Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein-protein interactions, protein-ligand interactions, and protein-nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems. Key features • Established procedures for loading and sealing tubes in an anaerobic chamber for subsequent analysis. • Sodium dithionite (NaDT) could easily be substituted with one electron-reduced 1,1\'-bis(3-sulfonatopropyl)-4,4\'-bipyridinium [(SPr)2V•] to perform sensitive biophysical assays on oxygen-sensitive proteins like the MoFe protein. • Established MST as an experimental tool to quantify binding affinities in novel enzyme-quantum dot biohybrid complexes that are extremely oxygen-sensitive.

Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent\'s DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.

This study explores the principles of resonance energy transfer and adsorption modulation using composites of Cu2S-MPA/NGODs. These composites can efficiently control the quenching process of electrochemiluminescence (ECL). Mercaptopropionic acid (MPA) was added during the synthesis of Cu2S-MPA to enhance its attachment to nitrogen-doped graphene quantum dots (NGODs). The UV absorption peaks of NGODs coincided with the emission peaks of luminol ECL, enabling resonance energy transfer and enhancing the quenching capability of Cu2S-MPA. Meanwhile, there is another quenching strategy. When the readily reducible Cu+ ions underwent partial reduction to Cu when they were bound to NGODs. This weakened the electrocatalytic effect on reactive oxygen species (ROS) and had a detrimental impact on electron transfer. Under optimal conditions, the immunosensor ECL intensity decreased linearly with the logarithm of carcinoembryonic antigen (CEA) concentration in the range of 0.00001-40 ng/mL, with a detection limit of 0.269 fg/mL. The sensor was effectively utilized for the identification of CEA in actual serum samples.

Multi-photon reduction (MPR) based on femtosecond laser makes rapid prototyping and molding in micro-nano scale feasible, but is limited in material selectivity due to lack of the understanding of the reaction mechanism in MPR process. In this paper, additively manufacturing of complex silver-based patterns through MPR is demonstrated. The effects of laser parameters, including laser pulse energies and scanning speeds, on the structural and chemical characteristics of the printed structures are systematically investigated. The results show that the geometric size of printed cubes deviates from the designed size further by increasing laser pulse energy or decreasing scanning speed. The reaction mechanism of MPR is revealed by studying the elemental composition and chemical structures of printed cubes. The evolution of Raman spectra upon the laser processing parameters suggests that the MPR process mainly includes two processes: reduction and decomposition. In the MPR process, silver ions are reduced and grow into particles by accepting the electrons from ethonal molecules; meanwhile carboxyl groups in polyvinylpyrrolidone are decomposed and form amorphous carbon that is attached on the surface of silver particles. The conductivity of silver wires fabricated by MPR reaches 2 × 105S m-1and stays relatively constant as varying their cross section area, suggesting excellent electrical conduction. The understanding of the MPR process would accelerate the development of MPR technology and the implementation of MPR in micro-electromechanical systems could therefore be envisioned.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2\'s requirement for activation.

BACKGROUND: Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two families of persistent organic pollutants that are dangerous as they remain in the atmosphere for long periods and are toxic for humans and animals. They are found all over the world, including the penguins of Antarctica. One of the mechanisms that explains the toxicity of these compounds is related to oxidative stress. The main idea of this theoretical research is to use conceptual density functional theory as a theory of chemical reactivity to analyze the oxidative stress that PCBs and PBDEs can produce. The electron transfer properties as well as the interaction with DNA nitrogenous bases of nine PCBs and ten PBDEs found in Antarctic penguins are investigated. From this study, it can be concluded that compounds with more chlorine or bromine atoms are more oxidizing and produce more oxidative stress. These molecules also interact directly with the nitrogenous bases of DNA, forming hydrogen bonds, and this may be an explanation for the toxicity. Since quinone-type metabolites of PCBs and PBDEs can cause neurotoxicity, examples of quinones are also investigated. Condensed Fukui functions are included to analyze local reactivity. These results are important as the reactivity of these compounds helps to explain the toxicity of PCBs and PBDEs.
METHODS: All DFT computations were performed using Gaussian16 at M06-2x/6-311 + g(2d,p) level of theory without symmetry constraints. Electro-donating (ω-) and electro-accepting (ω +) powers were used as global response functions and condensed Fukui functions as local parameters of reactivity.

The addition of biochar resulted in a 31.5 % to 44.6 % increase in decolorization efficiency and favorable decolorization stability. Biochar promoted extracellular polymeric substances (EPS) secretion, especially humic-like and fulvic-like substances. Additionally, biochar enhanced the electron transfer capacity of anaerobic sludge and facilitated surface attachment of microbial cells. 16S rRNA gene sequencing analysis indicated that biochar reduced microbial species diversity, enriching fermentative bacteria such as Trichococcus. Finally, a machine learning model was employed to establish a predictive model for biochar characteristics and decolorization efficiency. Biochar electrical conductivity, H/C ratio, and O/C ratio had the most significant impact on RR2 anaerobic decolorization efficiency. According to the results, the possible mechanism of RR2 anaerobic decolorization enhanced by different types of biochar was proposed.

DNA crosslinking agents such as cisplatin and related platinum(II) analogs are effective drugs to treat solid tumors. However, these therapeutics can cause high toxicity in the body, and tumors can develop resistance to them. To develop less toxic and more effective DNA crosslinkers, medicinal chemists have focused on tuning the ligands in square planar platinum(II) complexes to modulate their bioavailability, targeted cell penetration, and DNA binding rates. Unfortunately, linking in vitro DNA binding capacity of DNA crosslinkers with their in vivo efficacy has proven challenging. Here we report an electrochemical biosensor strategy that allows the study of platinum(II)-DNA binding in real time. Our biosensors contain a purine-rich deoxynucleotide sequence, T6 (AG)10 , modified with a 5\' hexylthiol linker for easy self-assembly onto gold electrodes. The 3\' terminus is functionalized with the redox reporter methylene blue. Electron transfer from methylene blue to the sensor is a function of platinum(II) compound concentration and reaction time. Using these biosensors, we resolve DNA binding mechanisms including monovalent and bivalent binding, as well as base stacking. Our approach can measure DNA binding kinetics in buffers and in 50 % serum, offering a single-step, real-time approach to screen therapeutic compounds during drug development.

The dehaloperoxidase-hemoglobin (DHP), first isolated from the coelom of a marine terebellid polychaete, Amphitrite ornata, is an example of a multi-functional heme enzyme. Long known for its reversible oxygen (O2) binding, further studies have established DHP activity as a peroxidase, oxidase, oxygenase, and peroxygenase. The specific reactivity depends on substrate binding at various internal and external binding sites. This study focuses on comparison of the binding and reactivity of the substrate 2,4-dichlorophenol (DCP) in the isoforms DHPA and B. There is strong interest in the degradation of DCP because of its wide use in the chemical industry, presence in waste streams, and particular reactivity to form dioxins, some of the most toxic compounds known. The catalytic efficiency is 3.5 times higher for DCP oxidation in DHPB than DHPA by a peroxidase mechanism. However, DHPA and B both show self-inhibition even at modest concentrations of DCP. This phenomenon is analogous to the self-inhibition of 2,4,6-trichlorophenol (TCP) at higher concentration. The activation energies of the electron transfer steps in DCP in DHPA and DHPB are 19.3 ± 2.5 and 24.3 ± 3.2 kJ/mol, respectively, compared to 37.2 ± 6.5 kJ/mol in horseradish peroxidase (HRP), which may be a result of the more facile electron transfer of an internally bound substrate in DHPA. The x-ray crystal structure of DHPA bound with DCP determined at 1.48 Å resolution, shows tight substrate binding inside the heme pocket of DHPA (PDB 8EJN). This research contributes to the studies of DHP as a naturally occurring bioremediation enzyme capable of oxidizing a wide range of environmental pollutants.

Time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study photosystem I (PSI) from Synechocystis sp. PCC 6803 with four high-potential, 1,4-naphthoquinones (NQs) incorporated into the A1 binding site. The incorporated quinones are 2-chloro-NQ (2ClNQ), 2-bromo-NQ (2BrNQ), 2,3-dichloro-NQ (Cl2NQ), and 2,3-dibromo-NQ (Br2NQ). For completeness 2-methyl-NQ (2MNQ) was also incorporated and studied. Previously, PSI with the same quinones incorporated were studied in the, so-called, anion spectral region between 1550 and 1400 cm-1 (Agarwala et al. in Biochim Biophys Acta 1864(1):148918, 2023). Here we focus on spectra in the previously unexplored 1400-1200 cm-1 spectral region. In this region several bands are identified and assigned to the neutral state of the incorporated quinones. This is important as identification of neutral state quinone bands in the regular 1700-1600 cm-1 region has proven difficult in the past. For neutral PhQ in PSI a broad, intense band appears at ~ 1300 cm-1. For the symmetric di-substituted NQs (Cl2NQ/Br2NQ) a single intense neutral state band is found at ~ 1280/1269 cm-1, respectively. For both mono-substituted NQs, 2ClNQ and 2BrNQ, however, two neutral state bands are observed at ~ 1280 and ~ 1250 cm-1, respectively. These observations from time-resolved spectra agree well with conclusions drawn from absorption spectra of the NQs in THF, which are also presented here. Density functional theory based vibrational frequency calculations were undertaken allowing an identification of the normal modes associated with the neutral state quinone bands.