Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.National Institutes of Health (U.S.) (NIH Grant R01-DA28299 )National Institutes of Health (U.S.) (NIH NRSA Fellowship (Award F32-GM087102))California Institute of Technology (Caltech Jacobs Grant

Effect of Mass Azithromycin Distributions on Childhood Growth in Niger: A Cluster-Randomized Trial.

Importance: Mass azithromycin distributions may decrease childhood mortality, although the causal pathway is unclear. The potential for antibiotics to function as growth promoters may explain some of the mortality benefit. Objective: To investigate whether biannual mass azithromycin distributions are associated with increased childhood growth. Design, Setting, and Participants: This cluster-randomized trial was performed from December 2014 until March 2020 among 30 rural communities in Boboye and Loga departments in Niger, Africa, with populations from 200 to 2000 individuals. Communities were randomized in a 1:1 ratio to biannual mass distributions of azithromycin or placebo for children ages 1 to 59 months. Participants, field-workers, and study personnel were masked to treatment allocation. Height and weight changes from baseline to follow-up at 4 years were compared between groups. Data were analyzed from June through November 2021. Interventions: Participants received azithromycin at 20 mg/kg using height-based approximation or by weight for children unable to stand every 6 months at the participants' households. Placebo contained the vehicle of the azithromycin suspension. Main Outcomes and Measures: Longitudinal anthropometric assessments were performed on a random sample of children before the first treatment and then annually for 5 years. Height and weight were the prespecified primary outcomes. Results: Among 3936 children enrolled from 30 communities, baseline characteristics were similar between 1299 children in the azithromycin group and 2637 children in the placebo group (mean 48.2% [95% CI, 45.5% to 50.8%] girls vs 48.0% [95% CI, 45.7% to 50.3%] girls; mean age, 30.8 months [95% CI, 29.5 to 32.0 months] vs 30.6 months [95% CI, 29.2 to 31.6 months]). Baseline anthropometric assessments were performed among 2230 children, including 985 children in the azithromycin group and 1245 children in the placebo group, of whom follow-up measurements were available for 789 children (80.1%) and 1063 children (85.4%), respectively. At the prespecified 4-year follow-up visit, children in the azithromycin group gained a mean 6.7 cm (95% CI, 6.5 to 6.8 cm) in height and 1.7 kg (95% CI, 1.7 to 1.8 kg) in weight per year and children in the placebo group gained a mean 6.6 cm (95% CI, 6.4 to 6.7 cm) in height and 1.7 kg (95% CI, 1.7 to 1.8 kg) in weight per year. Height at 4 years was not statistically significantly different between groups when adjusted for baseline height (0.08 cm [95% CI, -0.12 to 0.28 cm] greater in the azithromycin group; P = .45), and neither was weight when adjusted for height and baseline weight (0.02 kg [95% CI, -0.10 to 0.06 kg] less in the azithromycin group; P = .64). However, among children in the shortest quartile of baseline height, azithromycin was associated with a 0.4 cm (95% CI, 0.1 to 0.7 cm) increase in height compared with placebo. Conclusions and Relevance: This study did not find evidence of an association between mass azithromycin distributions and childhood growth, although subgroup analysis suggested some benefit for the shortest children. These findings suggest that the mortality benefit of mass azithromycin distributions is unlikely to be due solely to growth promotion. Trial Registration: ClinicalTrials.gov Identifier: NCT02048007

Probing intermetallic coupling in dinuclear N-heterocyclic carbene ruthenium(II) complexes

A series of bimetallic N-heterocyclic carbene (NHC) ruthenium(II) complexes were synthesized, which comprise two [RuCl₂(cymene)(NHC)] units that are interlinked via the NHC nitrogens by alkyl chains of different length. Electrochemical characterization revealed two mutually dependent oxidation processes for the complex with a methylene linker, indicating moderate intramolecular electronic coupling of the two metal centers (class II system). The degree of coupling decreases rapidly upon increasing the number of CH₂ units in the linker and provides essentially decoupled class I species when propylene or butylene linkers are used. Electrochemical analyses combined with structural investigations suggest a through-bond electronic coupling. Replacement of the alkyl linker with a p-phenylene group afforded cyclometalated complexes, which were considerably less stable. The electronic coupling in the methylene-linked complex and the relatively robust NHC–ruthenium bond may provide access to species that are switchable on the molecular scale

Structurally Characterized Cationic Silver(I) and Ruthenium(II) Carbene Complexes of 1,2,3-Triazol-5-ylidenes

A novel 1,3,4-substituted 1,2,3-triazolium salt was found to function as an effective precursor for the synthesis of the first structurally characterized cationic silver(I) and ruthenium(II) carbene complexes of overall 1:2 ligand-to-metal stoichiometry. The Ag(I) complex crystallized in the form of an eight silver atom containing cluster, whereas the Ru(II) complex proved to be a discrete species and was found to be capable of initiating the ring-opening metathesis polymerization of norbornene upon activation with (trimethylsilyl)diazomethane