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Inorder to investigate experimentally observed phototautomerizationof gas-phase cytosine, several excited-state tautomerization mechanismswere characterized at the EOM-CCSD and TDDFT levels. All pathwaysthat took place exclusively on the S1surface were foundto have significant barriers that were much higher than the barriersinvolved in radiationless decay of cytosine tautomers through conicalintersections back to the ground state; tautomerization in this fashioncannot compete with radiationless relaxation. However, an alternativepossibility is that the conical intersections that facilitate radiationlessdecay could also facilitate tautomerization. Barrierless pathwaysindicate that it is energetically possible that bifurcation at theconical intersections can lead to a subset of the population reachingdifferent tautomers. This could be an explanation for the observedtautomerization of keto cytosine after exposure to low-energy UV light. [ABSTRACT FROM AUTHOR]

Resistance to cancer therapy is driven by both cell-intrinsic and microenvironmental factors. Previous work has revealed that multiple resistant cell fates emerge in melanoma following treatment with targeted therapy and that, in vitro , these resistant fates are determined by the transcriptional state of individual cells prior to exposure to treatment. What remains unclear is whether these resistant fates are shared across different genetic backgrounds and how, if at all, these resistant fates interact with the tumor microenvironment. Through spatial transcriptomics and single-cell RNA sequencing, we uncovered distinct resistance programs in melanoma cells shaped by both intrinsic cellular states and the tumor microenvironment. Consensus non-negative matrix factorization revealed shared intrinsic resistance programs across different cell lines, highlighting the presence of universal and unique resistance pathways. In patient samples, we demonstrated that these resistance programs coexist within individual tumors and associate with diverse immune signatures, suggesting that the tumor microenvironment and distribution of resistant fates are closely connected. Single-cell resolution spatial transcriptomics in xenograft models revealed both intrinsically determined and extrinsically influenced resistant fates. Overall, this work demonstrates that each therapy resistant fate coexists with a distinct immune microenvironment in tumors and that, in vivo , tissue features, such as regions of necrosis, can influence which resistant fate is adopted., Competing Interests: Declaration of interests A.R. receives royalties related to Stellaris RNA FISH probes. J.A.W. is an inventor on US patent application no. PCT/US17/53.717 submitted by the University of Texas MD Anderson Cancer Center, which covers methods to enhance immune checkpoint blockade responses by modulating the microbiome. J.A.W. reports compensation for the speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, Exelixis and Bristol Myers Squibb; and has served as a consultant and/or advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol Myers Squibb, Micronoma, OSE therapeutics, Merck and Everimmune. J.A.W. receives stock options from Micronoma and OSE therapeutics. All other authors declare no competing interests.

The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively low-throughput such that collecting enough data to fully characterize populations of cells is challenging. Flow cytometry avoids this drawback, and is a powerful tool that allows for rapid, high-throughput measurement of fluorescent readouts in individual cells. When combined with pH-sensitive fluorescent reporters, it can be used to characterize the intracellular pH of large populations of cells at the single-cell level. We adapted microscopy and flow-cytometry based methods to measure the intracellular pH of yeast. Cells can be grown under near-native conditions up until the point of measurement, and the protocol can be adapted to single-point or dynamic (time-resolved) measurements during changing environmental conditions., Competing Interests: Competing interestsThe authors declare they do not have any competing interests., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)