What Is the Structure of the E4 Intermediate in Nitrogenase?

Nitrogenase is the only enzyme that can cleave the strong triple bond inN2. The active site contains a complicated MoFe7S9C cluster. It is believed that itneeds to accept four protons and electrons, forming the E4 state, before it can bind N2.However, there is no consensus on the atomic structure of the E4 state. Experimentalstudies indicate that it should contain two hydride ions bridging two pairs of Fe ions,and it has been suggested that both hydride ions as well as the two protons bind on thesame face of the cluster. On the other hand, density functional theory (DFT) studieshave indicated that it is energetically more favorable with either three hydride ions or with a triply protonated carbide ion, depending on the DFT functional. We have performed a systematic combined quantum mechanical and molecular mechanical (QM/MM) study of possible E4 states with two bridging hydride ions. Our calculations suggest that the most favorable structure has hydride ions bridging the Fe2/6 and Fe3/7 ion pairs. In fact, such structures are 14 kJ/mol more stable than structures with three hydride ions, showing that pure DFT functionals give energetically most favorable structures in agreement with experiments. An important reason for this finding is that we have identified a new type of broken-symmetry state that involves only two Fe ions with minority spin, in contrast to the previously studied states with three Fe ions with minority spin. The energetically best structures have the two hydride ions on different faces of the FeMo cluster, whereas better agreement with ENDOR data is obtained if they are on the same face; such structures are only 6−22 kJ/mol less stable

Putative reaction mechanism of nitrogenase after dissociation of a sulfide ligand

We have investigated the implications of the recent crystallographic findings that the m2-bridging S2B sulfide ligand may reversibly dissociate from the active-site FeMo cluster of nitrogenase. We show with combined quantum mechanical and molecular mechanical (QM/MM) calculations that once S2B has dis- sociated, N2 may bind in that position and can be protonated to two NH3 groups by thermodynamically favourable steps. The substrate forms hydrogen bonds with two protein ligands, Gln-191 and His-195. For all steps, we have studied three possible protonation states of His-195 (protonated on either ND1, NE2 or both). We find that the thermodynamically favoured path involves an end-on NNH2 structure, a mixed side-on/end-on H2NNH structure, a side-on H2NNH2 structure, a bridging NH2 structure and a bridging NH3 structure. In all cases, His-195 seems to be protonated on the NE2 atom. Dissociation of the NH3 pro- duct is often unfavourable and requires either further reduction or protonation of the cluster or rebinding of S2B. In conclusion, our calculations show that dissociation of S2B gives rise to a natural binding and reaction site for nitrogenase, between the Fe2 and Fe6 atoms, which can support an alternating reaction mechanism with favourable energetics

Assessment of Severe Accident Depressurization Valve Activation Strategy for Chinese Improved 1000 MWe PWR

To prevent HPME and DCH, SADV is proposed to be added to the pressurizer for Chinese improved 1000 MWe PWR NPP with the reference of EPR design. Rapid depressurization capability is assessed using the mechanical analytical code. Three typical severe accident sequences of TMLB’, SBLOCA, and LOFW are selected. It shows that with activation of the SADV the RCS pressure is low enough to prevent HPME and DCH. Natural circulation at upper RPV and hot leg is considered for the rapid depressurization capacity analysis. The result shows that natural circulation phenomenon results in heat transfer from the core to the pipes in RCS which may cause the creep rupture of pipes in RCS and delays the severe accident progression. Different SADV valve areas are investigated to the influence of depressurization of RCS. Analysis shows that the introduction of SADV with right valve area will delay progression of core degradation to RPV failure. Valve area is to be optimized since smaller SADV area will reduce its effect and too large valve area will lead to excessive loss of water inventory in RCS and makes core degradation progression to RPV failure faster without additional core cooling water sources

Protonation States of Homocitrate and Nearby Residues in Nitrogenase Studied by Computational Methods and Quantum Refinement

Nitrogenase is the only enzyme that can break the triple bond in N2 to form two molecules of ammonia. The enzyme has been thoroughly studied with both experimental and computational methods, but there is still no consensus regarding the atomic details of the reaction mechanism. In the most common form, the active site is a MoFe7S9C(homocitrate) cluster. The homocitrate ligand contains one alcohol and three carboxylate groups. In water solution, the triply deprotonated form dominates, but because the alcohol (and one of the carboxylate groups) coordinate to the Mo ion, this may change in the enzyme. We have performed a series of computational calculations with molecular dynamics (MD), quantum mechanical (QM) cluster, combined QM and molecular mechanics (QM/MM), QM/MM with Poisson-Boltzmann and surface area solvation, QM/MM thermodynamic cycle perturbations, and quantum refinement methods to settle the most probable protonation state of the homocitrate ligand in nitrogenase. The results quite conclusively point out a triply deprotonated form (net charge -3) with a proton shared between the alcohol and one of the carboxylate groups as the most stable at pH 7. Moreover, we have studied eight ionizable protein residues close to the active site with MD simulations and determined the most likely protonation states

Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

Abstract: Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract: [Figure not available: see fulltext.

Major emerging and re-emerging zoonoses in China: a matter of global health and socioeconomic development for 1.3 billion

SummaryEmerging and re-emerging zoonoses are a significant public health concern and cause considerable socioeconomic problems globally. The emergence of severe acute respiratory syndrome (SARS), highly pathogenic avian influenza (HPAI) H5N1, avian influenza H7N9, and severe fever with thrombocytopenia syndrome (SFTS), and the re-emergence of rabies, brucellosis, and other zoonoses have had a significant effect on the national economy and public health in China, and have affected other countries. Contributing factors that continue to affect emerging and re-emerging zoonoses in China include social and environmental factors and microbial evolution, such as population growth, urbanization, deforestation, livestock production, food safety, climate change, and pathogen mutation. The Chinese government has devised new strategies and has taken measures to deal with the challenges of these diseases, including the issuing of laws and regulations, establishment of disease reporting systems, implementation of special projects for major infectious diseases, interdisciplinary and international cooperation, exotic disease surveillance, and health education. These strategies and measures can serve as models for the surveillance and response to continuing threats from emerging and re-emerging zoonoses in other countries

Comparison of Copper Leaching from Alkaline Copper Quat Type-D Treated Chinese Fir and Mongolian Scots Pine after Different Posttreatments

Chinese fir (Cunninghamia lanceolata Hook.) and Mongolian Scots pine (Pinus sylvestris Linn. var. mongolica Litv.) sapwood were treated with alkaline copper quat type D (ACQ-D) solutions at two concentration levels (0.5 and 1.1%) followed by three posttreatments: oven-drying, conditioning in a humidity chamber, or exposed to boiling water. The effects of treatment on copper leaching from ACQ-D-treated wood were investigated by performing tests according to AWPA E11-06. Analysis of chemical composition and pH values were determined to evaluate the effects of wood species on copper leaching. The results showed that copper leaching from ACQ-D-treated Mongolian Scots pine without posttreatment was lower than that of treated Chinese fir. However, after posttreatment, more copper leaching was observed from ACQ-D-treated Mongolian Scots pine than Chinese fir. For example, after conditioning posttreatment, the percentage of copper leached from 1.1% ACQ-D-treated Chinese fir decreased 58.6 to 1.8%, while for Mongolian Scots pine, it decreased 49.5 to 19.4%. The difference was considered to be related to different chemical compositions and pH levels

An Equal-Size Hard EM Algorithm for Diverse Dialogue Generation

Open-domain dialogue systems aim to interact with humans through natural language texts in an open-ended fashion. Despite the recent success of super large dialogue systems such as ChatGPT, using medium-to-small-sized dialogue systems remains the common practice as they are more lightweight and accessible; however, generating diverse dialogue responses is challenging, especially with smaller models. In this work, we propose an Equal-size Hard Expectation--Maximization (EqHard-EM) algorithm to train a multi-decoder model for diverse dialogue generation. Our algorithm assigns a sample to a decoder in a hard manner and additionally imposes an equal-assignment constraint to ensure that all decoders are well-trained. We provide detailed theoretical analysis to justify our approach. Further, experiments on two large-scale open-domain dialogue datasets verify that our EqHard-EM algorithm generates high-quality diverse responses.Comment: Accepted by ICLR 202

Developing a fluorescence-based tool to measure noradrenaline transporter function in cardiovascular tissue

Sympathetic noradrenergic transmission in both the heart and vasculature is modulated at central, ganglionic, and end-organ sites. It is ultimately within the neuroeffector junction where the dynamic interplay between noradrenaline (NAd) release and its subsequent reuptake by the noradrenaline transporter (NAT) that predominately determines the junctional availability of the neurotransmitter. However, despite the fact that NAT dysfunction is implicated in many cardiovascular diseases, the mechanisms that govern transporter modulation remain poorly defined due to the limited methodologies available. This study demonstrates the development and optimisation of a novel, fluorescence-based technique, NTUA (Neurotransmitter Transporter Uptake Assay), that permits dynamic measurements of NAT function at high spatiotemporal resolutions in whole organ preparations ex vivo. This technique was then used to explore putative NAT regulators. It was discovered that several known release-inhibiting modulators, such as the muscarinic acetylcholine receptor, ⍺2-adrenoceptor (⍺2AR), and cannabinoid type I receptor (CB1R), also suppress NAT function. Importantly, the neuromodulatory roles of ⍺2AR and CB1R were also uncovered during bouts of sympathetic neuronal activity, which provides further insight into the complexity of net noradrenergic transmission. Moreover, the future potential for NTUA as an intravital stain to monitor disease progression in vivo was demonstrated by its use to simultaneously detect for changes in NAT function and sympathetic nerve density in a pre-clinical animal model of obstructive sleep apnoea, a disease with complex cardiovascular implications. These data, amongst others, show that NTUA will help future research into junctional NAd availability and hence the study of sympathetic transmission in the cardiovascular system

Chinese-Americans in the U.S., 1848-1979

Attracted by gold rush, many immigrants from China began to enter the U.S to seek fortune in 1848. However, they received miserable treatment and experienced deep prejudices from native-born American whites. The anti- Chinese sentiment among the whites reached climax when the Chinese Exclusion Act was enacted in 1882. Further Chinese immigration was thus barred. In order to shun from outside hostility, the Chinese retreated to inner-city regions and developed the Chinatowns. Like most immigrants, Chinese-American parents also faced the problem of educating their children and helping them to adjust to the new culture while still keeping them clinging to their traditional Chinese culture. But the young generation was different from their parental generation. They wanted to get integrated into the host society and were more inclined to have their voices heard. In the 1970s, they launched the Yellow Power Movement to fi ght for equal rights.Key words: Immigrant; Anti-Chinese; Chinatown; Yellow powe