Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.

Summary MDM2 associates with ribosomal protein S7 and this interaction is required to inhibit MDM2’s E3 ligase activity leading to stabilization of MDM2 and p53. Notably, the MDM2 homologue MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits Mdm2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.

Continuous and real‐time sensoring has received much attention in biomarker monitoring, toxicity assessment, and therapeutic agent tracking. However, its on‐site application is seriously limited by several stubborn defects including liability to fouling, signal drifting, short service life, poor repeatability, etc. Additionally, most current methods require extra sample pretreatment, delaying timely acquisition of testing results. To address these issues, MXene‐Ti3C2Tx based screen‐printed electrode incorporated with a dialysis microfluidic chip is constructed for a direct and continuous multicomponent analysis of whole blood. Dual‐function of MXene is developed and allows for simultaneous quantification of different target compounds through one device. Importantly, ratiometric sensing tactic is easily implemented in the system, which greatly alleviates signal drifting. As a proof of concept, this novel sensor is applied in hemodialysis, and continuous assay of urea, uric acid, and creatinine levels in human blood is realized. This work paves a new path for 2D MXene in biomedical and sensing applications.

We analyzed 18 high-quality waveform doublets with time separations of up to 35 years in the South Sandwich Islands region, for which the seismic signals have traversed the inner core as PKP(DF). The doublets show a consistent temporal change of travel times at up to 58 stations in and near Alaska, and they show a dissimilarity of PKP(DF) coda. Using waveform doublets avoids artifacts of earthquake mislocations and contamination from small-scale heterogeneities. Our results confirm that Earth's inner core is rotating faster than the mantle and crust at about 0.3 degrees to 0.5 degrees per year.

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2 + /p63 + CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2 + /p63 + CTB stem-like cells. These cells can be replated and further differentiated into STB-and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G + EVTs in an hypoxiainducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.human pluripotent stem cells | cytotrophoblast | extravillous trophoblast | syncytiotrophoblast | placenta

Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well known are GA, GB, GD, GF, VX and VR. All of these compounds contain a chiral phosphorus center with the S P -enantiomers being significantly more toxic than the R P -enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R P -enantiomers. A series of enantiomerically pure chiral nerve agent analogues has been developed containing the relevant phosphoryl centers found in GB, GD, GF, VX and VR. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed stereoselectivity, have been identified. A number of variants showed dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S P -enantiomers. Improvements of up to three orders of magnitude relative to the wild type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.Organophosphorus compounds have been utilized for more than 50 years as insecticides for the protection of agricultural crops (1) and similar compounds have been developed as chemical warfare agents (2). The structures of these latter compounds are presented in Scheme 1 and include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX and VR. GA has a cyanide leaving group, the three remaining G-agents (GB, GD, and GF) have a fluoride leaving group, and the two versions of VX have a thiolate leaving group. The toxicity of these organophosphonates is due to the inactivation of acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine at neural synapses, through the phosphonylation of an active site serine residue (3). GA, GB, GF, VX, and VR contain a chiral phosphorus center and thus each of these nerve agents has two stereoisomers, while soman has four stereoisomers because of an additional chiral center within the pinacolyl substituent. The enantiomers are differentially toxic; the S Pstereoisomer of sarin reacts with AChE approximately ~10 4 times faster than the R Pstereoisomer and the two S P -stereoisomers of soman react ~10 5 times faster than the two † This work was supported by the NIH (GM 68550).

ObjectivesEarly placement of transjugular intrahepatic portosystemic shunt (TIPS) has been shown to improve survival in high-risk patients (Child-Pugh B plus active bleeding at endoscopy or Child-Pugh C 10–13) with cirrhosis and acute variceal bleeding (AVB). However, early TIPS criteria may overestimate the mortality risk in a significant proportion of patients, and the survival benefit conferred by early TIPS in such patients has been questioned. Alternative criteria have been proposed to refine the criteria used to identify candidates for early TIPS. Nevertheless, the true survival benefit provided (or not) by early TIPS compared with standard treatment in the different risk categories has not been investigated in specifically designed comparative studies.DesignWe collected data on 1425 consecutive patients with cirrhosis and AVB who were admitted to 12 university hospitals in China between December 2010 and June 2016. Of these, 206 patients received early TIPS, and 1219 patients received standard treatment. The Fine and Gray competing risk regression model was used to compare the outcomes between the two groups that were stratified based on the currently available risk stratification systems after adjusting for liver disease severity and other potential confounders.ResultsOverall, early TIPS was associated with an 80% relative risk reduction (RRR) in mortality at 6 weeks (adjusted HR=0.20; 95% CI: 0.10 to 044; p<0.001) and 51% RRR at 1 year (adjusted HR=0.49, 95% CI: 0.32 to 0.73; p<0.001) compared with standard treatment. In stratification analyses, the RRRs in mortality did not significantly differ among the risk categories. However, the absolute risk reductions (ARRs) of mortality were more pronounced in high-risk patients. The ARRs at 6 weeks were −2.1%, −10.2% and −32.4% in Model for End-stage Liver Disease (MELD) ≤11, 12–18 and ≥19 patients and were −1.5%, −9.1% and −23.2% in Child-Pugh A, B and C patients, respectively (interaction tests, p<0.001 for both criteria). The ARRs for mortality at 1 year were −1.7%, −5.4% and −32.7% in MELD ≤11, 12–18 and ≥19 patients, respectively, and −3.6%, −5.2% and −20.3% in Child-Pugh A, B and C patients, respectively (interaction tests, p<0.001 for both criteria). After adjusting for liver disease severity and other potential confounders, a survival benefit was observed in MELD ≥19 or Child-Pugh C patients but not in MELD ≤11 or Child-Pugh A patients. In MELD 12–18 patients, a survival benefit was observed within 6 weeks but not at 1 year. In Child-Pugh B patients, a survival benefit was observed in those with active bleeding but not those without active bleeding. However, the evaluation of active bleeding was associated with a high interobserver variability. Furthermore, early TIPS was associated with a significantly reduced incidence of failure to control bleeding or rebleeding and new or worsening ascites, without increasing the risk of overt hepatic encephalopathy.ConclusionsEarly TIPS was associated with improved survival in patients with MELD ≥19 or Child-Pugh C ...

Objective: To evaluate the impact of nutrition education in kindergartens and to promote healthy dietary habits in children. Design: Prospective cohort study. Four kindergartens with 1252 children were randomized to the intervention group and three with 850 children to the control group. The personal nutritional knowledge, attitudes and dietary behaviours of the parents were also investigated. Each month, children and parents in the intervention group participated in nutrition education activities. The main outcome measures were anthropometrics and diet-related behaviours of the children and the nutritional knowledge and attitudes of the parents at baseline, 6 months (mid-term) and 1 year (post-test). Baseline demographic and socio-economic characteristics were also collected. Setting: Seven kindergartens from Hefei, the capital city of Anhui Province, eastern China. Subjects: Two thousand one hundred and two 4-to 6-year-old pre-schoolers from seven kindergartens participated. Results: The prevalence of children's unhealthy diet-related behaviours decreased significantly and good lifestyle behaviours increased in the group receiving nutrition education compared with controls. Parental eating habits and attitudes to planning their children's diets also changed appreciably in the intervention group compared with the control group (P , 0?05). However, there were no statistically significant differences in children's height, weight, height-for-age Z-score or weight-for-age Z-score between the two groups. Conclusions: Kindergarten-based nutrition education improves pre-schoolers' lifestyle behaviours and brings about beneficial changes in parents' attitudes to planning their children's diets and their own personal eating habits.