Ammonia synthesis via an engineered nitrogenase assembly pathway in .

Heterologous expression of nitrogenase has been actively pursued because of the far-reaching impact of this enzyme on agriculture, energy and environment. Yet, isolation of an active two-component, metallocentre-containing nitrogenase from a non-diazotrophic host has yet to be accomplished. Here, we report the heterologous synthesis of an active Mo-nitrogenase by combining genes from and in .

Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N at its surface-exposed L-cluster ([FeSC]), a structural/functional homolog of the M-cluster (or cofactor; [(-homocitrate)MoFeSC]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N reduction.

Implementation and Evaluation of a Trainee Orientation Manual in an Intensive Care Unit Rotation.

Background: The intensive care unit (ICU) rotation places trainees in a fast-paced, high-intensity environment that requires complex patient care and multidisciplinary coordination. Trainees seek continuous medical knowledge acquisition while tasked with learning ICU-specific workflows, procedures, and policies. The unfamiliarity with rotation logistics and administrative roles and expectations could hinder the ICU rotation learning experience.

Attention-deficit/hyperactivity disorder and dopamine receptor D4 (DRD4) exon 3 variable number of tandem repeats (VNTR) 2-repeat allele.

To investigate the association of attention-deficit/hyperactivity disorder (ADHD) with the 48-base pair (bp) variable number of tandem repeats (VNTR) in exon 3 of the dopamine receptor D4 (DRD4) gene, we genotyped 240 ADHD patients and their parents from Hong Kong. The 4R allele was most common, followed by 2R. We examined association between the 2R allele (relative to 4R) and ADHD by Transmission Disequilibrium Test (TDT).

ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N to NH at its active-site cofactor. Previous studies of an N-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N species on Av1* remains elusive. Here we report a study of ATP-independent, Eu/SO -driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques.

Heterologous expression of a fully active nitrogenase Fe protein in .

The heterologous expression of a fully active Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in .

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([FeS]) and M-cluster core (or L-cluster; [FeSC]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of , , , and genes, and that of an L-cluster-containing NifB protein upon coexpression of , and genes alongside the gene, in .

Light-driven Transformation of Carbon Monoxide into Hydrocarbons using CdS@ZnS : VFe Protein Biohybrids.

Enzymatic Fisher-Tropsch (FT) process catalyzed by vanadium (V)-nitrogenase can convert carbon monoxide (CO) to longer-chain hydrocarbons (>C2) under ambient conditions, although this process requires high-cost reducing agent(s) and/or the ATP-dependent reductase as electron and energy sources. Using visible light-activated CdS@ZnS (CZS) core-shell quantum dots (QDs) as alternative reducing equivalent for the catalytic component (VFe protein) of V-nitrogenase, we first report a CZS : VFe biohybrid system that enables effective photo-enzymatic C-C coupling reactions, hydrogenating CO into hydrocarbon fuels (up to C4) that can be hardly achieved with conventional inorganic photocatalysts. Surface ligand engineering optimizes molecular and opto-electronic coupling between QDs and the VFe protein, realizing high efficiency (internal quantum yield >56 %), ATP-independent, photon-to-fuel production, achieving an electron turnover number of >900, that is 72 % compared to the natural ATP-coupled transformation of CO into hydrocarbons by V-nitrogenase.

Metal-binding peptides and their potential to enhance the absorption and bioavailability of minerals.

Minerals including calcium, iron, zinc, magnesium, and copper have several human nutritional functions due to their metabolic activities. Body tissues require sufficient levels of a variety of micronutrients to maintain their health. To achieve these micronutrient needs, dietary consumption must be adequate.

Enzymatic Fischer-Tropsch-Type Reactions.

The Fischer-Tropsch (FT) process converts a mixture of CO and H into liquid hydrocarbons as a major component of the gas-to-liquid technology for the production of synthetic fuels. Contrary to the energy-demanding chemical FT process, the enzymatic FT-type reactions catalyzed by nitrogenase enzymes, their metalloclusters, and synthetic mimics utilize H and e as the reducing equivalents to reduce CO, CO, and CN into hydrocarbons under ambient conditions. The C chemistry exemplified by these FT-type reactions is underscored by the structural and electronic properties of the nitrogenase-associated metallocenters, and recent studies have pointed to the potential relevance of this reactivity to nitrogenase mechanism, prebiotic chemistry, and biotechnological applications.

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO or CO to hydrocarbons.

Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor.

The Mo-nitrogenase catalyses the ambient reduction of N to NH at the M-cluster, a complex cofactor that comprises two metal-sulphur partial cubanes ligated by an interstitial carbide and three belt-sulphurs. A recent crystallographic study suggests binding of N via displacement of the belt-sulphur(s) of the M-cluster upon turnover. However, the direct proof of N binding and belt-sulphur mobilization during catalysis remains elusive.

Incorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.

Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe S C]), to reduce atmospheric N to bioaccessible NH . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe S (SEt) ] . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe S (SH)] as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold.

Should We Use High-Flow Nasal Cannula in Patients Receiving Gastrointestinal Endoscopies? Critical Appraisals through Updated Meta-Analyses with Multiple Methodologies and Depiction of Certainty of Evidence.

(1) Background: High-flow nasal cannula (HFNC) therapy or conventional oxygen therapy (COT) are typically applied during gastrointestinal (GI) endoscopic sedation. (2) Methods: We conducted a rigorous systematic review enrolling randomized controlled trials (RCTs) from five databases. Risk of bias was assessed using Cochrane's RoB 2.

The Early Dynamic Change in Cardiac Enzymes and Renal Function Is Associated with Mortality in Patients with Fulminant Myocarditis on Extracorporeal Membrane Oxygenation: Analysis of a Single Center's Experience.

(1) Background: Fulminant myocarditis (FM) could result in hemodynamic derangement and fatal arrhythmia. Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) is used to maintain organ perfusion in FM patients complicating cardiogenic shock. The present study aims to assess the static and dynamic factors in association with mortality in FM patients on V-A ECMO (2) Methods: Twenty-eight patients were enrolled between 2013 to 2019 for analysis (3) Results: In-hospital survival rate was 78.

Radical SAM-dependent formation of a nitrogenase cofactor core on NifB.

Nitrogenase is a versatile metalloenzyme that reduces N, CO and CO at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFeSC], and it is assembled through the generation of a unique [FeSC] core prior to the insertion of Mo and homocitrate. NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly.

Targeting foodborne pathogens via surface-functionalized nano-antimicrobials.

The incorporation of antibiotics and bioactive compounds into non-toxic nanoparticles has been popularly used to produce effective antimicrobial nanocarriers against foodborne pathogens. These systems can protect antimicrobials against harsh environments, control their release, and increase their antimicrobial activities; however, their functions can be decreased by some major barriers. Intracellular localization of bacteria protects them from the host immune system and antimicrobial agents.

Tumor Lysis Syndrome in Patients With Hepatocellular Carcinoma: A Systematic Review of Published Case Reports.

Tumor lysis syndrome (TLS) is a life-threatening oncologic emergency. It is characterized by massive tumor cell death leading to metabolic derangements and multiple organ failure. It is a rare complication of hepatocellular carcinoma (HCC) with only a few cases have been reported in the literature to date.

Phycocyanin, a super functional ingredient from algae; properties, purification characterization, and applications.

Phycocyanins (PCYs) are a group of luxuriant bioactive compounds found in blue-green algae with an estimated global market of about US$250 million within this decade. The multifarious markets of PCYs noted by form (e.g.

Tracing the incorporation of the "ninth sulfur" into the nitrogenase cofactor precursor with selenite and tellurite.

Molybdenum nitrogenase catalyses the reduction of N to NH at its cofactor, an [(R-homocitrate)MoFeSC] cluster synthesized via the formation of a [FeSC] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [FeSC] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the 'ninth sulfur' in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon 'ninth sulfur' insertion remain elusive.

Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins.

The Fe protein of nitrogenase reduces two C1 substrates, CO and CO, under ambient conditions when its [FeS] cluster adopts the all-ferrous [FeS] state. Here, we show disparate reactivities of the - and -encoded Fe proteins from (designated NifH and VnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that VnfH adopts the all-ferrous state at a more positive reduction potential than NifH, which could account for the weaker reactivity of the VnfH toward C1 substrates than NifH.

Temporary coronary sinus pacing to improve ventricular dyssynchrony with cardiogenic shock: A case report.

Background: Temporary transvenous pacing through the coronary sinus is a novel approach rarely used in treating unstable bradycardia. This modality could provide cardiac pacing while achieving better ventricular synchrony. We present a case who received cardiac pacing through the coronary sinus and provide a summary of evidence in the current literature.

Response to Comment on "Structural evidence for a dynamic metallocofactor during N reduction by Mo-nitrogenase".

Peters comment on our report of the dynamic structure of the nitrogenase metallocofactor during N reduction. Their claim that their independent structural refinement and consideration of biochemical data contradict our finding is incorrect and is strongly refuted by our biochemical and structural data that collectively and conclusively point to the binding of dinitrogen species to the nitrogenase cofactor.