ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.

Cystathionine b-synthase (CBS) catalyzes the formation of L-cystathionine from L-serine and L-homocysteine. The resulting L-cystathionine is decomposed into L-cysteine, ammonia, and aketobutylic acid by cystathionine c-lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H 2 S). In some bacteria, including the plant-derived lactic acid bacterium Lactobacillus plantarum, the CBS-and CGL-encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H 2 S production in bacteria; interestingly, it has been shown that H 2 S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O-acetyl-L-serine sulfhydrylase (OASS) that catalyzes the generation of L-cysteine from O-acetyl-L-serine (L-OAS) and H 2 S. The L. plantarum CBS shows L-OAS-and L-cysteine-dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H 2 S in the presence of L-cysteine and L-homocysteine, together with the formation of L-cystathionine. The high affinity toward L-cysteine as a first substrate and tendency to use L-homocysteine as a second substrate might be associated with its enzymatic ability to generate H 2 S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H 2 S-generating activity.

This paper reviews the NTIRE 2020 challenge on real image denoising with focus on the newly introduced dataset, the proposed methods and their results. The challenge is a new version of the previous NTIRE 2019 challenge on real image denoising that was based on the SIDD benchmark. This challenge is based on a newly collected validation and testing image datasets, and hence, named SIDD+. This challenge has two tracks for quantitatively evaluating image denoising performance in (1) the Bayer-pattern rawRGB and (2) the standard RGB (sRGB) color spaces. Each track ∼250 registered participants. A total of 22 teams, proposing 24 methods, competed in the final phase of the challenge. The proposed methods by the participating teams represent the current state-of-the-art performance in image denoising targeting real noisy images. The newly collected SIDD+ datasets are publicly available at: https://bit.ly/siddplus_data. A. Abdelhamed (kamel@eecs.yorku.ca, York University), M. Afifi, R. Timofte, and M.S. Brown are the NTIRE 2020 challenge organizers, while the other authors participated in the challenge. Appendix A contains the authors' teams and affiliations. NTIRE webpage: arXiv:2005.04117v1 [cs.CV] 8 May 2020

We report extensive conformational searches of the gas-phase neutral nicotine, nornicotine, and their protonated analogs and the pathways and barriers for the interconversion between their various isomers that are based on ab initio second-order Møller-Plesset perturbation (MP2) electronic structure calculations. Initial searches were performed with the 6-31G(d,p), and the energetics of the most important structures were further refined from geometry optimizations with the larger aug-cc-pVTZ basis set. On the basis of the calculated free energies at T = 298 K for the gas-phase molecules, neutral nicotine has two dominant trans conformers, whereas neutral nornicotine is a mixture of several conformers. For nicotine, the protonation on both the pyridine and the pyrrolidine sites is energetically competitive, whereas nornicotine prefers protonation on the pyridine nitrogen. The protonated form of nicotine is mainly a mixture of two pyridine-protonated trans conformers and two pyrrolidine-protonated trans conformers, whereas the protonated form of nornicotine is a mixture of four pyridine-protonated trans conformers. Nornicotine is conformationally more flexible than nicotine; however, it is less protonated at the biologically important pyrrolidine nitrogen site. The lowest energy isomers for each case were found to interconvert via low (<6 kcal/mol) rotational barriers around the pyridine-pyrrolidine bond. These barriers are much lower than previous estimates based on lower levels of theory obtained without relaxation of the structure along the path. Nicotine was found to bind more strongly to tryptophan (Trp) than nornicotine, a finding that is consistent with nicotine's enhanced affinity in the nicotinic acetylcholide receptor.

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