Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

Two birds one stone: ß-fluoropyrrolyl-cysteine SNAr chemistry enabling functional porphyrin bioconjugation.

Bioconjugation, a synthetic tool that endows small molecules with biocompatibility and target specificity through covalent attachment of a biomolecule, holds promise for next-generation diagnosis or therapy. Besides the establishment of chemical bonding, such chemical modification concurrently allows alteration of the physicochemical properties of small molecules, but this has been paid less attention in designing novel bioconjugates. Here, we report a "two birds one stone" methodology for irreversible porphyrin bioconjugation based on ß-fluoropyrrolyl-cysteine SNAr chemistry, in which the ß-fluorine of porphyrin is selectively replaced by a cysteine in either peptides or proteins to generate novel ß-peptidyl/proteic porphyrins. Notably, due to the distinct electronic nature between fluorine and sulfur, such replacement makes the Q band red-shift to the near-infrared region (NIR, >700 nm). This facilitates intersystem crossing (ISC) to enhance the triplet population and thus singlet oxygen production. This new methodology features water tolerance, a fast reaction time (15 min), good chemo-selectivity, and broad substrate scope, including various peptides and proteins under mild conditions. To demonstrate its potential, we applied porphyrin ß-bioconjugates in several scenarios, including (1) cytosolic delivery of functional proteins, (2) metabolic glycan labeling, (3) caspase-3 detection, and (4) tumor-targeting phototheranostics.

Designing Artificial Metalloenzymes by Tuning of the Environment beyond the Primary Coordination Sphere.

Metalloenzymes catalyze a variety of reactions using a limited number of natural amino acids and metallocofactors. Therefore, the environment beyond the primary coordination sphere must play an important role in both conferring and tuning their phenomenal catalytic properties, enabling active sites with otherwise similar primary coordination environments to perform a diverse array of biological functions. However, since the interactions beyond the primary coordination sphere are numerous and weak, it has been difficult to pinpoint structural features responsible for the tuning of activities of native enzymes. Designing artificial metalloenzymes (ArMs) offers an excellent basis to elucidate the roles of these interactions and to further develop practical biological catalysts. In this review, we highlight how the secondary coordination spheres of ArMs influence metal binding and catalysis, with particular focus on the use of native protein scaffolds as templates for the design of ArMs by either rational design aided by computational modeling, directed evolution, or a combination of both approaches. In describing successes in designing heme, nonheme Fe, and Cu metalloenzymes, heteronuclear metalloenzymes containing heme, and those ArMs containing other metal centers (including those with non-native metal ions and metallocofactors), we have summarized insights gained on how careful controls of the interactions in the secondary coordination sphere, including hydrophobic and hydrogen bonding interactions, allow the generation and tuning of these respective systems to approach, rival, and, in a few cases, exceed those of native enzymes. We have also provided an outlook on the remaining challenges in the field and future directions that will allow for a deeper understanding of the secondary coordination sphere a deeper understanding of the secondary coordintion sphere to be gained, and in turn to guide the design of a broader and more efficient variety of ArMs.

Social anxiety and associated factors among graduate students under the normalization of epidemic prevention and control / 中国学校卫生

Objective@#To understand social anxiety and relevant factors among graduate students under the normalization stage of COVID-19 epidemic prevention and control.@*Methods@#Using convenience sampling method, an online questionnaire survey on graduate students from 5 universities in Jiangsu Province was conducted. Measurements used in the survey includes General Self Efficacy Scale (GSES), General Alienation Scale (GAS), Interaction Anxiousness Scale (IAS) and self made survey for basic information and household living conditions.@*Results@#The overall score of graduate students self efficacy was (2.58±0.50). Average score was (30.68±6.22) for alienation, and (47.55±8.77) for interaction anxiety, with detection rate of social anxiety being 43.96%. Increased dependence on smartphones and electronic devices ( OR=1.86, 95%CI =1.32-2.61) and high alienation score (medium level: OR=2.06, 95%CI =1.45-2.92; high level: OR=5.19, 95%CI =1.00-27.00) were positively correlated with social anxiety. Increased communication with friends ( OR=0.65, 95%CI =0.47-0.90 and high self efficacy (medium level: OR= 0.37 , 95%CI =0.21-0.66; high level: OR=0.15, 95%CI =0.08-0.30) were negatively correlated with social anxiety.@*Conclusion@#At the normalization stage of COVID-19 epidemic prevention and control, social anxiety of graduate students is one of the mental health issues which need further attention. Participation in peer support helps prevent social anxiety through developing self efficacy, alleviating individual alienation, and reducing dependence on electronic devices among graduate students.

Fluorescence lifetime imaging of upper gastrointestinal pH in vivo with a lanthanide based near-infrared τ probe.

Time-resolved fluorescence lifetime imaging (FLIM) in the near-infrared region of 900-1700 nm not only allows a deep tissue penetration depth but also offers the unique benefit of the quantitative visualization of molecular events in vivo and is independent of local luminescence intensity and fluorophore concentration. Herein, we report the design of a wide-range pH sensitive molecular probe based on Yb3+ porphyrinate. The Yb3+ probe shows increasing NIR emission and lifetime with pK a values of ca. 6.6 from pH 9.0 and 5.0 and also displays an elongated lifetime from ca. 135 to 170 µs at lower pH values (5.0-1.0) due to aggregation and reduced exposure to water at low pH values. Importantly, the probe is able to monitor a wide range of in vivo gastrointestinal pH values in mice models and the potential applications in imaging-guided gastrointestinal diagnostics and therapeutics were revealed. This study shows that lifetime contrast is important for preclinical imaging; lanthanide complexes could be successfully used in the design of stimuli-responsive NIR τ probes for advanced in vivo imaging.