The ability to induce endothelial cell (EC) damage in the mouse brain with high spatial precision is invaluable for mechanistic studies of brain capillary injury and repair. Here, we introduce an optical method, termed Endothelial Cell guided obliteration (ECgo), that utilizes a two-photon-excitable porphyrin-based photosensitizer (Ps2P) to selectively obliterate single ECs within the brain microvascular network. Using the developed approach, we were able to induce occlusions of single capillaries with high spatiotemporal control, while preserving the surrounding tissue. Combined with longitudinal two-photon imaging, ECgo enables studies of morphological and functional consequences of targeted single capillary EC injury in vivo under healthy and diseased conditions.

Dipyrrins form a group of versatile chromophores, which find use as laser dyes as well as in light harvesting and bioimaging applications. The mode of the central ion coordination and the ensuing molecular geometry play a key role in the photophysics of dipyrrins, whereby some complexes are brightly fluorescent and some completely lack emissivity. However, the relationship between the structure and excitation dynamics in dipyrrins is still poorly understood. Here, we used a range of spectroscopic methods to investigate the photophysics of Zn(II) complexes of meso-Ar-2,2'-di-tert-butoxycarbonyl-dibenzodipyrrins (BDP; Ar = 4-MeO2C-C6H4). In particular, two-dimensional electronic spectroscopy (2DES) was used to characterize the initial excited states in a homoleptic bis-dipyrrinate Zn(BDP)2, in which two dipyrrin ligands are oriented in a nonorthogonal geometry. From the position of the peaks in the 2DES spectra and spectral modeling, the initial excited states of Zn(BDP)2 were assigned to excitonic states. The low oscillator strength, associated with excitation to the lower excitonic state, is responsible in part for the weak emissivity of Zn(BDP)2, contrasting the bright fluorescence of mono-dippyrinate Zn(BDP)X. Femtosecond (fs-), nanosecond (ns-) transient absorption (TA), and time-resolved fluorescence spectroscopies were used to monitor the solvent-dependent evolution of the excitonic states, which appear to evolve into an intermediate state possibly with charge transfer character. Taken together, our findings reveal a significant impact of both structural and environmental factors on the photophysics of dipyrrins and present the first example of the application of 2DES to investigate excitonic states in a system where the interacting chromophores are held together via coordination of an optically neutral metal ion. On a broader scale, we demonstrate that nonorthogonal bis-dipyrrin complexes constitute a versatile model for studying exciton coupling and associated energy and charge dynamics.

Correction for 'Charge engineering controls cooperative assembly and loading in protein host-guest complexes' by Zhiheng Wang et al., J. Mater. Chem. B, 2025, https://doi.org/10.1039/D5TB01202C.

Voltage-sensitive probes based on donor-acceptor dyads, whose fluorescence is modulated by photoinduced electron transfer (PET) in response to changes in membrane potential, are known as PET molecular wires. PET wires have been widely used in cultured cells; however, their applications in tissue-level imaging have been hampered by their inadequate aqueous solubility, necessitating pre-dissolution in organic solvents that potentially can cause toxic effects. Here we present the synthesis, electronic structure analysis, photophysical characterization, and initial demonstration of water-soluble amphiphilic molecular wires, consisting of a rosamine, as a fluorescent PET acceptor, and a PET donor consisting of a conjugated bridge terminated by dimethylaniline (DMA). The rosamine moiety is extended by several carboxylates or polyethylene glycol (PEG) groups, which render the probes highly water-soluble and facilitate labeling of phospholipid membranes, positioning molecules in proper orientation. The probes produced functional responses in electrically stimulated mouse cardiomyocytes and as well as in intact neurohypophysis, and in glucose-stimulated murine islets of Langerhans. The new molecular wires make up a useful addition to the toolkit of optical reporters for membrane potential imaging.

Previously, we have reported the synthesis and photophysical properties of free-base (H 2 ), Zn(II), Pt(II) and Pd(II) of di-arylphthalimidoporphyrins (DAPIP) and tetra-arylphthalimidoporphyrins (TAPIP), including their multiphoton absorption spectra. Using a combination of phosphorescent Pt complexes of TAPIP and DAPIP we are designing a sensor for pH and oxygen, termed pHOx, which operates by measuring ratios of phosphorescence lifetimes. Unlike all existing pH sensors, measurements by pHOx are not affected by optical heterogeneities of the medium and provide unbiased pH readings in vivo simultaneously with pO 2 . Pd complexes of TAPIP have been shown to emit both phosphorescence and thermally activated E-type delayed fluorescence, providing means for optical sensing of temperature. However, to make the temperature readings unobstructed by tissue optical heterogeneities, a PdTAPIP-based probe is supplemented by a probe system, based on H 2 DAPIP, which emits prompt fluorescence at the same wavelengths and can serve as a correction standard for temperature measurements.

Controlling cargo loading in self-assembling protein capsules remains a key challenge in supramolecular chemistry. Inspired by nature's capacity for host-guest recognition, we engineered supercharged green fluorescent protein (GFP) cargo for controlling its encapsulation by Archaeoglobus fulgidus ferritin. Guided by molecular dynamics (MD) simulations and computational protein design, experiments confirmed that GFP charge magnitude and distribution dictate capsule assembly and loading efficiency. These data provide the first example of cooperative assembly with a ferritin capsule. Finally, we established a strategy for generating stoichiometric 1 : 1 protein host-guest complexes, confirmed by time-resolved fluorescence anisotropy. This provides a blueprint for designing ferritin host-guest complexes with enhanced homogeneity and functionality.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

Three-photon microscopy shows potential to probe much deeper brain structures than previously possible with two-photon microscopy. Here we demonstrate the first intravascular measurements of pO2 in subcortical vasculature in awake mice using three-photon phosphorescence lifetime microscopy (3PLM) and a phosphorescent probe Oxyphor 2P.

Identifying high quality chemical starting points is a critical and challenging step in drug discovery, which typically involves screening large compound libraries or repurposing of compounds with known mechanisms of actions (MoAs). Here we introduce a novel cheminformatics approach that mines existing large-scale, phenotypic high throughput screening (HTS) data. Our method aims to identify bioactive compounds with distinct and specific MoAs, serving as a valuable complement to existing focused library collections. This approach identifies chemotypes with selectivity across multiple cell-based assays and characterized by persistent and broad structure activity relationships (SAR). We prospectively demonstrate the validity of the approach in broad cellular profiling assays (cell painting, DRUG-seq, Promotor Signature Profiling) and chemical proteomics experiments where the compounds behave similarly to known chemogenetic libraries, but with a bias towards novel protein targets and required no synthetic effort to improve compound properties. A public set of such compounds is provided based on the PubChem BioAssay dataset for use by the scientific community.

Pulmonary arterial hypertension (PAH) is a devastating rare disease, which despite currently available treatments, still represents a high unmet medical need. Specific E3 ubiquitin protein ligase 1 (SMURF1) is a HECT E3 ligase that ubiquitinates key signaling molecules from the TGFβ/BMP pathways, which are of great relevance in the pathophysiology of PAH. Herein, the design and synthesis of novel potent small-molecule SMURF1 ligase inhibitors are described. Lead molecule 38 has demonstrated good oral pharmacokinetics in rats and significant efficacy in a rodent model of pulmonary hypertension.

Background: Reduced expression of bone morphogenetic protein receptor 2 (BMPR2) predisposes to pulmonary vascular remodelling and the development of pulmonary arterial hypertension (PAH). The HECT E3 ligase SMURF1 is a key negative regulator of this pathway that is overexpressed in the pulmonary vasculature of patients with PAH. However, the absence of an active-site pocket renders E3s undruggable. Hypothesis: We hypothesized that a large, unbiased screen would be needed to identify SMURF1 inhibitors and that a SMURF1 inhibition would augment BMP signaling and treat experimental PAH. Aims: We aimed to: identify SMURF1 inhibitors through a large, unbiased screen; determine mechanism of action; examine the effects on BMP signalling, cell phenotype and experimental models of PAH. Methods: We constructed a time-resolved fluorescence resonance energy transfer-based assay reporting SMURF1 self-ubiquitylation and undertook a 1.1 million compound screen. Primary hits were rationalised based on biochemical selectivity and cell-based assays designed to prioritize and optimize molecules for specific inhibition of SMURF1. Crystal structure of SMURF1 and SMURF2 were determined in the bound and unbound state at 2.05-2.75 Å. Target interaction was examined in a split-CAT based system. The effect on signaling and phenotype was examined in primary pulmonary artery endothelial and smooth muscle cells, and in rats with PAH induced by monocrotaline and sugen hypoxia. Results: Screening identified lead compounds that selectively inhibited SMURF1. Structures reveal that inhibitor binding induces an α-helix elongation over an invariant glycine-containing hinge, limiting a motion mandatory for catalytic activity. We affirmed this structure-based model with mutants engineered to resist inhibition and demonstrated that inhibition prevents direct BMPR2 ubiquitylation, normalized BMP signaling and restored homeostasis in pulmonary vascular cells from patients with PAH. Finally, we demonstrated that SMURF1 inhibition reverses established, experimental PAH. Conclusions: Our structural understanding enables design of HECT and other glycine-hinge protein inhibitors opening a new druggable space.

PURPOSE:The goal of our study was to characterize the dynamics of intracellular oxygen during application of radiation at conventional (CONV) and FLASH dose rates and obtain evidence for or against the oxygen depletion hypothesis as a mechanism of the FLASH effect. METHODS AND MATERIALS:The measurements were performed by the phosphorescence quenching method using probe Oxyphor PtG4, which was delivered into the cellular cytosol by electroporation. RESULTS:Intracellular radiochemical oxygen depletion (ROD) g-value for a dose rate of 100 Gy/s in the normoxic range was found to be 0.58 ± 0.03 μM/Gy. Intracellular ROD g-values for FLASH and CONV dose rates in the normoxic range were found to be nearly equal. As in solution-based studies, intracellular ROD was found to exhibit strong dependence on oxygen concentration in the range of 0 to ∼40 μM [O2]. CONCLUSIONS:Depletion of oxygen in cells in vitro by a clinical dose of proton radiation delivered as FLASH is unable to produce a transient state of hypoxia and, therefore, unable to induce radioprotection. The difference between ROD g-values for FLASH and CONV dose rates, detected previously in solutions-based experiments, disappears when measurements are conducted inside cells. Understanding this phenomenon should provide additional insight into the role of oxygen in FLASH radiation therapy and help to decipher the mechanism of the FLASH effect.

The ability to quantify partial pressure of oxygen (pO2) is of primary importance for studies of metabolic processes in health and disease. Here, we present a protocol for imaging of oxygen distributions in tissue and vasculature of the cerebral cortex of anesthetized and awake mice. We describe in vivo two-photon phosphorescence lifetime microscopy (2PLM) of oxygen using the probe Oxyphor 2P. This minimally invasive protocol outperforms existing approaches in terms of accuracy, resolution, and imaging depth. For complete details on the use and execution of this protocol, please refer to Esipova et al. (2019).

We will discuss a family of recently developed tetraarylphalimidoporphyrins (TAPIP) and diarylphalimidoporphyrins (DAPIP), which possess unique photophysical properties and suggest new approaches to the design of superior biological sensors for pH and temperature in addition to their already established ability as sensors for oxygen. The synthesis of TAPIP has already been developed by our group previously. Here we present a synthetic route to asymmetric diarylphthalimidoporphyrins (DAPIP) based on [2+2] condensation of the corresponding dipyrromethanes, followed by oxidative aromatization. We report the properties of free-base (H2), Zn(II), Pt(II) and Pd(II) DAPIP's, including their two-photon absorption (2PA) spectra. Using a combination of phosphorescent Pt complexes of TAPIP and DAPIP we are designing a phosphorescent sensor for pH and oxygen, termed pHOx, which operates by measuring ratios of phosphorescence lifetimes. Unlike all existing optical pH sensors, measurements by pHOx are not affected by optical heterogeneities of the medium and provide unbiased pH readings in vivo simultaneously with pO2. At the same time, Pd complexes of TAPIP have been shown to emit both phosphorescence and thermally activated E-type delayed fluorescence, providing a functional element for optical sensing of temperature. However, to make the temperature readings unobstructed by optical heterogeneities of biological tissue, a PdTAPIP-based probe is supplemented by another probe system, based on H2DAPIP, which emits prompt fluorescence at the same wavelengths and can serve as a correction standard for unbiased temperature measurements.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

Aromatically π-extended porphyrins possess exceptionally intense one-photon (1P) and sometimes two-photon (2P) absorption bands, presenting interest for construction of optical imaging probes and photodynamic agents. Here we investigated how breaking the molecular symmetry affects linear and 2PA properties of π-extended porphyrins. First, we developed the synthesis of porphyrins fused with two phthalimide fragments, termed syn-diarylphthalimidoporphyrins (DAPIP). Second, the photophysical properties of H2, Zn, Pd, and Pt DAPIP were measured and compared to those of fully symmetric tetraarylphthalimidoporphyrins (TAPIP). The data were interpreted using DFT/TDDFT calculations and sum-over-states (SOS) formalism. Overall, the picture of 2PA in DAPIP was found to resemble that in centrosymmetric porphyrins, indicating that symmetry breaking, even as significant as by syn-phthalimido-fusion, induces a relatively small perturbation to the porphyrin electronic structure. Collectively, the compact size, versatile synthesis, high 1PA and 2PA cross sections, and bright luminescence make DAPIP valuable chromophores for construction of imaging probes and other bioapplications.

Glycine betaine (GB) is a naturally occurring osmolyte that has been widely recognized as a protein protectant. Since GB consists of a methylated ammonium moiety, it can engage in strong cation-π interactions with aromatic amino acid sidechains. We hypothesize that such specific binding interactions would allow GB to decrease the stability of proteins that are predominantly stabilized by a cluster of aromatic amino acids. To test this hypothesis, we investigate the effect of GB on the stability of two β-hairpins (or mini-proteins) that contain such a cluster. We find that for both systems the stability of the folded state first decreases and then increases with increasing GB concentration. Such non-monotonic dependence not only confirms that GB can act as a protein denaturant, but also underscores the complex interplay between GB's stabilizing and destabilizing forces toward a given protein. While stabilizing osmolytes all have the tendency to be excluded from the protein surface which is the action underlying their stabilizing effect, our results suggest that in order to quantitatively assess the effect of GB on the stability of any given protein, specific cation-π binding interactions need to be explicitly considered. Moreover, our results show, consistent with other studies, that cation methylation can strengthen the respective cation-π interactions. Taken together, these findings provide new insight into the mechanism by which amino acid-based osmolytes interact with proteins.

Histamine H3 receptor (H3R) inverse agonists that have been in clinical trials for the treatment of excessive sleep disorders, have been plagued with insomnia as a mechanism-based side effect. We focused on the identification of compounds that achieve high receptor occupancy within a short time, followed by rapid disengagement from the receptor, a target profile that could provide therapeutic benefits without the undesired side effect of insomnia. This article describes the optimization work that led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18 b, LML134).