Hole-scavenging in photo-driven N2 reduction catalyzed by a CdS-nitrogenase MoFe protein biohybrid system.

The light-driven reduction of dinitrogen (N2) to ammonia (NH3) catalyzed by a cadmium sulfide (CdS) nanocrystal­nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N2 reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N2 reduction to NH3 in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N2 reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N2 reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N2 reduction by the biohybrid complex to the standard reduction potential (Eo) of the SEDs reveals that Eo is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N2 reduction reaction catalyzed by a CdS QD-MoFe protein hybrid.

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.

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.

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.

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.

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.

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.

Alleviative effect of poly-ß-hydroxybutyrate on lipopolysaccharide-induced oxidative stress, inflammation and cell apoptosis in Cyprinus carpio.

In this study, the alleviative effects of poly-ß-hydroxybutyrate (PHB) in bioflocs on oxidative stress, inflammation and apoptosis of common carp (Cyprinus carpio) induced by lipopolysaccharide (LPS) were evaluated. Common carp were irregularity divided into 5 groups and fed five diets with 0 % (CK), 2 %, 4 %, 6 % and 8 % PHB. After 8-week feeding trial, LPS challenge was executed. Results showed that appropriate level of PHB enhanced serum immune function by reversing LPS-induced the decrease of C3, C4, IgM, AKP, ACP and LZM in serum, alleviated LPS-induced intestinal barrier dysfunction by decreasing the levels of 5-HT, D-LA, ET-1 and DAO in serum, increasing ZO-1, Occludin, Claudin-3 and Claudin-7 mRNA, improving intestinal morphology. Moreover, dietary PHB reversed LPS-induced the decrease of AST and ALT in hepatopancreas, while in serum exhibited the opposite trend. Suitable level of PHB reversed LPS-induced the reduction of GSH-PX, CAT, T-SOD and T-AOC in intestines and hepatopancreas, whereas MDA showed the opposite result. PHB alleviated LPS-induced the decrease of Nrf2, HO-1, CAT, SOD and GSH-PX mRNA, the increase of Keap1 mRNA. Appropriate level of PHB alleviated LPS-induced inflammation and apoptosis by up-regulating TGF-ß, IL-10 and Bcl-2 mRNA, down-regulating NF-κB, TNF-α, IL-6, Bax, Caspase-3, Caspase-8 and Caspase-9 mRNA. Furthermore, PHB inhibited activation of NLRP3 inflammasomes by reducing the levels of NLRP3, Caspase-1, ASC, IL-1ß and IL-18 mRNA and protein. In addition, the increases of dietary PHB linearly and quadratically affected LPS-induced adverse effects on common carp. Summary, this study suggested that appropriate level of dietary PHB alleviated LPS-induced oxidative stress, inflammation, apoptosis and the activation of NLRP3 inflammasome in common carp. And the appropriate level of PHB in common carp diets was 4 %.

A new, feasible, and convenient method based on semantic segmentation and deep learning for hemoglobin monitoring.

Objective: Non-invasive methods for hemoglobin (Hb) monitoring can provide additional and relatively precise information between invasive measurements of Hb to help doctors' decision-making. We aimed to develop a new method for Hb monitoring based on mask R-CNN and MobileNetV3 with eye images as input. Methods: Surgical patients from our center were enrolled. After image acquisition and pre-processing, the eye images, the manually selected palpebral conjunctiva, and features extracted, respectively, from the two kinds of images were used as inputs. A combination of feature engineering and regression, solely MobileNetV3, and a combination of mask R-CNN and MobileNetV3 were applied for model development. The model's performance was evaluated using metrics such as R2, explained variance score (EVS), and mean absolute error (MAE). Results: A total of 1,065 original images were analyzed. The model's performance based on the combination of mask R-CNN and MobileNetV3 using the eye images achieved an R2, EVS, and MAE of 0.503 (95% CI, 0.499-0.507), 0.518 (95% CI, 0.515-0.522) and 1.6 g/dL (95% CI, 1.6-1.6 g/dL), which was similar to that based on MobileNetV3 using the manually selected palpebral conjunctiva images (R2: 0.509, EVS:0.516, MAE:1.6 g/dL). Conclusion: We developed a new and automatic method for Hb monitoring to help medical staffs' decision-making with high efficiency, especially in cases of disaster rescue, casualty transport, and so on.

Serum Soluble Vascular Endothelial Growth Factor Receptor 1 as a Potential Biomarker of Hepatopulmonary Syndrome.

Background and Aims: The results of basic research implicate the vascular endothelial growth factor (VEGF) family as a potential target of hepatopulmonary syndrome (HPS). However, the negative results of anti-angiogenetic therapy in clinical studies have highlighted the need for markers for HPS. Therefore, we aimed to determine whether VEGF family members and their receptors can be potential biomarkers for HPS through clinical and experimental studies. Methods: Clinically, patients with chronic liver disease from two medical centers were enrolled and examined for HPS. Patients were divided into HPS, intrapulmonary vascular dilation [positive contrast-enhanced echocardiography (CEE) and normal oxygenation] and CEE-negative groups. Baseline information and perioperative clinical data were compared between HPS and non-HPS patients. Serum levels of VEGF family members and their receptors were measured. In parallel, HPS rats were established by common bile duct ligation. Liver, lung and serum samples were collected for the evaluation of pathophysiologic changes, as well as the expression levels of the above factors. Results: In HPS rats, all VEGF family members and their receptors underwent significant changes; however, only soluble VEGFR1 (sFlt-1) and the sFlt-1/ placental growth factor (PLGF) ratio were changed in almost the same manner as those in HPS patients. Furthermore, through feature selection and internal and external validation, sFlt-1 and the sFlt-1/PLGF ratio were identified as the most important variables to distinguish HPS from non-HPS patients. Conclusions: Our results from animal and human studies indicate that sFlt-1 and the sFlt-1/PLGF ratio in serum are potential markers for HPS.

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.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

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.

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.

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.

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.

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.

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.

A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization.

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity1. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster.

Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.