A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells.

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.

Unlocking the potential of N-acetylcysteine: Improving hepatopancreas inflammation, antioxidant capacity and health in common carp (Cyprinus carpio) via the MAPK/NF-κB/Nrf2 signalling pathway.

N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.

High Affinity Electrostatic Interactions Support the Formation of CdS Quantum Dot:Nitrogenase MoFe Protein Complexes.

Nitrogenase MoFe protein can be coupled with CdS nanocrystals (NCs) to enable photocatalytic N2 reduction. The nature of interactions that support complex formation is of paramount importance in intermolecular electron transfer that supports catalysis. In this work we have employed microscale thermophoresis to examine binding interactions between 3-mercaptopropionate capped CdS quantum dots (QDs) and MoFe protein over a range of QD diameters (3.4-4.3 nm). The results indicate that the interactions are largely electrostatic, with the strength of interactions similar to that observed for the physiological electron donor. In addition, the strength of interactions is sensitive to the QD diameter, and the binding interactions are significantly stronger for QDs with smaller diameters. The ability to quantitatively assess NC protein interactions in biohybrid systems supports strategies for understanding properties and reaction parameters that are important for obtaining optimal rates of catalysis in biohybrid systems.

Potential protective effects of sodium butyrate on glycinin-induced oxidative stress, inflammatory response, and growth inhibition in Cyprinus carpio.

Investigated mitigating effects of sodium butyrate (SB) on the inflammatory response, oxidative stress, and growth inhibition of common carp (Cyprinus carpio) (2.94 ± 0.2 g) are caused by glycinin. Six isonitrogenous and isoenergetic diets were prepared, in which the basal diet was the control diet and the Gly group diet contained 80 g/kg glycinin, while the remaining 4 diets were supplemented with 0.75, 1.50, 2.25, and 3.00 g/kg SB, respectively. The feeding trial lasted for 8 weeks, and the results indicated that supplementing the diet with 1.50-2.25 g/kg of SB significantly improved feed efficiency and alleviated the growth inhibition induced by glycinin. Hepatopancreas and intestinal protease activities and the content of muscle crude protein were significantly decreased by dietary glycinin, but supplement 1.50-2.25 g/kg SB partially reversed this result. SB (1.50-2.25 g/kg) increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hepatopancreas and reduced the activities of AST and ALT in the serum. Glycinin significantly reduced immune and antioxidant enzyme activities, whereas 1.50-2.25 g/kg SB reversed these adverse effects. Furthermore, compared with the Gly group, supplement 1.50-2.25 g/kg SB eminently up-regulated the TGF-ß and IL-10 mRNA, and down-regulated the IL-1ß, TNF-α, and NF-κB mRNA in hepatopancreas, mid-intestine (MI), and distal intestine (DI). Meanwhile, supplement 1.50-2.25 g/kg SB activated the Keap1-Nrf2-ARE signaling pathway and upregulate CAT, SOD, and HO-1 mRNA expression in hepatopancreas, MI, and DI. Summarily, glycinin induced inflammatory response, and oxidative stress of common carp ultimately decreased the digestive function and growth performance. SB partially mitigated these adverse effects by activating the Keap1-Nrf2-ARE signaling pathway and inhibiting the NF-κB signaling pathway.

The Fe Protein Cycle Associated with Nitrogenase Catalysis Requires the Hydrolysis of Two ATP for Each Single Electron Transfer Event.

A central feature of the current understanding of dinitrogen (N2) reduction by the enzyme nitrogenase is the proposed coupling of the hydrolysis of two ATP, forming two ADP and two Pi, to the transfer of one electron from the Fe protein component to the MoFe protein component, where substrates are reduced. A redox-active [4Fe-4S] cluster associated with the Fe protein is the agent of electron delivery, and it is well known to have a capacity to cycle between a one-electron-reduced [4Fe-4S]1+ state and an oxidized [4Fe-4S]2+ state. Recently, however, it has been shown that certain reducing agents can be used to further reduce the Fe protein [4Fe-4S] cluster to a super-reduced, all-ferrous [4Fe-4S]0 state that can be either diamagnetic (S = 0) or paramagnetic (S = 4). It has been proposed that the super-reduced state might fundamentally alter the existing model for nitrogenase energy utilization by the transfer of two electrons per Fe protein cycle linked to hydrolysis of only two ATP molecules. Here, we measure the number of ATP consumed for each electron transfer under steady-state catalysis while the Fe protein cluster is in the [4Fe-4S]1+ state and when it is in the [4Fe-4S]0 state. Both oxidation states of the Fe protein are found to operate by hydrolyzing two ATP for each single-electron transfer event. Thus, regardless of its initial redox state, the Fe protein transfers only one electron at a time to the MoFe protein in a process that requires the hydrolysis of two ATP.

Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E4(2N2H) Intermediate.

A critical step in the mechanism of N2 reduction to 2NH3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H2 or going forward with N2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H2 to form the E4(2N2H) state. E4(2N2H) can relax to E4(4H) by the oxidative addition (oa) of H2 and release of N2 or can be further reduced in a series of catalytic steps to release 2NH3. If the H2 re/oa mechanism is correct, it requires that oa of H2 be associative with E4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E2 and E4(2N2H), as well as signals suggesting the formation of E6 or E8. It is shown that in the frozen state when pN2 is much greater than pH2, the E4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H2 to the E4(2N2H) state follows an associative reaction mechanism.

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation.

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e-/H+] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e-/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e-/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e-/H+] generates E4(4H) with Fe2-H-Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E2(2H) containing Fe3-H-Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E2(2H) contains Fe2-H-Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.

Correction: Noncovalent wedging effect catalyzed the cis to syn transformation of a surface-adsorbed polymer backbone toward an unusual thermodynamically stable supramolecular product.

Correction for 'Noncovalent wedging effect catalyzed the cis to syn transformation of a surface-adsorbed polymer backbone toward an unusual thermodynamically stable supramolecular product' by Zhi-Xuan Liu et al., Phys. Chem. Chem. Phys., 2022, 24, 30010-30016, https://doi.org/10.1039/D2CP04184G.

Dynamic Nomogram for Predicting the Risk of Perioperative Neurocognitive Disorders in Adults.

BACKGROUND: Simple and rapid tools for screening high-risk patients for perioperative neurocognitive disorders (PNDs) are urgently needed to improve patient outcomes. We developed an online tool with machine-learning algorithms using routine variables based on multicenter data. METHODS: The entire dataset was composed of 49,768 surgical patients from 3 representative academic hospitals in China. Surgical patients older than 45 years, those undergoing general anesthesia, and those without a history of PND were enrolled. When the patient's discharge diagnosis was PND, the patient was in the PND group. Patients in the non-PND group were randomly extracted from the big data platform according to the surgical type, age, and source of data in the PND group with a ratio of 3:1. After data preprocessing and feature selection, general linear model (GLM), artificial neural network (ANN), and naive Bayes (NB) were used for model development and evaluation. Model performance was evaluated by the area under the receiver operating characteristic curve (ROCAUC), the area under the precision-recall curve (PRAUC), the Brier score, the index of prediction accuracy (IPA), sensitivity, specificity, etc. The model was also externally validated on the multiparameter intelligent monitoring in intensive care (MIMIC) Ⅳ database. Afterward, we developed an online visualization tool to preoperatively predict patients' risk of developing PND based on the models with the best performance. RESULTS: A total of 1051 patients (242 PND and 809 non-PND) and 2884 patients (6.2% patients with PND) were analyzed on multicenter data (model development, test [internal validation], external validation-1) and MIMIC Ⅳ dataset (external validation-2). The model performance based on GLM was much better than that based on ANN and NB. The best-performing GLM model on validation-1 dataset achieved ROCAUC (0.874; 95% confidence interval [CI], 0.833-0.915), PRAUC (0.685; 95% CI, 0.584-0.786), sensitivity (72.6%; 95% CI, 61.4%-81.5%), specificity (84.4%; 95% CI, 79.3%-88.4%), Brier score (0.131), and IPA (44.7%), and of which the ROCAUC (0.761, 95% CI, 0.712-0.809), the PRAUC (0.475, 95% CI, 0.370-0.581), Brier score (0.053), and IPA (76.8%) on validation-2 dataset. Afterward, we developed an online tool (https://pnd-predictive-model-dynnom.shinyapps.io/ DynNomapp/) with 10 routine variables for preoperatively screening high-risk patients. CONCLUSIONS: We developed a simple and rapid online tool to preoperatively screen patients' risk of PND using GLM based on multicenter data, which may help medical staff's decision-making regarding perioperative management strategies to improve patient outcomes.

Low-temperature trapping of N2 reduction reaction intermediates in nitrogenase MoFe protein-CdS quantum dot complexes.

The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.

Enabling Quick Response to Nitrogen Dioxide at Room Temperature and Limit of Detection to Ppb Level by Heavily n-Doped Graphene Hybrid Transistor.

Sensitive detection of nitrogen dioxide (NO2) is of significance in many areas for health and environmental protections. In this work, we developed an efficient NO2 sensor that can respond within seconds at room temperature, and the limit of detection (LOD) is as low as 100 ppb. Coating cyano-substituted poly(p-phenylene vinylene) (CN-PPV) films on graphene (G) layers can dope G sheets effectively to a heavy n state. The influences of solution concentrations and annealing temperatures on the n-doping effect were investigated in detail. The CN-PPV-G transistors fabricated with the optimized parameters demonstrate active sensing abilities toward NO2. The n-doping state of CN-PPV-G is reduced dramatically by NO2, which is a strong p-doping compound. Upon exposure to 25 ppm of NO2, our CN-PPV-G sensors react in 10 s, indicating it is almost an immediate response. LOD is determined as low as 100 ppb. The ultrahigh responding speed and low LOD are not affected in dry air. Furthermore, cycling use of our sensors can be realized through simple annealing. The superior features shown by our CN-PPV-G sensors are highly desired in the applications of monitoring the level of NO2 in situ and setting immediate alarms. Our results also suggest that transfer curves of transistors can react very promptly to the stimulus of target gas and, thus, are very promising in the development of fast-response sensing devices although the response values may not reach maximum as a tradeoff.

13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe-C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6 ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6 Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe-C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe-C bonding of the CFe6 core indicate that it does not provide a functionally dynamic (hemilabile) "beating heart"─instead it acts as "a heart of steel", stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2.

The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4(+) T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the ß strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.

The One-Electron Reduced Active-Site FeFe-Cofactor of Fe-Nitrogenase Contains a Hydride Bound to a Formally Oxidized Metal-Ion Core.

The nitrogenase active-site cofactor must accumulate 4e-/4H+ (E4(4H) state) before N2 can bind and be reduced. Earlier studies demonstrated that this E4(4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H2. The state having acquired 2e-/2H+ (E2(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e-/H+ (E1(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E1(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E1(H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity, 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E1(H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E1(H)* relaxing to E1(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E1(H)/E1(H)* photointerconversion. These observations indicate that the addition of the first e-/H+ to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

Reduction of Substrates by Nitrogenases.

Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to support food and chemical production and the heavy reliance of the industrial Haber-Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.

Noncovalent wedging effect catalyzed the cis to syn transformation of a surface-adsorbed polymer backbone toward an unusual thermodynamically stable supramolecular product.

The significant influence of noncovalent interactions on catalytic processes has been recently appreciated but is still in its infancy. In this report, it is found that wedging Me-PTCDI (small-molecule) between the alkyl chains of PffBT4T-2OD (polymer) and a graphite substrate can reduce the energy barrier of flipping over the surface-adsorbed alkylthiophene group from the cis to syn conformation, revealing the catalytic role of Me-PTCDI via a noncovalent wedging effect. The wedging of Me-PTCDI brings the interactions between the alkyl chains and substrate to a very weak level by lifting up the alkyl chains, which eliminates the major hindrance of the flipping process to one main factor: the torsion of the dihedral angles of the thiophene group. The Me-PTCDI/syn PffBT4T-2OD arrangement shows unusual stability compared to the cis one because the syn conformation allows the alkyl chains to construct dense lamella and facilitates interactions between Me-PTCDI and the syn PffBT4T-2OD backbones. The results are helpful for boosting the development of noncovalent catalysis and bottom-up fabrications toward devices functionalized at a molecular level.

Learning to predict in-hospital mortality risk in the intensive care unit with attention-based temporal convolution network.

BACKGROUND: Dynamic prediction of patient mortality risk in the ICU with time series data is limited due to high dimensionality, uncertainty in sampling intervals, and other issues. A new deep learning method, temporal convolution network (TCN), makes it possible to deal with complex clinical time series data in ICU. We aimed to develop and validate it to predict mortality risk using time series data from MIMIC III dataset. METHODS: A total of 21,139 records of ICU stays were analysed and 17 physiological variables from the MIMIC III dataset were used to predict mortality risk. Then we compared the model performance of the attention-based TCN with that of traditional artificial intelligence (AI) methods. RESULTS: The area under receiver operating characteristic (AUCROC) and area under precision-recall curve (AUC-PR) of attention-based TCN for predicting the mortality risk 48 h after ICU admission were 0.837 (0.824 -0.850) and 0.454, respectively. The sensitivity and specificity of attention-based TCN were 67.1% and 82.6%, respectively, compared to the traditional AI method, which had a low sensitivity (< 50%). CONCLUSIONS: The attention-based TCN model achieved better performance in the prediction of mortality risk with time series data than traditional AI methods and conventional score-based models. The attention-based TCN mortality risk model has the potential for helping decision-making for critical patients. TRIAL REGISTRATION: Data used for the prediction of mortality risk were extracted from the freely accessible MIMIC III dataset. The project was approved by the Institutional Review Boards of Beth Israel Deaconess Medical Center (Boston, MA) and the Massachusetts Institute of Technology (Cambridge, MA). Requirement for individual patient consent was waived because the project did not impact clinical care and all protected health information was deidentified. The data were accessed via a data use agreement between PhysioNet, a National Institutes of Health-supported data repository (https://www.physionet.org/), and one of us (Yu-wen Chen, Certification Number: 28341490). All methods were carried out in accordance with the institutional guidelines and regulations.

Role of pyroglutamic acid in cumulus cells of women with polycystic ovary syndrome.

PURPOSE: Polycystic ovary syndrome is a complex heterogeneous endocrine disorder associated with established metabolic abnormalities and is a common cause of infertility in females. Glutathione metabolism in the cumulus cells (CCs) of women with PCOS may be correlated to the quality of oocytes for infertility treatment; therefore, we used a metabolomics approach to examine changes in CCs from women with PCOS and oocyte quality. METHODS: Among 135 women undergoing fertility treatment in the present study, there were 43 women with PCOS and 92 without. CCs were collected from the two groups and levels of pyroglutamic acid were measured using LC-MS/MS followed by qPCR and Western blot analysis to examine genes and proteins involved in pyroglutamic acid metabolism related to glutathione synthesis. RESULTS: Women with PCOS showed increased levels of L-pyroglutamic acid, L-glutamate, and L-phenylalanine and decreased levels of Cys-Gly and N-acetyl-L-methionine. Gene expression of OPLAH, involved in pyroglutamic synthesis, was significantly increased in women with PCOS compared with those without. Gene expression of GSS was significantly decreased in women with PCOS and synthesis of glutathione synthetase protein was decreased. Expression of nuclear factor erythroid 2-related factor 2, involved in resistance to oxidative stress, was significantly increased in women with PCOS. CONCLUSIONS: CCs of women with PCOS showed high concentrations of pyroglutamic acid and reduced glutathione synthesis, which causes oxidative stress in CCs, suggesting that decreased glutathione synthesis due to high levels of pyroglutamic acid in CCs may be related to the quality of oocytes in women with PCOS.

Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted 13C Labeling and ENDOR Spectroscopy.

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.