Abstract Riboflavin (vitamin B2) is an important visible chromophore in flavoproteins and a popular photosensitizer in organic photocatalysis. Despite its importance, a complete understanding of riboflavin’s full photocycle remains undetermined, specifically the factors that govern the relative yields of its singlet and triplet excited states─both playing important roles in electron transfer chemistry. A combination of transient absorption spectroscopy and time-resolved infrared spectroscopy is used to address this problem, tracking the photoexcited states of riboflavin over 9 decades of time under the same experimental conditions. These studies reveal that the triplet quantum yield increases by a factor of 2.7 by merely changing the solvent from DMSO to H2O. Electronic structure calculations show that this enhancement is not likely to originate from a change in the associated singlet and triplet state potential surfaces. Instead, we discern solvent-dependent branching, with more efficient internal conversion in DMSO, likely mediated by its stronger hydrogen bonding acceptor character than water. This strong modulation shows that the triplet branching, and thus the reservoir of long-lived states required for electron transfer reactions, can be carefully tuned by the immediate environment of the flavin chromophore.

Driven by deep-learning approaches, computational protein design is advancing rapidly, and it is now possible to generate many de novo protein structures quickly and robustly. This sets new frontiers for the field, including designing proteins that bind small molecules tightly and specifically, and understanding the non-covalent interactions that underpin such designs to make binding predictable and tunable. Here we address these challenges with a rapid physics-based computational method to generate isosteric and chemically complementary binding pockets for small-molecule targets in de novo designed proteins. We test this experimentally by constructing and characterizing binding proteins for several synthetic and natural chromophores. By evaluating only single-digit numbers of designs, the pipeline delivers stable proteins with pre-organized binding sites confirmed by X-ray crystallography, which bind the targets selectively with micromolar affinities or better. To illustrate the scope and applications of this approach, we incorporate distinct and coupled chromophore-binding sites in a two-domain de novo protein enabling controlled energy transfer between the two sites, and we develop a small de novo binding protein that can be used in live mammalian cells to visualize sub-cellular structures. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/X003027/1, BB/T008741/1 Engineering and Physical Sciences Research Council, EP/T012455/1 Leverhulme Trust, RGP-2021-049 Royal Society, URF\R\201007

Nitroaromatic compounds are found in brown carbon aerosols emitted to the Earth's atmosphere by biomass burning, and are important organic chromophores for the absorption of solar radiation. Here, transient absorption spectroscopy spanning 100 fs-8 mu s is used to explore the pH-dependent photochemical pathways for aqueous solutions of p-nitrophenol, chosen as a representative nitroaromatic compound. Broadband ultrafast UV-visible and infrared probes are used to characterize the excited states and intermediate species involved in the multistep photochemistry, and to determine their lifetimes under different pH conditions. The assignment of absorption bands, and the dynamical interpretation of our experimental measurements are supported by computational calculations. After 320 nm photoexcitation to the first bright state, which has 1 pi pi* character in the Franck-Condon region, and ultrafast (similar to 200 fs) structural relaxation in the adiabatic S1 state to a region with 1n pi* electronic character, the S1 p-nitrophenol population decays on a time scale of similar to 12 ps. This decay involves competition between direct internal conversion to the S0 state (similar to 40%) and rapid intersystem crossing to the triplet manifold (similar to 60%). Population in the T1-state decays by excited-state proton transfer (ESPT) to the surrounding water and relaxation of the resulting triplet-state p-nitrophenolate anion to its S0 electronic ground state in similar to 5 ns. Reprotonation of the S0-state p-nitrophenolate anion recovers p-nitrophenol in its electronic ground state. Overall recovery of the S0 state of aqueous p-nitrophenol via these competing pathways is close to 100% efficient. The experimental observations help to explain why nitroaromatic compounds such as p-nitrophenol resist photo-oxidative degradation in the environment.

Abstract The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell–dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. Significance: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5

PURPOSE:To increase awareness, outline strategies, and offer clinical guidance on navigating the complexities of treatment planning amid antineoplastic drug shortages.METHODS:A multidisciplinary panel of oncologists, ethicists, and patient advocates was assembled to provide rapid clinical guidance to help providers navigate appropriate patient care in cases where rationing or alternative therapies must be considered. The groups of content experts developed general principles for resource allocation during shortages and clinical guidance on alternative therapies for specific disease sites. The recommendations are supported by evidence when available.RESULTS:A total of 44 volunteers with content expertise formed the Advisory Group that developed general guidance on the prioritization of antineoplastic agents in limited supply. Disease site-specific clinical guidance was then produced by subgroups on the basis of members' specialties and expertise. The majority of alternative treatment options were developed in consideration of cisplatin and carboplatin shortages. All guidance is posted on ASCO's website.RECOMMENDATIONS:The prioritization of antineoplastic agents in limited supply should be based on specific goals of the therapy where evidence-based medicine has shown survival outcome and life-extending benefit in both early and advanced stages. Recommendations for specific disease sites are presented. While management options vary according to the disease site, alternatives are presented. For settings in which there are no alternatives with comparable efficacy and safety, it is recommended that patients are referred to an area where the necessary drug is available or can be obtained.Additional information is available at asco.org/drug-shortages.

ABSTRACTDe novoprotein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses α-helical barrel (αHB) peptide assemblies, which comprise 5 or more α-helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, αHBs are analogous to ‘molecular flasks’ made in supramolecular, polymer, and materials chemistry. Using Förster resonance energy transfer as a readout, we demonstrate that specific αHBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an αHB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the αHB.

The photoionization dynamics of indole, the ultraviolet-B chromophore of tryptophan, were explored in water and ethanol using ultrafast transient absorption spectroscopy with 292, 268, and 200 nm excitation. By studying the femtosecond-to-nanosecond dynamics of indole in two different solvents, a new photophysical model has been generated that explains many previously unsolved facets of indole's complex solution phase photochemistry. Photoionization is only an active pathway for indole in aqueous solution, leading to a reduction in the fluorescence quantum yield in water-rich environments, which is frequently used in biophysical experiments as a key signature of the protein-folded state. Photoionization of indole in aqueous solution was observed for all three pump wavelengths but via two different mechanisms. For 200 nm excitation, electrons are ballistically ejected directly into the bulk solvent. Conversely, 292 and 268 nm excitation populates an admixture of two (1)pi pi* states, which form a dynamic equilibrium with a tightly bound indole cation and electron-ion pair. The ion pair dissociates on a nanosecond time scale, generating separated solvated electrons and indole cations. The charged species serve as important precursors to triplet indole production and greatly enhance the overall intersystem crossing rate. Our proposed photophysical model for indole in aqueous solution is the most appropriate for describing photoinduced dynamics of tryptophan in polypeptide sequences; tryptophan in aqueous pH 7 solution is zwitterionic, unlike in peptides, and resultantly has a competitive excited state proton transfer pathway that quenches the tryptophan fluorescence.

Figure S5. AMG 160 engages NHP PSMA and CD3 in vitro.

Figure S1. AMG 160 has high binding affinity.

Recent claims of achieving exponential quantum advantage have attracted attention to Gaussian boson sampling (GBS), a potential application of which is dense subgraph finding. We investigate the effects of sources of error including loss and spectral impurity on GBS applied to dense subgraph finding algorithms. We find that the effectiveness of these algorithms is remarkably robust to errors, to such an extent that there exist efficient classical algorithms that can simulate the underlying GBS. These results imply that the speedup of GBS-based algorithms for the dense subgraph problem over classical approaches is at most polynomial, though this could be achieved on a quantum device with dramatically less stringent requirements on loss and photon purity than general GBS.

Ocean warming is increasingly affecting marine ecosystems across the globe. Reef-building corals are particularly affected by warming, with mass bleaching events increasing in frequency and leading to widespread coral mortality. Yet, some corals can resist or recover from bleaching better than others. Such variability in thermal resilience could be critical to reef persistence; however, the scientific community lacks standardized diagnostic approaches to rapidly and comparatively assess coral thermal vulnerability prior to bleaching events. We present the Coral Bleaching Automated Stress System (CBASS) as a low-cost, open-source, field-portable experimental system for rapid empirical assessment of coral thermal thresholds using standardized temperature stress profiles and diagnostics. The CBASS consists of four or eight flow-through experimental aquaria with independent water masses, lighting, and individual automated temperature controls capable of delivering custom modulating thermal profiles. The CBASS is used to conduct daily thermal stress exposures that typically include 3-h temperature ramps to multiple target temperatures, a 3-h hold period at the target temperatures, and a 1-h ramp back down to ambient temperature, followed by an overnight recovery period. This mimics shallow water temperature profiles observed in coral reefs and prompts a rapid acute heat stress response that can serve as a diagnostic tool to identify putative thermotolerant corals for in-depth assessments of adaptation mechanisms, targeted conservation, and possible use in restoration efforts. The CBASS is deployable within hours and can assay up to 40 coral fragments/aquaria/day, enabling high-throughput, rapid determination of thermal thresholds for individual genotypes, populations, species, and sites using a standardized experimental framework.

Figure S2. Enzalutamide increases PSMA expression levels and may enhance AMG 160-dependent cytotoxicity.

Reninomas are exceedingly rare renin-secreting kidney tumours that derive from juxtaglomerular cells, specialised smooth muscle cells that reside at the vascular inlet of glomeruli. They are the central component of the juxtaglomerular apparatus which controls systemic blood pressure through the secretion of renin. We assess somatic changes in reninoma and find structural variants that generate canonical activating rearrangements of, NOTCH1 whilst removing its negative regulator, NRARP. Accordingly, in single reninoma nuclei we observe excessive renin and NOTCH1 signalling mRNAs, with a concomitant non-excess of NRARP expression. Re-analysis of previously published reninoma bulk transcriptomes further corroborates our observation of dysregulated Notch pathway signalling in reninoma. Our findings reveal NOTCH1 rearrangements in reninoma, therapeutically targetable through existing NOTCH1 inhibitors, and indicate that unscheduled Notch signalling may be a disease-defining feature of reninoma.

Despite many decades of study, the excited state photophysics of polyenes remains controversial. In diphenylpolyenes with conjugated backbones that contain between 2 and 4 double carbon-carbon bonds, the first two excited electronic states are nearly degenerate but of entirely different character, and their energy splitting is strongly dependent on solvent polarizability. To examine the interplay between these different states, steady-state and time-resolved fluorescence spectroscopies were used to undertake a comprehensive investigation of diphenylocatetraene's (DPO) excited state dynamics in 10 solvents of different polarizabilities and polarities, ranging from weakly interacting alkanes to polar hydrogen-bonding alcohols. These data revealed that photopreparation of the optically bright 1B(u) state resulted in fast (<170 ps) internal conversion to the lower-lying optically dark 2A(g) state. The 2A(g) state is responsible for almost all the observed DPO fluorescence and gains oscillator strength via vibronic intensity stealing with the near-degenerate 1B(u) state. The fluorescence lifetime associated with the 2A(g) state decayed monoexponentially (4.2-7.2 ns) in contrast to prior biexponential decay kinetics reported for similar polyenes, diphenylbutadiene and diphenylhexatriene. An analysis combining the measured fluorescence lifetimes and fluorescence quantum yields (the latter varying between 7 and 21%) allowed for a 190 cm(-1) Herzberg-Teller vibronic coupling constant between the 1B(u) and 2A(g) states to be determined. The analysis also revealed that the ordering of electronic states remains constant in all the solvents studied, with the 2A(g) state minimum always lower in energy than that of the 1B(u) state, thus making it a relatively simple polyene compared to structurally similar diphenylhexatriene.

Figure S4. Faster clearance of AMG 160 in the NSG B2M mouse strain.

Supplementary methods, tables, and references.

The persistence of Pacific coral reefs is threatened by large-scale mortality resulting from heat stress, storms, overfishing, disease, and land-based pollution. While reefs have demonstrated the ability to recover from these disturbances, recovery potential is highly variable; in part driven by the high spatial variability in the abundance of coral juveniles. However, our understanding of the patterns and predictors of juvenile coral density is hampered by the limited geographic scope of previous studies. Our objectives in this study were to: (1) explore the spatial and temporal patterns of juvenile (1- < 5cm in diameter) colony density across the U.S.-affiliated Pacific islands and atolls; and (2) identify the potential role of ecological and environmental factors which may influence coral juvenile abundance. Juvenile density was quantified as part of NOAA's National Coral Reef Monitoring Program between 2013 and 2019 using a stratified sampling design across 1,405 forereef sites on 34 islands and atolls. Regional mean juvenile density varied from 1.4 to 10.5 colonies m(-2) with the highest densities observed in the northern Mariana Islands, Northwestern Hawaiian Islands, and southern Mariana Islands. Juvenile density significantly increased between sampling periods in the Main Hawaiian Islands, Line Islands and northern Mariana Islands. Survey-weighted generalized linear modeling demonstrated that juvenile density was best predicted by the interaction between time since a heat stress event and heat stress severity, depth, site and sector-level percent coral cover, human density, percent unconsolidated substrate, macroalgal cover, herbivore biomass, and the interaction of sector coral cover area and heat stress, and the interaction of wave power (combination of wave height and period) and heat stress. With the unique scale and gradients across the U.S. Pacific, our study found that the high latitude regions have enhanced juvenile density compared to their lower latitude counterparts. Our results also suggest density is most dependent on the timing and severity of heat stress, depth, stock-recruitment with an open adult population, availability of suitable habitat at the site-scale, and the number of humans in the system.

Figure S3. PSMA-11 does not compete with AMG 160 for PSMA binding.