possesses an allele or alleles that substantially increases nicotine consumption independent of Chrna5 genotype. Future studies with the CSSChrna5KO and CSS WT mice of interest can take advantage of the unique structure of the CSS .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Japanese cedar pollinosis is a major seasonal allergy in Japan, and Japanese cypress pollinosis is a growing concern because the cypress pollen season follows the cedar pollen season and cross-reactivity among allergens occurs between these closely related species. Allergens purified from pollen under unspecified collecting conditions can potentially heterogenous allergens profiles and batch to batch variability, and amino acid sequence variants in allergens possibly exist among trees. Polymorphisms have not been investigated for the cypress pollen major allergens, Cha o 1 and Cha o 2. Our aim was to examine the homogeneity of allergen amino acid sequences. DNA sequences of Cha o 1 and Cha o 2 from pollen collected from Chiba and Ibaraki prefectures and from needles of 47 plus trees located at seed orchards in Chiba Prefecture were examined by amplicon sequencing and amino acid substitutions were deduced. Sequence analysis of the pollen samples revealed that eight and seven residues of Cha o 2 were polymorphic, respectively. Thirteen residues in Cha o 2, including those residues identified in pollen, were deduced to be polymorphic for the plus trees. Cha o 2 expressed by the 47 plus trees included amino acid differences when compared with that of isoallergen Cha o 2.0101. No substitution was deduced in Cha o 1 for pollen taken from the two prefectures. One conservative amino acid substitution was deduced in Cha o 1 for the plus trees. Of the 47 plus trees examined, 38 were deduced to express only the isoallergen Cha o 1.0101 isoform, whereas eight trees were heterozygous and a single tree was homozygous for the non-synonymous mutation, which indicates relative uniformity of Cha o 1. Cha o 2 was found to be a heterogeneous allergen which suggests that studies using pollen from different trees may not give the same results. Cupressus arizonica) and Mediterranean cypress (Cupressus sempervirens) release pollen that cause allergic reactions, and a major allergen from Mediterranean cypress is the most common allergen among 75 allergens found in Italy .–31.29–31We found 1 and 13 amino acid substitutions in Cha o 1 and Cha o 2, respectively, from plus trees located at nurseries in Chiba Prefecture and pollen collected in Chiba and Ibaraki prefectures. These polymorphisms in Cha o 2 may affect mAb affinity toward Cha o 2 when the antibody against Cha o 2 has been raised against a particular Cha o 2 amino acid sequence where one or more of the 13 substitutions resides in the antibody epitope.


A novel calcium carbonate cement system that mimics the naturally occurring mineralization process of carbon dioxide to biogenic or geologic calcium carbonate deposits was developed utilizing carbon dioxide-containing flue gas and high-calcium industrial solid waste as raw materials. The calcium carbonate cement reaction is based on the polymorphic transformation from metastable vaterite to aragonite and can achieve >40 MPa compressive strength. Due to its unique properties, the calcium carbonate cement is well suited for building materials applications with controlled factory manufacturing processes that can take advantage of its rapid curing at elevated temperatures and lower density for competitive advantages. Examples of suitable applications are lightweight fiber cement board and aerated concrete. The new cement system described is an environmentally sustainable alternative cement that can be carbon negative, meaning more carbon dioxide is captured during its manufacture than is emitted. The cementing reaction is based on the polymorphic transformation from metastable amorphous CaCO3 and vaterite to aragonite or calcite through a dissolution–reprecipitation process in an aqueous medium. However, limited production scale and mechanical properties (3–13 MPa compressive strength) have restricted larger scale applications in the construction industry. Nevertheless, the results obtained by Combes et al. provide promising insights for the development of a nature inspired, synthetic CaCO3 cement system at scale from anthropogenic CO2 with properties suitable for various building materials applications.Inspired by the biological use of CaCOe et al.  and Combe et al. ,16 succe3 cement: CO2, calcium, and alkalinity. CO2 can originate from CO2-containing industrial flue gas, e.g., thermal power plants, chemical plants, and cement kilns. Calcium and alkalinity can come from industrial waste streams that contain both calcium and alkalinity in the form of calcium oxide (CaO), calcium hydroxide (Ca(OH)2), and calcium silicates. Examples of calcium and alkalinity-rich industrial waste streams include: carbide lime sludge, an impure Ca(OH)2 by-product of producing acetylene gas (C2H2) from coal and limestone, slag, a Ca/Mg-rich aluminosilicate glass by-product of iron and steel production, and cement kiln dust, an impure CaCO3/CaO by-product of Portland cement manufacturing that is often recycled in the cement kiln feed [4Cl), ammonium sulfate ((NH4)2SO4), and ammonium nitrate (NH4NO3) have been shown to be effective acids in the production of vaterite from CO2 because they can bring the calcium into solution (Equation (1)), absorb CO2 from a gas stream (Equation (2)), and then be regenerated during the formation of CaCO3 (Equation (3)) [Industrially, three feedstocks are required to produce CaCOiln feed ,18. An aion (3)) ,19,20.23 over calcite and aragonite, precipitation is performed at high reactant concentrations, needed to achieve high supersaturation and kinetic stabilization [2 across the gas–liquid interface, (2) the rate at which CO2 converts into carbonate, and (3) the carbonate–bicarbonate equilibrium. The rate of mass transfer can be increased by increasing the flow rate of gas or by increasing the gas–liquid interface, for example by high shear mixing. Subsequent conversion of CO2 into carbonate can occur through the reaction with water (H2O) or hydroxyl ions (OH−), but because the latter reaction is 107 times faster, higher rates can be achieved by performing the reaction at higher pH. Higher pH also favors higher equilibrium concentrations of carbonate over bicarbonate. Except for high gas flow rates, these strategies also improve the CO2 absorption efficiency.To promote the formation of metastable forms of CaCOlization . The con3 cement system that mimics the naturally occurring mineralization process of CO2 to biogenic or geologic CaCO3 deposits is described [3 cement is produced by capturing CO2-containing industrial flue gas and utilizing a calcium and alkalinity-rich industrial waste stream [2 and energy footprint compared to traditional inorganic binders, such as Portland cement. Furthermore, the CaCO3 cement offers processing and performance advantages in a variety of building materials applications over traditional inorganic binders.Herein, a novel CaCOescribed ,22. The e stream . The res2− and SO42−, see 2-containing flue gas used was generated from a boiler simulator  burning propane. NH4Cl  was used for solubilizing Ca(OH)2.The carbide lime sludge  contained 25 wt% solids and had silica, alumina, and mixed oxidation state sulfur impurities, e.g., S3 cement, the carbide lime sludge is first solubilized with aqueous NH4Cl then passed through a leaf filter to remove insoluble impurities resulting in an aqueous solution of CaCl2 and ammonia (NH3), see Equation (1). NH3 dissolved in water is in equilibrium with ammonium hydroxide (NH4OH). CO2-containing flue gas (11 vol%) is then contacted with the solution in a three-phase continuous-stirred tank reactor (CSTR) controlled at below 40 °C (Equation (2)), resulting in the precipitation of CaCO3 cement (Equation (3)). The simulated flue gas was blown from the boiler simulator to the absorber via a 20 cm steel pipe using a 600 cfm roots style blower. Unlike some other carbon capture technologies, the CO2 does not need to be concentrated or compressed to high pressures for storage, which is energy intensive. The CaCO3 cement is then mechanically dewatered by a filter press  and thermally dried in a swirl fluidizer  to a free-flowing powder. The NH4Cl solution is recovered during dewatering and is recycled for the solubilization of additional carbide lime sludge. Insoluble impurities accounted for 7% of the dry mass of the carbide lime sludge and were composed of 70% mixed oxides and 30% carbon, see 3 cement manufacturing process. The CaCO3 cement pilot plant is shown in To produce CaCO3 cement. Measurements were carried out with test samples dispersed in isopropyl alcohol using refractive indexes of 1.378 for isopropyl alcohol and 1.58 for CaCO3.A Horiba LA-950V2 Laser Scattering Particle Size Distribution Analyzer (two light sources: 650 nm wavelength laser and 405 nm wavelength LED) was used to study the particle size distribution of the CaCO3 cement. The loss on ignition (LOI) was determined by measuring the mass loss after heating the samples in an electric muffle furnace  at 950 °C for 60 min.An ARL QUANT’X Energy Dispersive X-ray Fluorescence Spectrometer  was used to evaluate the chemical composition of the oven-dried raw materials and the CaCO3 cement and the transformed cement pastes. The instrument was operated at 40 kV and 30 mA  using a scan range of 5–65° 2θ, a step size of 0.02°, and dwell time of 4 s. The resulting spectra were analyzed using Jade 9 software  for qualitative phase composition information and MAUD software for quantitative Rietveld analysis (QXRD) [A X’Pert Pro X-ray Diffractometer  was used to determine the phase composition of the CaCOs (QXRD) .3 cement and the transformed cement paste. The samples were coated with platinum/palladium using a sputter coater  prior to secondary electron imaging.A SU-6600 Field Emission Scanning Electron Microscope (SEM)  was used to collect images of the CaCO3 cement was evaluated at 4 h, 10 h, 1 day, 3 days, 7 days, and 14 days. Paste cubes (50 × 50 × 50 mm3) with a solution-to-solid ratio (w/c) of 0.35 were utilized. The solution contained 0.1 M magnesium and strontium chloride. The pastes were mixed according to ASTM C305 and then cast and compressed according to ASTM C109 [Compressive strength development of the CaCOSTM C109 ,26. The 3 cement has a median particle size of 15 with a 4 µm standard deviation. The D90 and D10 for the CaCO3 cement are 19 and 11 µm, respectively. The CaCO3 cement. The alumina and silica impurities found in the carbide lime sludge are not found in the resulting cement because they are relatively insoluble impurities that were removed during the filtering process  and NH3 gas. The HCl and NH3 gas are recovered while scrubbing the flue gas. A portion of the HCl gas contacts water on the drying CaCO3, forming hydrochloric acid, and is neutralized by the CaCO3 cement, leaving a residual amount of CaCl2 [3 cement produced is mainly composed of metastable vaterite (>99 wt%) with a minor amount of calcite (<1 wt%), see 3 is deliberately avoided as it is difficult to stabilize and process on an industrial scale. Once the CaCO3 cement is dried, it is stable under ambient conditions. process ; howeverof CaCl2 . The CaC3 cement is the transformation of vaterite (CaCO3) to aragonite (CaCO3) or calcite (CaCO3). Vaterite dissolves and aragonite and/or calcite precipitates. Since the cementing reaction is an aqueous dissolution-reprecipitation process, curing the CaCO3 cement requires high humidity to maintain the transformation of vaterite to aragonite and/or calcite. If the humidity of the curing environment is not controlled, the material will desiccate, and the cementing reaction will cease, similar to Portland cement. Additionally, like Portland cement, the cementing reaction of CaCO3 cement can be accelerated by increasing the curing temperature. 3 cementing reaction and compressive strength development. The ultimate compressive strength of the material is 40 MPa, which is 3–13× higher than the 3–13 MPa compressive strength, previously reported for CaCO3 cement developed for medical applications [3 cement paste does not show strength development through 1 day and takes 14 days to achieve the full strength. By elevating the temperature, full strength can be achieved by 7 or 3 days for 60 and 80 °C, respectively. Additionally, at elevated temperature, CaCO3 cement pastes have developed enough strength by 4 to 10 h  to be demolded and handled. Fast early strength is important for manufactured building materials with the need to transfer materials between initial and final curing steps, e.g., from pre-curing in molds at lower temperatures to autoclaving in stacks at elevated temperatures.The cementing reaction in CaCOications ,15. At 42, MgSO4, Mg(C2H3O2)2), which are known to inhibit calcite growth [2, Sr(C2H3O2)2), which are known to promote aragonite formation [During the dissolution of vaterite, aragonite is the preferred precipitate over the most thermodynamically stable calcite, as aragonite exhibits an acicular morphology which can lead to an interconnected microstructure. The formation of aragonite can be controlled through the use of magnesium-based additives  and heterogeneous structure. At all curing temperatures, aragonite crystals nucleate on the surface of vaterite particles, then grow outward, which results in a dense and interconnected honeycomb microstructure . This ph3 cement (vaterite) to aragonite in the hardened cement paste. Comparing 3 cement is entirely dependent on the vaterite to aragonite transformation, which is less complex than traditional cements and can be easily accelerated through temperature without negative effects on performance. For instance, at 80 °C, the CaCO3 cement  has fully transformed to 98% aragonite and 2% calcite by 3 days of curing; whereas, at 40 °C, the hardened cement paste is composed of 12% vaterite, 87% aragonite and 1% calcite at 14 days of curing.2 on a dry basis with minor impurities such as carbon, silica, alumina, iron, and sulfur species depending on the composition of coal and limestone. Carbide lime sludge is used in China to replace limestone as a raw material for Portland cement production which can reduce CO2 emissions yet increase energy demand depending on the additional energy required to dry the carbide lime [Annual global production of carbide lime sludge was estimated at approximately 25 million tons on a dry basis, where >95% is generated in China . Carbideide lime . However2 could be captured from industrial facilities and approximately 230 million tons of CaCO3 cement could be produced.Iron and steel slag production is, on the contrary, more evenly distributed worldwide. It is estimated that about 510–664 million tons of iron and steel slag were generated annually in 2019 . Iron an3 cement is well suited for building materials applications with controlled factory manufacturing processes that are typically made from inorganic binders, such as Portland cement or gypsum. The CaCO3 cement can provide processing and performance advantages over Portland cement, e.g., shorter production cycles, lighter weight, and flexibility on fillers and reinforcement elements. Furthermore, CaCO3 cement is white, which makes it easy to color and texturize for decorative purposes.With its unique properties, CaCO3 cement is not suitable for structural applications that require mild steel reinforcement but is suited for composite systems development with the flexibility to incorporate a wide range of reinforcement elements for superior toughness and flexural properties. The neutral pore solution of the CaCO3 cement also makes it amenable for use in environmentally sensitive areas and applications that require biocompatibility.Because of its neutral pore solution, CaCO3 cement’s porosity has inherent advantages and disadvantages. The advantage is the ability to formulate lightweight products. For example, suitable potential applications are lightweight fiber cement board and autoclaved aerated concrete due to their manufacture in controlled environments that utilize elevated curing temperatures to accelerate property development. In other applications, the porosity could be a disadvantage, such as concrete in severe freeze–thaw or physical salt attack environments. The durability of CaCO3 cement in a range of situations and applications should be investigated in future research projects.CaCO3 cement manufacturing process  and thermal energy for drying at approximately 130 °C. Based on pilot plant data, production of CaCO3 cement is calculated to require 2.8 to 3.9 MJ/kg when considering a range of dry and wet raw materials. This compares to the 4.6 to 5.6 MJ/kg energy consumed during Portland cement production, mainly from the calcination of limestone at 900 °C and the sintering process at 1450 °C [3 cement from waste feedstocks does not require high temperature processes and can capture and store CO2 from industrial emissions, CaCO3 cement has a significantly lower CO2 footprint compared to Portland cement. The energy and carbon footprint for CaCO3 cement manufacturing will vary for different sites and plant scales due to factors such as electricity mix, fuel sources, and operational efficiency.The CaCO process  requires 1450 °C . Since p3 cement), the electrical energy consumption is 92 kWh/metric ton of CaCO3 cement, and the natural gas consumption is 3.57 GJ/metric ton of CaCO3 cement. The plant operations that consume the most electricity are carbide lime sludge dissolution and filtration (39%) and CO2 absorption including flue gas compression (38%). 88% of the natural gas is consumed in the drying of the feedstock and product. Using a 2019 California grid carbon intensity of 175 kg CO2/MWh, the electricity results in emissions of 16 kg CO2/metric ton of CaCO3 cement. With an emission factor of 56 kg CO2/GJ, the natural gas combustion produces 200 kg CO2/metric ton of CaCO3 cement [2/metric ton of CaCO3 cement. 3 cement production to the CO2 emitted during Portland cement production. Since every metric ton of CaCO3 cement produced captures 440 kg of CO2 and manufacturing CaCO3 cement releases 216 kg of CO2, CaCO3 cement has a net capture of 224 kg of CO2/metric ton of CaCO3 cement produced. Substituting CaCO3 cement for Portland cement, results in a reduction of 882 kg of CO2/metric ton of cement utilized due to net emissions of 658 kg of CO2 avoided from Portland cement and a net capture of 224 kg of CO2 during the manufacture of CaCO3 cement when utilizing a waste Ca feedstock.For the highest energy consumption case  to heat from 20 to 80 °C. End of life uses for CaCO3 cement composites would be similar to those of Portland cement composites with the dominant recycling scheme being crushed into aggregate.If curing is conducted at elevated temperature, additional energy input is required to heat the cementitious mixture to the curing temperature. For example, 1 m2 emissions into CaCO3 cement at a pilot scale. The CaCO3 cement gains approximately 40 MPa of strength during its conversion from vaterite to aragonite, which is 3× higher than previously reported results. The cement paste hardens and gains strength through the formation of a network of interlocking aragonite needles. The conversion and strength gain can be accelerated by elevated curing temperatures, e.g., full conversion and strength in 3 days at 80 °C. The CaCO3 cement system is particularly well-suited in applications that can take advantage of the inherent porosity, low pH, or rapid kinetics. The new cement system described is an environmentally sustainable alternative cement that can be carbon negative, meaning more CO2 can be captured during its manufacture than is emitted when utilizing a waste Ca feedstock.We have described a method of converting anthropogenic CO


Translational Psychiatry 10.1038/s41398-021-01494-5, published online 05 July 2021Correction to: The original version of this article unfortunately contained a mistake in Table 1. All blank boxes (☐) in Table 1 columns 3 and 4 should be replaced with the horizontal solid arrow (→). The remaining dashed horizontal arrow (-->) in the bottom cell of column 3 of Table 1 should be replaced with the horizontal solid arrow (→) as well. We apologize for this error caused by the typesetter. The original article has been corrected.


Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model. GATA2 is a zinc finger transcription factor expressed widely across a number of tissues including vascular, lymphatic, urogenital, cardiovascular, neural, and hematopoietic systems. It is a master regulator of hematopoiesis with the majority of knockout mice succumbing to death from anemia before embryonic day E10.5 . Its impGermline mutations in GATA2 show phenotypes across all ages. However, are particularly enriched in children and teenagers presenting with myelodysplastic syndrome (MDS) and congenital neutropenia . This hiGATA2 gene termed gata2a and gata2b. Both genes are highly homologous to the human GATA2 gene, particularly in the zinc finger domains where they are 98% and 95% identical at the amino acid level, respectively (gata2a and gata2b show synteny with regions of human Chromosome 3 where GATA2 is located (itga2b:eGFP) zebrafish show that in adult hematopoiesis, both Gata2a and Gata2b are expressed in HSPC in an “or” fashion  stocks were maintained according to standard procedures in UK Home Office approved aquaria (um27gata2a carrying a 10 bp deletion in Gata2a (a gift from Nathan Lawson)7, Tg(lyzC:nfsB-mCherry) (a gift from Stephen Renshaw), la116Tg(kdrl:GFP), la2TgTg(itga2b:GFP) la2Tg .2, 0.33 mM MgSO4) in Petri dishes. Pigment formation was avoided by supplementing E3 medium with phenylthiourea (PTU) (Sigma) 0.2 mM from 24 hpf or removed by bleaching post in situ hybridization by incubating in 5% v/v deionized formamide, 5% v/v H2O2, 0.5x SSC. For live experiments and prior to fixation, embryos were anesthetized with ethyl 3-aminobenzoate methanesulfonate . Embryos and larvae were staged according to Embryos were raised at 28.5°C in E3 medium  as previously described . SB posiO-dianisidine staining was used to label hemoglobinised erythrocytes in zebrafish larvae as previously described (escribed .la2TgTg(itga2b:GFP) larvae were analyzed by flow cytometry at 3 dpf as described previously , and TOPO cloned into pcDNA3.1V5-His. Forward orientation was confirmed by sequencing. mRNA was synthesized using Ambion mMessage mMachine  Sp6 or T7 ultra kit, respectively.c-myb expression in runx1 in Fixed embryos and larvae were imaged either in 1x PBST or 80% v/v glycerol. Brightfield and epifluorescence images were acquired using a Leica M205 FA fluorescent dissecting stereoscope equipped with a Leica DFC 365 FX camera and LAS 4.0 software. Images were processed using Fiji version v1.49u software . Quantifum27 fish were genotyped by PCR and restriction enzyme (RE) digest using MspA1I, which is abolished by the 10 bp deletion, or Kompetitive allele specific PCR (KASP). Gu5008ata2b genotyping was also conducted using PCR and RE digest with FauI enzyme, which abolished by the deletion, or by KASP. Additional details can be located in Gata2a® software and similarities assessed using BLOSUM62 matrix. Synteny was assessed using the comparative genomics tools-region comparison on ENSEMBL for each GATA2 gene independently and subsequently for 10 genes upstream and downstream of each.GATA2 (human and zebrafish) protein sequences were aligned using GeneiousTg(itga2b:eGFP); um27/+Gata2a× u5008/+Tg(lyzC:mCherry);Gata2b embryos were raised in a single tank and genotyped just prior to analysis to mitigate for any tank specific effects. Adult zebrafish kidneys were dissected from terminally anaesthetized animals, dissociated in PBS with 1% FBS using a gentleMACs tissue dissociator (Milteyni) and analyzed by flow cytometry. Flow analysis was conducted using FlowJoTM.For these experiments a clutch of p < 0.05 and all tests used were two-tailed. Data are presented as mean ± standard deviation. Data from each independent experiment was normalized to the mean of its wildtype group, and experiments were pooled for analysis. No normality assumptions were made, and data was analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons post hoc test or two-way ANOVA with Sidak’s post hoc test. In um27/um27gata2a embryos scored according to their circulation were tested for equal distribution with contingency Chi-squared test.Statistical analysis was performed using GraphPad Prism version 8.1.2 for OS X software . The probability level for statistical significance was um27gata2a mutant embryos over time. The earliest hemato-vascular progenitors express Lmo2 which is required for expression of downstream erythroid and myeloid progenitors. We analyzed the expression of lmo2 in the anterior lateral plate mesoderm (ALPM) at 10ss (14 hpf) in Gata2aum27 mutants to assess hematopoietic progenitors with myeloid potential. Lmo2 expression in the ALPM was not affected by Gata2a loss indicating Gata2a is not required for anterior hemato-vascular cell specification  around this time, the origin of which is not clearly delineated and thought to be distinct from that of anterior derived macrophages  which emerge around this time, however, we did not assess EMP specifically using other markers. In summary, loss of Gata2a does not impact specification of early hematopoietic progenitors but results in loss of both anterior and posterior cells of the myeloid lineage during primitive hematopoiesis.We next assessed primitive myeloid development in and tail  Scheme fucocytes . l-plastnd trunk . A subserophages . Quantif2aum27/+ . Further mutants . At thisc-myb and runx1. Expression of both runx1 and c-myb was reduced in Gata2a mutants . We further quantified HSPC using epifluorescent imaging of gata2aum27 mutants crossed to Tg(itga2b:GFP) transgenic animals where static GFPlo cells mark HSPC . By contrast GFPlo cells are virtually absent in gata2aum27/um27. We hypothesized that the near absence of stem cells in Gata2aum27 homozygous animals is related to its loss of trunk circulation lo cells  transgenic animals, we noted a proportion of um27/um27Tg(itga2b:GFP); gata2a embryos had GFPlo cells in the CHT and circulating GFPhi thrombocytes  line, a small proportion of um27/um27gata2a mutants show expression of myeloid cells in the CHT suggesting those embryos that escape circulatory defects and develop HSPC that are able to undergo differentiation to mature granulocytes, however, it was not possible to quantify this further in view of the vascular defects.We went on to analyze the effects of Gata2a loss on differentiated myeloid and erythroid cells using SB and observed  quantifim27/um27 . As obseum27/um27 ogata2a-dianisidine stained embryos at 28 hpf . We assessed HSPC numbers further at 72 hpf using the Tg(itga2b:GFP) line crossed to u5008gata2b  cells in the CHT at 60 hpf and 4 dpf. As observed in single heterozygotes, HSPC numbers recover and show no difference between genotypes at this timepoint  as well as in u5008gata2b (in definitive hematopoiesis). Using SB as a readout, we did not observe any difference between the number of granulocytes in +/um27; gata2b+/u5008gata2a double heterozygotes compared to single heterozygotes or WT at 2 dpf  at 12 and 15 months post of age. Blood populations were assess based on forward and side scatter characteristics, with the addition of back-gating GFP and mCherry expressing populations . No chantrophils . Therefo4/i4 (i4 phenotype is a tissue specific knockdown of Gata2a in hematopoietic cells and occurs in homozygous gata2ai4/i4, therefore the level of Gata2a is likely significantly lower than in our mutants that are not viable as homozygotes. Also gata2a i4 mutants have reduced levels of Gata2b, potentially adding additional impact to their phenotype. Thus our data supports the fact that double heterozygous mutants exhibit features of GATA2 deficiency, but that there may be compensatory mechanisms from the other ohnolog that ameliorate some of the phenotypes.We did not, however, observe any leukemias in our double heterozygous mutants, or any overt features of defective lymphatic function as observed in Embergers syndrome. Our phenotype is notably more subtle that that observed in gata2ai4/i4 . We thinWe sought to develop a model of human GATA2 haploinsufficiency using zebrafish to further understand the etiology and spectrum of disease related to effects on developmental hematopoiesis. To do this we proposed analysis of loss of Gata2a and Gata2b alone and in combination in zebrafish would recapitulate a range of phenotypes that may contribute to disease evolution and may further infer structure-function relationships inherent from the sequence similarities and differences between these two highly similar ohnologs. In addition, we hypothesized that we may uncover important redundancies between these and potentially other GATA factors.We showed that both Gata2 genes are syntenic with human chromosome 3, indicating that these genes arose from an ancestral additional round of duplication of this region. The expression pattern of Gata2a and Gata2b genes are disparate with limited overlap during development .um27/um27gata2a mutants have severe vascular defects (um27/um27gata2a homozygotes are capable of recovering and developing HSPCs in a few cases where blood flow was recovered or truncal vasculature remained intact. These observations are consistent with the recovery of HSPC development reported in fish with mutations in a conserved enhancer that drives endothelial expression of Gata2a (u5008/u5008Gata2b mutants have normal vascular development but have a reduction in HSPCs in the aorta from 36 hpf. This defect is no longer evident by 4 dpf in the CHT and notably overall their phenotype is milder than that observed in gata2b morphants (um27/+gata2a heterozygotes, consistently with the role of Gata2a upstream of Gata2b, regulating runx1 and gata2b expression in the hemogenic endothelium (Our studies show that loss of Gata2a or Gata2b result in a reduction of HSPC. Prior studies have shown that  defects  which arf Gata2a . Other rf Gata2a . In our orphants . Interesothelium .um27gata2a mutants shows a role for Gata2a during primitive myelopoiesis while analysis of u5008gata2b mutants show that Gata2b is dispensable for primitive myelopoiesis but is required for definitive myelopoiesis. This phenotype can be observed at 4 dpf and is consistent with decreased myeloid cells in the whole kidney marrow of adult Gata2b mutants (gata2b derived promoter elements that the majority of adult myeloid cells are derived from gata2b+ cells. We propose that the role for Gata2b in myelopoiesis is independent from HSPC production, as experiments on heterozygote +/u5008gata2b fish show normal HSPC numbers as soon as 60 hpf. Additionally, u5008Gata2b mutants exhibit normal definitive erythropoiesis, suggesting that Gata2b is required specifically for granulocyte development. This is consistent with findings of neutropenia in patients carrying GATA2 mutations (Analysis of  mutants . Butko eutations .gata2a is expressed as early as 8ss in the PLM, while gata2b expression is much more spatially restricted and can only be detected from 18ss (Our results show that both Gata2a and Gata2b are required for myeloid and HSPC development, each at different developmental stages. A possible explanation could be the sub-functionalization of these Gata2 ohnologs, by exclusion of territories of expression. Expression analysis shows that rom 18ss . Consistum27/um27 homozygotes to a similar level as Gata2a. Rescue studies in Gata2b morphants conversely could not be rescued by constitutive expression of Gata2a (The differing contributions of Gata2a and Gata2b could also be explained by expression exclusion without substantial changes in protein function. The observed non-synergistic effects on HSPC development in compound heterozygotes suggest that over time, one WT copy of each Gata2 gene is sufficient to compensate the lack of the other. This supports the possibility that the differing phenotypes are a product of expression exclusion rather than different protein function. Further support to this comes from circulation rescue experiments which show that Gata2b overexpression is able to partially rescue blood flow in the trunk of Gata2af Gata2a , althougf Gata2a .um27/+;gata2bu5008/+gata2a animals show defects in the myeloid compartment which we first observe at 12 months of age consistent with features of GATA2 deficiency. However, the range of phenotypes and rate if onset is less florid than an alternative published zebrafish Gata2 disease model (Adult se model , where aIn summary, we describe the sequential and complementary defects resulting from loss of Gata2a and Gata2b in zebrafish during development and in adults, further expanding our understanding of the complex inter-regulation of these GATA factors during hematopoiesis, and their role in developmental hematopoiesis relevant to human GATA2 deficiency.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the UK Home Office.OP designed and performed the experiments, interpreted data, wrote, and edited the manuscript. AL, JR, YH, NK, and HL designed, performed the experiments, and interpreted data. CM analyzed and interpreted the experiments. CU performed the experiments and interpreted data. EP conceived the study, directed the experimental design, provided resources, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The incidence is increasing in elderly, diabetic or immunocompromised patients and can be fatal without ‘appropriate’ antibiotic therapy.3 This novel study evaluated the use of erythrocyte sedimentation rate (ESR) and pain scores as measures of response to determine the dose and duration of antibiotic therapy in patients diagnosed with NOE over a 4 year period from November 2015.CT, MRI, and technetium 99 bone scans are often performed but poorly correlate to the clinical response to empirical treatment.Patients diagnosed with NOE were evaluated using pain scores (0–10/10 where 0 = no pain and 10 = worst pain) and ESR at presentation and then at subsequent outpatient visits. Patients received 6 weeks of IV antibiotics  followed by oral ciprofloxacin. Initially ciprofloxacin was prescribed at 750 to 500 mg twice daily and the dose and duration of treatment was personalized according to pain scores and ESR. No ethical approval was required since this was an observational study of standard management of this condition at the Trust.Pseudomonas aeruginosa which in four patients was resistant to gentamicin and in two of these patients also to ciprofloxacin. At presentation pain score was 10/10 in all but three patients (5/10 in one patient and 8/10 in two patients) and ESR values ranged from 31 to 82 (median 66). Both values fell within 1–2 weeks of commencing IV antibiotics and after 6 weeks pain scores were 0/10 in eight patients, 3/10 in the others. ESR at this stage fell to 10–33 (median 15). One patient developed diarrhoea and stopped ciprofloxacin after only 2 weeks remaining well at 6 months follow-up. Eight patients required 3 to 12 months ciprofloxacin. Four patients required restarting or increasing ciprofloxacin dose to control rising pain scores which reflected an increase in their ESR values but in one of these patients having grown Pseudomonas resistant to ciprofloxacin, two further 6 week courses of piperacillin/tazobactam were required. Three patients currently remain on ciprofloxacin at the lowest dose to keep their pain scores and ESR under controlTwelve patients  aged 49 to 91 years (median 74.5 years) were treated. Five had diabetes mellitus and three had been treated for a malignancy. The time of first symptoms to diagnosis ranged from 3 weeks to 32 months (median 3 months). Six had developed facial nerve palsies; three of whom had additional cranial nerve involvement. All had swabs positive for Pain scores with ESR values are invaluable in determining the activity of NOE thereby personalizing the antibiotic dose and duration in patients with this life-threatening infection.


RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene  GCN5 ortholog acetyltransferase  dependent loci in yeast  methylation pathway, with multiple independent hits in SET1, BRE1, and BRE2. We also show results that known mediators of H3K4 repression are not involved and that the RNA Polymerase III complex remains actively bound at the HMR-tDNA in rpd3Δ strains.To address this question in more detail and identify additional potential effectors of this Selected transposon mutant strains isolated in the screen that were further characterized in this study are listed in HMR-ADE2 rpd3 reporter strains DDY3133 and DDY2973  containing 45 µg/ml adenine  due to increased spreading of silencing that represses the ectopic ADE2 allele at HMR. This sufficient but suboptimal level of adenine is critical to the colony color assay, as lower levels (less than 15 µg/ml) of adenine activate the ADE2 promoter to overcome silencing and higher levels (over 90 µg/ml) feedback inhibit the pathway, both leading to white colony growth. Colonies typically were grown for 3 days at 30°C, then plates were held at 4°C for 3–4 days to obtain optimum pigmentation color prior to photographing on a dissecting microscope. Slow-growing rpd3 hst1 hos2 mutants were grown for 5 days at 25°C to obtain optimum pigmentation.Parent  DDY2973  were conubstrate . HMR-ADELEU2 marked transposon insertion, and complete cosegregation of Leu+ Kan+ markers with white and Leu− Kan+ with red colony phenotypes.The yeast transposon mutagenized library was obtained from Mike Snyder (Stanford University), and mutagenesis was performed as described . TransfoSET1, BRE1, and BRE2 was performed by standard yeast genetics procedures by amplifying the LEU2 gene from plasmid pRS405  plates to isolate Leu+ colonies that lost the URA3-marked RPD3 plasmid. These 5-FOARrpd3Δ isolates were confirmed for proper SET1, BRE1, or BRE2 gene deletion by PCR on each end (confirmation oligos listed in RPD3 from ∼240 bp upstream of the ORF to ∼350 bp downstream with oligos DDO-2155 and DDO-2156 and Q5 polymerase (New England Biolabs), using an RPD3-containing genomic library plasmid (with a 12 kilobase insert) as template. The PCR product was digested with Xho I and Not I then cloned into URA3 vector pRS416 . We previously demonstrated that rpd3 mutants result in spread of silencing beyond the boundary, resulting in repression of ADE2 and growth as red colonies on 15% ade media , as complete loss of silencing  results in a nonmating phenotype. Out of 71 primary white colony isolates, 18 were nonmating. Two randomly selected nonmating isolates were found to have independent transposon insertions within the SIR4 gene (DDY5640 and DDY5641). Transposon insertion sites were determined by the anchor bubble ligation-vectorette PCR method , plus two randomly selected nonmating isolates. Interestingly, most of the identified mutants were in the histone H3K4 methylation pathway, including multiple hits in each BRE1, BRE2, and SET1. An additional insertion in NGG1, which codes for a component of the SAGA coactivator complex resulted in a very light pink and variegated phenotype, suggesting a partial suppression of increased rpd3Δ mediated silencing. However, this insertion also resulted in a slow growth phenotype, and it is not clear whether slow growth affects the accumulation of the pigment in this reporter assay. SIR4 gene. Identification of white colony isolates containing insertions within the SIR genes was expected, as they completely abolish HMR silencing. Additional expected insertions were identified in adenine biosynthesis pathway genes, including ADE3, ADE4, ADE5,7, and ADE6, as second adenine pathway mutations are known to suppress the ade2 red colony phenotype .To verify single transposon insertions and cosegregation of the insertion with the white colony phenotype, selected primary isolates were backcrossed to strain DDY814 which contains the rpd3Δ::KanMX allele grew as red colonies on 15% ade media, and those with both rpd3Δ::KanMX and Tn:LEU2 insertions in SET1, BRE1, or BRE2 grew as white colonies  maintained the red colony color of the parent strain.As an additional verification of suppression, we directly deleted rpd3Δ mediated silencing at HMR and was not simply affecting the ADE2 promoter of the reporter gene or the expression of other ADE pathway genes, we used a silencing-dependent mating assay that relies on the repression of a different promoter , and this phenotype was reversed in a strain containing mutations in both rpd3 and bre2 which became nonmating (DDY5679).As a final verification that mutations in the SET1/COMPASS pathway specifically suppress the increased henotype , DDY282.SET1 pathway has been demonstrated to repress target genes by promoting the recruitment of alternative HDACs, namely Hst3p, or a combination of Hst1p and Hos2p . We also performed Northern blot analysis to assay relative transcription levels of the HMR-tDNA using strains specifically marked with a 19 base pair extension to distinguish HMR-tRNA transcripts from those encoded by the seven other copies of this tRNAThr isoacceptor in S. cerevisiae , consistent with our findings reported here.Enhancement of Sir-protein mediated silencing by loss of SET1 encodes the sole H3K4 methyltransferase activity in S. cerevisiae which is responsible for mono-, di-, and trimethylation of this residue. BRE2 encodes a key structural factor within this complex that is also required for methylation  is a conserved multiprotein complex recruited to chromatin to methylate H3K4 (and possibly other substrates) and contains both Set1p and Bre2p . SET1 enhylation . While ihylation . Interesrpd3Δ mediated extended silencing at HMR by mutations that affect the SET1 pathway. rpd3 mutations as suppressors of defective silencing in a catalytically inactive sir2N345A mutant. In that study, restored silencing was mediated by the SIR2 related Sirtuin HDAC Hst3p, as rpd3 hst3 mutants did not suppress the sir2 mutation. We deleted HST3 in our HMR-ADE2 rpd3Δ strains, but the resulting strains maintained the red colony phenotype  product of Sirtuin deacetylases which has been implicated in promoting silencing (SIR-mediated silencing can propagate via deacetylation by a nonsirtuin HDAC in the absence of local OAADPR production (HMR. We are currently constructing yeast strains to confirm this potential association by conventional ChIP. Since SIR-mediated silencing in S. cerevisiae is modeled to propagate by sequential Sir2p deacetylation followed by Sir3p and Sir4p binding (HMR-tDNA. The subsequent loss of RPD3 function would then allow Sir2p to promote the propagation of silencing on both sides of the tDNA by attaching to and deacetylating the nucleosomes normally targeted by Rpd3p. Why subsequent spreading of silencing in rpd3 mutants is then dependent on the SET1 pathway remains to be determined.So how does loss of oduction  that weaThese studies are significant to gain a deeper understanding of the interplay and crosstalk of chromatin writers, erasers, and readers  but may All yeast strains and plasmids described in this study are available on request. The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article, its tables and figures and the cited literature.


A blue shiftof the spectral response of the photoinduced absorbance signal inthe first picosecond after the pump excitation is attributed to thedynamical formation of small polarons with a characteristic time of330 fs. A further important result of our work is that the combineduse of steady-state and ultrafast transient absorption allows us topropose a revised value for the optical gap for ceria (Eog = 4 eV), significantly larger than usually reported.Theultrafast dynamics of excited states in cerium oxide are investigatedto access the early moments of polaron formation, which can influencethe photocatalytic functionality of the material. UV transient absorbancespectra of photoexcited CeO The electrons can be used to promote reductionprocesses while the holes can be employed in oxidations, dependingon whether the catalyst is used as photocathode or photoanode. Theefficiency of these processes strongly depends on carrier lifetimeand mobility.Transitionmetal oxides (TMOs)are candidates for efficient photoelectrochemical catalysts of reactionssuch as water splitting and reduction of CO2, TiO2, and Fe2O3 are good catalysts because of their electronic structure,whichallows transition metal ions to undergo redox cycles quickly and repeatably.However, the use of TMOs in photocatalysis is hampered by the formationof small polarons that affect the lifetime mobility of both chargecarriers.7 A polaron is formed whenthe charge carriers polarize the lattice with the ensuing changesof the charge carrier energy.8 The polaronhas a larger effective mass and smaller mobility than the bare chargecarrier. Polarons are classified into two types depending on the spatialextension of the polarization field: large polarons are spread acrossseveral lattice unit cells while small polarons have the size of asingle or few unit cells. The formation of small polarons has beenobserved in several TMOs such as Fe2O3,9 TiO2,10 NiO,11 and Co3O4.12 The textbook case of the small polaronmodel was proposed to explain electron mobility in partially reducedcerium oxide (CeOx2–).5 In particular, the presence of small polaronsin CeO2 was suggested by the temperature dependence ofthe conductivity of single crystals and supported using the thermopower-conductivityrelation.13 The results were interpretedas being due to an enhanced polarization field following the removalof oxygen atoms from the lattice.16 In this picture, thefunctionality of cerium oxide is linked to the mobility of oxygenions, in turn entangled with carrier mobility via polaron hopping.17CeO6 only recently has theformation of small polaronsin photoexcited states been studied,20 in particular in hemeatite19 and NiO.11 Those works have shown that the small polaronis formed by coupling between photoexcited electrons and the longitudinaloptical (LO) phonons. For Fe2O3, a two-stepprocess was proposed2 that takes placeafter photoexcitation by transferring electron density from oxygento iron atoms. This initial coupling between excited electrons andoptical phonons is followed by the recombination of the phonons withthe hot electrons to form small polarons. The time constant for theprocess was found to be 660 fs in Fe2O319 and 0.3–1.7 ps in NiO.11While the transport mechanism of the ground-state polaronis established,2 basedon time-resolved optical absorption measurements. We have taken advantageof the peculiar electronic structure of CeO2, which involvesa valence band composed mainly of oxygen 2p states and a conductionband characterized by the cerium 5d states , we explore the dynamicsof the 4f states after the photoexcitation of CeO2. Weobserved a fast blue shift of the photoinduced transition from Ce4f to the empty Ce 5d band. This behavior is interpreted as a modificationof the band structure induced by small polaron formation  on quartz (SiO2) at room temperatureby evaporating metallic cerium in a partial pressure of oxygen (10–6 mbar).26 The film wascharacterized using UV–vis spectrophotometry. The absorbance A was estimated by measuring the fraction of transmittedlight T and of specular reflected light R (A = 1 – T – R), neglecting the scattered light. As shown in 2 exhibits a strong absorbance at energies higher than 3.2 eV witha shoulder at about 4 eV (highlighted by the dashed line). The analysisof Ce 3d XPS spectra taken in situ after the MBEdeposition reports a superficial concentration of Ce3+ lowerthan 5%, showing the good stoichiometry of the film (see the We used a 6 nm thick film of CeO see the .A. We probed the system using a visible (2.0–3.5eV) or a UV (3.5–4.3 eV) supercontinuum. The instrument responsefunction (IRF) has been evaluated in separate experiments to be characterizedby a Gaussian with a FWHM of 70 fs28 , which is far fromthe PB signal, suggests that the PIA slightly shifts to higher energiesin the first picosecond  substrate at roomtemperature by evaporating metallic cerium in a partial pressure ofoxygen (10–6 mbar). This procedure, already describedin previous works,26 was used to grow a6 nm film of CeO2 with almost full stoichiometry. The filmthickness was determined by using a cerium evaporation rate measuredby a quartz crystal microbalance. The film stoichiometry was evaluatedby in situ X-ray photoelectron spectroscopy (XPS) by fitting Ce 3dspectra using the procedure proposed by Skàla et al.37The cerium oxide film examined in this work was grownby molecularbeam epitaxy (MBE) on a quartz (SiOA by measuring the fraction of transmitted light T and of specular reflected light R (A = 1 – T – R), neglecting the scattered light.Steady-state UV–vis spectrophotometrymeasurements wereperformed using a white nonpolarized light source generated by a xenonlamp equipped with an ORIEL-MS257 monochromator and a silicon photodetector(with a 250–750 nm range of detection). We estimated the absorbance 2. In order to generate thewhite light supercontinuum that acts as the probe in the visible range(2.00–3.60 eV), a small portion of the amplified fundamental800 nm radiation (∼3 μJ) was focused into a rotatingCaF2 crystal.28 The second harmonicof the amplified fundamental (400 nm) was used to drive the supercontinuumprobe generation in the UV energy range (3.50–4.35 eV). Inthe transient absorbance maps presented in this work, the chirp ofthe probe pulse has been corrected. The instrument response function(IRF) has been evaluated in separate experiments to be Gaussian witha FHWM of 70 fs. Further details on the experimental setups are givenelsewhere.27Our setup for the transientabsorption spectroscopy is composedof a femtosecond laser system consisting of a chirped-pulse amplifier seeded by a Ti:Sa oscillator. As a pump,we used a 275 nm (4.5 eV) pulse generated by an optical parametricamplifier seeded by the amplifier. The fluence of the pump pulse wasestimated to be 11 μJ/cm


One-third of menstruating adolescents miss school or sports because of dysmenorrhea.2Features suggestive of secondary dysmenorrhea  should p3Box 1:Onset immediately with menarcheProgressively worsening dysmenorrheaAbnormal bleeding (including irregular bleeding) with painFamily or personal history of renal or other congenital anomalies Midcycle or acyclic painDyspareuniaFamily history of endometriosisNaproxen, ibuprofen and other NSAIDs are equally effective, with a number needed to treat (NNT) of 3 to achieve pain relief in people with primary dysmenorrhea.3Combined oral contraceptives have an NNT of 5 for treating primary dysmenorrhea.7Endometriosis is found in as many as 70% of adolescents who undergo laparoscopy for dysmenorrhea refractory to treatment with NSAIDs and hormonal therapy.


There is a growing appreciation for individual responses to diet. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed. In this study, we searched for genetic variants underlying differences in the responses to American and ketogenic diets between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains.Fmgq1) , Chr5 at 73.7 Mb  , and Chr7 at 40.5 Mb (Fmgq3) . Analysis of serum HDL cholesterol concentration identified a significant  QTL on Chr1 at 168.6 Mb (Hdlq1). Causal network inference suggests that HDL cholesterol and fat mass gain are both linked to Fmgq1.Genetic mapping of fat and lean mass gain revealed QTLs on Chromosome (Chr) 1 at 191.6 Mb (Fmgq2 and Lmgq1, which are also diet-dependent. Interestingly, Fmgq2 and Fmgq3 affect fat gain directly, while Fmgq1 influences fat gain directly and via an intermediate change in serum cholesterol. These results demonstrate how precision nutrition will be advanced through the integration of genetic variation and sex in physiological responses to diets varied in carbohydrate composition.Strong sex effects were identified at both  Efforts to provide individualized dietary recommendations based on genetic markers have been underway for several years. However, these efforts have largely detected alleles with small-effect sizes that are not clinically actionable –3. As wiRecently, our group observed strong mouse strain-specific differences in response to feeding different human-relevant diets –6. This To further investigate the genetic origin of differential response to carbohydrate restriction and sensitivity to high-fat diets, we generated an intercross population (F2) between B6 and FVB. All F2s were fed either an American  or a ketogenic  diet and changes to body composition and serum cholesterol concentrations were measured. In this population, we performed genome-wide linkage analysis to elucidate the genetic architecture contributing to differential responses to the specific diets. The data obtained provide evidence for genetic loci that not only directly affect body composition response but also loci that indirectly affect response through differences in serum cholesterol concentration. In addition, significant sex differences in the effects of the identified quantitative trait loci (QTL) were detected.Initially, we screened 6-week-old B6 and FVB mice for their response to American  and ketogenic  diets after a 6-month feeding trial. These strains have previously exhibited significantly different responses to these two diets [For the feeding trials, mice were randomly assigned to one of the two diet groups. Half of the B6, FVB, F1, and F2 mice were placed on American diet  and a half on the ketogenic diet . Researchers were not blinded to diet assignments. All animals were maintained in accordance with Texas A&M University Institution Animal Care and Use Committee guidelines at 22 °C under a 12-h light cycle. At the end of the feeding trial, mice were euthanized by carbon dioxide asphyxiation, blood was collected, and tissues were harvested and immediately flash-frozen in liquid nitrogen.Echo magnetic resonance spectroscopy (MRI)  was used to measure the fat and lean mass of all individuals. During the initial screen of the B6 and FVB strains, body weight and body composition were measured at a 3-month time point of the 6-month feeding trial. In the F1 population, body weight and body composition were measured at the beginning and end of the 3-month feeding trial. The fat percentage of total body weight was calculated at the 3-month timepoint for B6, FVB, and F1 populations. Fat percentage is the percentage of total body fat mass measured by MRI relative to body weight at the time of the MRI measurement. In the F2 population, body weight and body composition from before and after the 3-month feeding trial allowed for changes in fat and lean mass to be calculated. Fat and lean mass gains were calculated as the difference in fat and lean mass prior to the feeding trial and after 3 months on the assigned diet.In the F2 population, total cholesterol, HDL, and LDL fractions, as well as APOA2 were measured in serum obtained from blood at sacrifice at the end of the feeding trial. Total cholesterol, HDL, and LDL measurements were performed in duplicate using the EnzyChrom AF HDL and LDL/VLDL Assay kit . APOA2 measurements were performed in duplicate in a subset of the F2 population with the highest and lowest serum HDL cholesterol concentration , using the Mouse Apolipoprotein A2 ELISA kit .https://kbroman.org/MUGAarrays/new_annotations.html).The F2 population was genotyped on the Mouse Universal Genotyping Array (MUGA) that includes 7854 SNP markers [Broad-sense heritability for body fat percentage was calculated as the ratio of total genetic variance (variance in the F2 population) to environmental variance (variance in the F1 population).Outliers can have a strong influence on the results of QTL analyses. We observed biologically implausible errors in data that were greater than three standard deviations from the mean and suspect that these reflect the technical errors in the measurements. As such, outliers were defined as individuals with phenotypes that were more than three standard deviations away from the mean for each sex and phenotype in the F2 population. Outliers were set to missing. This procedure was repeated iteratively to discover all outliers in the data. Pearson’s correlation between phenotypes was determined after correcting for sex and diet effects  or highly significant . A LOD drop of 1.5 LOD from the top marker was used to determine the 95% confidence intervals, or support intervals, for each QTL. Linear models using ANOVA was used to check for any interactions between sex and/or diet with the top markers of each QTL. The variance explained by the top markers at each QTL was calculated by dividing the sum of squares of the model including the top marker by the total sum of squares of the model without QTL.The combined model includes all F2s, both sexes, and both diets (y~ sex * diet + marker). QTL peaks with a logarithm of the odds (LOD) greater than thresholds determined by 10,000 permutations were considered genome-wide significant  where P = α0.05/(number of tests – rank of ith hypothesis +1). A P value < 0.05 is considered significant. An initial pathway describing the relationship between T1 and T2 was defined based on the inferred causal networks, and the predicted causal models were compared with the Akaike’s information criterion (AIC) and Bayesian information criterion (BIC). A lower AIC and BIC indicates a better model.For traits with overlapping QTL we made causal inferences in networks based on methods described elsewhere . BrieflyStructural equation modeling was used to illustrate observed and unobserved relationships between the QTL models. The models were refined until all path coefficients were significantly different from zero. Linear models using ANOVA was used to check for the amount of variation explained by each predictor in the structural model. The proportion of variation explained with the predictors modeled was calculated by dividing the sum of squares of the model including the predictor by the total sum of squares without the predictor.B2m was used as a housekeeping gene to correct for starting amounts of cDNA in both gonadal fat and liver. Primers were purchased from Integrated DNA Technologies as custom DNA oligos and sequences are provided in Supplementary Table SRNA from flash-frozen gonadal fat and liver were extracted using the simplyRNA Tissue kit on a Maxwell AS3000 (Promega). cDNA was generated using the Transcriptor First Strand cDNA Synthesis kit (Roche). qPCRs were performed on a LightCycler 480 (Roche) and CFX384 (BioRad) with LightCycler 480 SYBR Green I Master reagent (Roche). P = 0.001, CI = 5.8–18.0%; Fig. The fat percentage of B6 males consuming an American diet was 1.77-fold higher compared to B6 males consuming a ketogenic diet , lower lean mass gain than males , and lower serum HDL cholesterol concentration than males .As expected, sex had a profound effect on the phenotypes used for analysis  and had lower serum HDL cholesterol concentration on the ketogenic diet compared to F2s on the American diet .Diet had a significant effect on lean mass gain and serum HDL cholesterol .A significant interaction of sex and diet , Chr5 at 73.7 Mb , and Chr7 at 40.5 Mb   accounts for 3.44% of the variance. Fmgq2 is a highly significant QTL where the FVB allele contributes to lower fat mass gain. The top marker (backupUNC050383757) accounts for 6.3% of the total variance in the F2 population  accounts for 3.4% of the variance in fat mass gain.In the combined analysis that incorporates all F2s on both diets (y~ sex * diet + marker), we detected QTL for fat mass gain on Chr1 at 191.6 Mb  affecting fat mass gain. The interaction effect was specific to males on the ketogenic diet. Males that are homozygous for the B6 allele at Fmgq2 and that carry at least one FVB allele at Fmgq1 gained about 5 g more fat on a ketogenic diet than males homozygous for the B6 allele at both loci   explains 3.3% of the variance in lean mass gain. In males on the ketogenic diet, the top marker (JAX00131700) explains 19.0% of the variation in lean mass gain while it only explaining 1.2% of the variation in females on the ketogenic diet and 8.2% and 4.1% of the variation in males and females on the American diet, respectively.In the combined model that includes male and female F2s on both diets (y~ sex * diet + marker), we detected a significant QTL for lean body mass gain during the 3-month feeding trial (b) Table . Lmgq1 oHdlq1) for serum HDL concentration after the 3-month feeding trial , we detected a highly significant QTL on Chr1 at 168.6 Mb , Ephx1 , Camk1g , and Ppp2r5a   and Cox7b2  , Ephx1 (liver), or Ppp2r5a   and observed no genotype-dependent differences  the presence or absence of carbohydrates in the high-fat diet; (2) the combination of alleles occurring in the study population; and (3) sex. In our experiment, B6 males have a lower percentage of body fat in response to the high fat, no carbohydrate, ketogenic diet, while in contrast, B6 females and FVB males and females do not have a differential response to American and ketogenic diets. This sex-specific response to carbohydrate restriction on ketogenic diet observed in B6 males persists in F1s males, and we observed that the combination of B6 and FVB alleles in the hybrids results in a higher percentage of body fat in response to the feeding trial. The difference in ages at the beginning of the parent and F1 feeding trials might also contribute to the higher percentage of body fat we observed in the F1s. In the F2 population, we observed a very high heritability for fat mass gain in both sexes and were able to identify significant QTLs for fat mass gain at Hdlq1 is significant in both males and females and contains Apoa2. Apoa2 has been described repeatedly for its relationship to serum HDL cholesterol concentration, especially in these two strains [Apoa2 locus affecting serum concentrations of HDL cholesterol was made in an association study of females using an advanced intercross line between B6 and NZB/BINJ [Hdlq1 affects HDL in males and females. We confirmed that FVB alleles at Hdlq1 drive higher concentrations of serum HDL cholesterol and serum APOA2.The QTL for serum HDL cholesterol concentration at  strains –14. The NZB/BINJ . Later, NZB/BINJ . B6 carrFmgq1. The close proximity of the Fmgq1 and Hdlq1 QTLs puts them in linkage disequilibrium and this makes their direct and indirect effects difficult to detangle. In general, we would have expected that serum HDL cholesterol concentration would have a negative relationship with fat mass gain given the well-established relationship between serum HDL cholesterol and obesity in humans. It is likely that the two traits are independently linked to Fmgq1 because of the linkage disequilibrium. However, the AIC and BIC scores for the models suggest that differences in serum HDL cholesterol concentration occur upstream of the differences we observe in fat mass gain at Fmgq1.In our causal network of the relationship between fat mass gain and serum HDL cholesterol concentration, we found that serum HDL cholesterol concentration and fat mass gain are independently linked to Fmgq1 and Fmgq2 onto metabolically relevant KEGG pathways revealed candidate genes involved in the biogenesis of cortisol (Hsd11b1), aldosterone (Camk1g), and testosterone (Srd5a3). Cholesterol falls upstream of the production of each of these steroid hormones. Ephx1 resides inside of Fmgq1 and plays a role in the transfer of bile acids to the liver, a process that is critical for the endogenous synthesis of cholesterol. Each of these candidate genes has in common, a relationship with cholesterol. This offers insight into the role these particular genes could play in the causal network we inferred that showed serum HDL cholesterol concentration occurring upstream of fat mass gain. The inferred network might be reflective of the relationship between fat mass gain and one of these candidate genes more so than a direct relationship between serum HDL cholesterol concentration and fat mass gain.Overlaying genes within Fmgq1 and Fmgq2. Our most prominent QTL at Fmgq2 harbors Srd5a3 that converts inactive testosterone to active dihydrotestosterone. We confirmed that F2s that are homozygous for FVB alleles at Fmgq2 express Srd5a3 less than F2s that are homozygous for B6 alleles. At this QTL, FVB alleles also drive lower fat mass gain. This suggests that more active dihydrotestosterone promotes more fat mass gain at Fmgq2. The functional interaction we observed between Fmgq1 and Fmgq2 showed us that male F2s that are homozygous B6 at Fmgq2 and carry at least one FVB allele at Fmgq1 gained the most fat mass. Interestingly, we observed that F2s that carry this interaction have a 1.59-fold increase in expression of Srd5a3 relative to animals that are homozygous B6 at both Fmgq1 and Fmgq2. This provides further evidence that more active dihydrotestosterone promotes more fat mass gain in this population. Ppp2r5a at Fmgq1 has been associated with polycystic ovary syndrome (PCOS) through pathway analysis of genes enriched in genome-wide association studies (GWAS). Ppp2r5a is a member of the oocyte meiosis KEGG pathway that was significantly associated with PCOS, a pathway affected by the hyperandrogenemia that frequently occurs with PCOS [of Ppp2r5a with PCOS makes Srd5a3, and Ppp2r5a primary candidate genes of interest for further investigation.Further, our candidate genes offer insight into the sex-specific interaction in males on the ketogenic diet that we observed between ith PCOS . The assSrd5a3 and active dihydrotestosterone levels. Hsd11b1 at Fmgq1 reversibly converts active cortisol to inactive cortisone. Epidemiological data in humans show that the ratio of androgens to glucocorticoids is critical to metabolic homeostasis and alterations in this ratio can lead to increased incidence of obesity and metabolic syndrome [Camk1g at Fmgq1 is a member of this pathway and people with obesity often experience hyperaldosteronism [Ephx1 at Fmgq1 has previously been associated with other Srd5a isoforms in GWAS of PCOS [Cox7b2 at Fmgq2, has been identified in another GWAS for PCOS [Ephx1 and Cox7b2 and abnormal androgen secretion.Each of the remaining candidate genes has a potential relationship with syndrome . In addisyndrome . Camk1g teronism . Ephx1 a of PCOS , and simfor PCOS . The assHsd11b1 and Srd5a3 does result in noncoding transcript variants that might be of interest in further investigation. These noncoding transcript variants would suggest differences in expression of relevant candidate genes like we observed for Srd5a3. However, we did not observe genotype-dependent differences in expression of Hsd11b1, but did find in the F2 population that mice that are B6/B6 at Fmgq2 and carry FVB/FVB alleles at Fmgq1 express Ppp2r5a 1.61-fold more than F2s that are B6/B6 at both loci.Sequence variation between B6 and FVB result in amino acid changes in EPHX1, CAMK1G, and PPP2R5A, sometimes at multiple locations within the proteins. In HSD11B1 and SRD5A3, no amino acid changes have been documented, but sequence variation in Overall, our data parallel a clinical trial showing a greater fat mass loss among men than women on very low energy, ketogenic diet –23. The Fmgq2 and Lmgq1 and serum HDL cholesterol concentration at Hdlq2 and Hdlq3 highlights the importance of sex-specific analyses during the development of individualized dietary guidelines. We identified a known QTL at Hdlq1 for serum HDL cholesterol concentration that harbors Apoa2. Interestingly, the QTLs at Fmgq2 and Fmgq3 affect fat gain directly while Fmgq1 seems to influence fat gain directly as well as via an intermediate physiological change in serum cholesterol related to the strain-specific Apoa2 phenotypes. We have shown that genotypes at Fmgq2 alter the expression of Srd5a3 in a sex- and genotype-specific manner. It remains unclear if these loci explain the differential response to carbohydrate restriction we observed in the initial parent strain screen or if instead, they characterize the increased fat mass gain that we observe after the parental genomes are combined in hybrid and F2 populations. Further investigation of the candidate genes presented here will elucidate their roles in response to carbohydrate restriction in the parent, hybrid, and F2 populations.It is possible that the genetic architecture for fat gain in response to high-fat diets is more complex in females than in males, and as such, the genetic component we are able to detect in our model contributes more to the overall response in males than in females. The observation of these sex-specific QTLs for fat and lean mass gain at Supplementary FiguresSupplementary Tables


Inactivation of the tumor suppressor p53 has been generally accepted as a hallmark of tumor. MDM2 and MDMX, the two closely related proteins are considered to be critical for negatively regulating p53 activity through inhibitory binding to and post-translational modification of the p53 protein. We have demonstrated that MDMX facilitates MDM2-mediated p53 ubiquitination and degradation via recruitment of the ubiquitin-conjugating enzyme UbcH5c to the MDM2-MDMX heterooligomers. Here, we discuss our new findings from genetically engineered mouse models and a potential therapeutic strategy. Most p53-activation compounds developed to date offer relatively specific MDM2 inhibition by targeting the p53-binding domain on MDM2, such as nutlin [The p53 stress response is an important regulator of cellular homeostasis, responding to a variety of stressors and eliciting hallmark effects on cell proliferation, apoptosis, and senescence . As suchs nutlin . Howevers nutlin . Howevers nutlin , 10. DruIn vitro experiments have demonstrated a greater affinity for the formation of MDM2-MDMX heterooligomers than either MDM2 or MDMX homooligomers alone [in vitro and in vivo studies.Recent studies from our lab and others’ have demonstrated that MDM2 and MDMX heterooligomerization through their RING domains is essential for p53 control, while the MDM2 E3 ligase function is dispensable for growth and development under unstressed conditions but becomes essential for survival after genotoxic stresses –13. Whilrs alone . A propors alone –17. Studrs alone . Consistrs alone  and rescrs alone . HoweverER). The ERp53 gene is expressed at physiological levels under control of the native promoter [ER activity can be rapidly switched between WT and knockout states by administration and withdrawal of 4-hydroxytamoxifen (4-OHT). We generated mice expressing inducible ERp53 under various MDM2 and MDMX deletion and mutation backgrounds and studied p53 regulation in mice and MEF cells. Unexpectedly, using the inducible p53ER system we found that in vivo MDMX is essential for p53 degradation, as we found that in the presence of WT MDM2 and absence of MDMX the degradation of p53 is hardly detected [in vivo MDM2 and MDMX can interact with p53 in the absence of each other, but they bind p53 more efficiently as a heterodimer; and that the MDM2-MDMX heterodimerization promotes p53 cytoplasmic localization, where p53 is degraded by the cytoplasmic proteasome. However, we believe the most likely mechanism for MDMX to enable MDM2 E3 ligase activity is its ability to bind UbcH5c and bring it to MDM2 proximity. Previous studies have shown that knockdown UbcH5c in MCF-7 cells increases p53 expression [ER system, we found that MDMX, but not MDM2, interacts with UbcH5c, and that the ability of MDMX to interact with both UbcH5c and MDM2 is essential for MDM2 mediated p53 degradation [A major obstacle of investigating individual functions of MDM2 and MDMX in mice is the early embryonic lethality caused by inactivation of either one while in the presence of p53. To solve this problem we took advantage of a previously developed system , in whicpromoter . The p53detected . Severalpression , suggestradation . We furtradation . Based o


Observational pain scales can help identify pain in persons with impaired cognition including dementia who may have difficulty expressing pain verbally. The Pain Assessment in Impaired Cognition-15 (PAIC15) observational pain scale covers 15 important items that are indicative of pain, but it is unclear how likely pain is for persons with each summed score . The goal of our study was to determine sensitivity and specificity of cut offs for probable pain on the PAIC15 against three possible standards. We determined cut offs against (1) self report when able, (2) the established Pain Assessment in Advanced Dementia (PAINAD) cut off of 2, and (3) observer’s overall estimate based on a series of systematic observations. We used data of 238 nursing home residents with dementia who were observed by their physician in training or nursing staff in the context of an evidence-based medicine (EBM) training study, with 137 residents assessed twice. The area under the ROC curve was excellent against the PAINAD cut off (□0.8) at both assessments, but acceptable or less than acceptable for the other two standards. Across standards and criteria for optimal sensitivity and specificity, cut offs at the PAIC15 could be 3 or 4. Guided by self report we recommend PAIC15 scores of 3 and higher to represent probable pain with sensitivity and specificity in the 0.5 to 0.7 range.