Psychiatric diagnoses are common after liver transplantation and are associated with increased health care utilization and patient financial burden.

Psychiatric disorders after liver transplantation (LT) are associated with worse patient and graft outcomes, which may be amplified by inadequate treatment. We aimed to characterize the burden of psychiatric disorders, treatment patterns, and associated financial burden among liver transplantation recipients (LTRs). IQVIA PharMetrics (R) Plus for Academics-a large health plan claims database representative of the commercially insured US population-was used to identify psychiatric diagnoses among adult LTRs and assess treatment. Multivariable logistic regression analysis identified factors associated with post-LT psychiatric diagnoses and receipt of pharmacotherapy. Patient financial liability was estimated using adjudicated medical/pharmacy claims for LTRs with and without psychiatric diagnoses. Post-LT psychiatric diagnoses were identified in 395 (29.5%) of 1338 LTRs, of which 106 (26.8%) were incident cases. Treatment varied, with 67.3% receiving pharmacotherapy, 32.1% psychotherapy, 21.0% combination therapy, and 21.5% no treatment. Among 340 LTRs on psychotropic medications before transplant, 24% did not continue them post-LT. Post-LT psychiatric diagnoses were independently associated with female sex, alcohol-associated liver disease (ALD), prolonged LT hospitalization (>2 wk), and pre-LT psychiatric diagnosis. Incident psychiatric diagnoses were associated with female sex, ALD, and prolonged LT hospitalization. Patients with a post-LT psychiatric diagnosis had higher rates of hospitalization (89.6% vs. 81.5%, p <0.001) and financial liability (median $5.5K vs. $4.6K USD, p =0.006). Having a psychiatric diagnosis post-LT was independently associated with experiencing high financial liability >$5K. Over 1 in 4 LTRs had a psychiatric diagnosis in a large national cohort, yet nearly a quarter received no treatment. LTRs with psychiatric diagnoses experienced increased health care utilization and higher financial liability. Sociodemographic and clinical risk factors could inform high-risk subgroups who may benefit from screening and mitigation strategies.

The financial burden after liver transplantation is significant among commercially insured adults: A large US National Cohort.

Liver transplantation (LT) is lifesaving for patients with cirrhosis; however, the resultant financial burden to patients has not been well characterized. We aimed to provide a nationally representative portrayal of patient financial burden after LT. Adult recipients of LT from 2006 to 2021 were identified using IQVIA PharMetrics® Plus for Academics-a large nationally representative claims database of commercially insured Americans. Patient financial liability (ie, what patients owe) was estimated using the difference between allowed and paid costs for adjudicated medical/pharmacy claims. Descriptive statistics were provided stratified by the financial liability group within 1 year after LT. Multivariable logistic regression modeling identified factors associated with high/extreme liability adjusting for covariates. Potential indirect costs of post-LT care were estimated based on hourly wages lost for care. Among 1412 recipients of LT, financial liability was heterogeneous-~3% had no liability and 21% had extreme liability > $10K for 1-year post-LT care; most (69%) paid between $1 and 10K, with 48% having liability >$5K. Factors associated with >$5K liability included older age, insurance/enrollment type, US region, history of HCC, and simultaneous liver-kidney transplant (for liability >$10K). Medication costs comprised ~30% of outpatient financial liability. Potential indirect costs from wages lost were $2,201-$6,073 per person, depending on an hourly wage. In a large national cohort of commercially insured recipients of LT, financial liability was highly variable across sociodemographic and clinical characteristics; nearly 1 out of 2 recipients of LT owed >$5K for 1 year of post-LT care. Transplant programs should help patients anticipate potential costs and identify vulnerable populations who would benefit from enhanced financial counseling.

Tuning of B12 photochemistry in the CarH photoreceptor to avoid radical photoproducts.

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Highlighter: An optogenetic system for high-resolution gene expression control in plants.

Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to function in the context of horticultural light environments. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter, a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system. Analysis of a re-engineered CcaS in Escherichia coli demonstrated green/red photoswitching with phytochromobilin, a chromophore endogenous to plants, but also revealed a blue light response likely derived from a flavin-binding LOV-like domain. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We implemented the system to demonstrate optogenetic control over plant immunity and pigment production through modulation of the spectral composition of broadband visible (white) light. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.

Essential elements of radical pair magnetosensitivity in Drosophila.

Many animals use Earth's magnetic field (also known as the geomagnetic field) for navigation1. The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field2. However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations2-8. Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.

Investigating radical pair reaction dynamics of B12 coenzymes 1: Transient absorption spectroscopy and magnetic field effects.

B12 coenzymes are vital to healthy biological function across nature. They undergo radical chemistry in a variety of contexts, where spin-correlated radical pairs can be generated both thermally and photochemically. Owing to the unusual magnetic properties of B12 radical pairs, however, most of the reaction and spin dynamics occur on a timescale (picoseconds-nanoseconds) that cannot be resolved by most measurement techniques. Here, we describe a method that combines femtosecond transient absorption spectroscopy with magnetic field exposure, which enables the direct scrutiny of such rapid processes. This approach should provide a means by which to investigate the apparently profound effect protein environments have on the generation and reactivity of B12 radical pairs.

Investigating radical pair reaction dynamics of B12 coenzymes 2: Time-resolved electron paramagnetic resonance spectroscopy.

The chemistry of B12 coenzymes is highly sensitive to the nature of their upper axial ligand and can be further tuned by their environment. Methylcobalamin, for example, generates RPs photochemically but undergoes non-radical biochemistry when bound to its dependent enzymes. Owing to the transient nature of the reaction intermediates, it remains a challenge to investigate how their environment controls reactivity. Here, we describe how to use time-resolved electron paramagnetic spectroscopy to directly monitor the generation and evolution of transient radicals that result from the photolysis of a B12 coenzyme. This method produces evolving, spin-polarized spectra that are rich in mechanistic detail.

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Histologic evaluation of therapeutic responses in ischemic myocardium elicited by dual growth factor delivery from composite glycosaminoglycan hydrogels.

In this project, the ability of dual growth factor-preloaded, silk-reinforced, composite hyaluronic acid-based hydrogels to elicit advantageous histologic responses when secured to ischemic myocardium was evaluated in vivo. Reinforced hydrogels containing both Vascular Endothelial Growth Factor (VEGF) and Platelet-derived Growth Factor (PDGF) were prepared by crosslinking chemically modified hyaluronic acid and heparin with poly(ethylene glycol)-diacrylate around a reinforcing silk mesh. Composite patches were sutured to the ventricular surface of ischemic myocardium in Sprague-Dawley rats, and the resulting angiogenic response was followed for 28 days. The gross appearance of treated hearts showed significantly reduced ischemic area and fibrous deposition compared to untreated control hearts. Histologic evaluation showed growth factor delivery to restore myofiber orientation to pre-surgical levels and to significantly increase elicited microvessel density and maturity by day 28 in infarcted myocardial tissue (p < 0.05). In addition, growth factor delivery reduced cell apoptosis and decreased the density of elicited mast cells and both CD68+ and anti-inflammatory CD163+ macrophages. These findings suggest that HA-based, dual growth factor-loaded hydrogels can successfully induce a series of beneficial responses in ischemic myocardium, and offer the potential for therapeutic improvement of ischemic myocardial remodeling.

Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems.

The use of organic based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limits their effectiveness and longevity bothin vitroandin vivo. As an alternative, lanthanide doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and RET sensing; they exhibit high photo- and chemical stability and utilise near infrared excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd2SO4:Yb:Er (PTIR545) and Gd2SO4:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface-accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide-thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.

Assessing the Covalent Attachment and Energy Transfer Capabilities of Upconverting Phosphors With Cofactor Containing Bioactive Enzymes.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Examples of biosensors utilizing the apparent energy transfer of UCPs and nanophosphors (UCNPs) with biomolecules have started to appear in the literature but very few exploit the covalent anchoring of the biomolecule to the surface of the UCP to improve the sensitivity of the systems. Here, we demonstrate a robust and versatile method for the covalent attachment of biomolecules to the surface of a variety of UCPs and UCNPs in which the UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate biomolecules with surface-accessible cysteines. Variants of BM3Heme, cytochrome C, glucose oxidase, and glutathione reductase were then attached via maleimide-thiol coupling. BM3Heme, glucose oxidase, and glutathione reductase were shown to retain their activity when coupled to the UCPs potentially opening up opportunities for biosensing applications.

Observation of the Δg mechanism resulting from the ultrafast spin dynamics that follow the photolysis of coenzyme B12.

Throughout nature, both free radicals and transient radical reaction intermediates are vital to many biological functions. Coenzyme B12 is a case in point. This organometallic cofactor generates a radical pair upon activation in its dependent enzymes by substrate binding and following photolysis. The resulting cob(ii)alamin/5'-deoxyadenosyl radical pair has unusual magnetic properties that present a challenge to detailed investigation at ambient temperatures. Here, we use femtosecond transient absorption spectroscopy adapted for magnetic field exposure to reveal that the spin dynamics of the B12 radical pair are sufficiently fast for magnetic field effects to be observed on the ultrafast reaction kinetics. Moreover, the large difference in g-values between the radicals of the pair means that effects of the Δg mechanism are observed for the first time for a radical pair system exposed to magnetic fields below 1 T. Spin dynamic simulations allow a value of the cob(ii)alamin radical g-value (2.105) at ambient temperature to be extracted and, because the spin dynamic time scale is faster than the diffusional rotation of the cob(ii)alamin radical, the observed value corresponds to the anisotropic g|| value for this radical.

Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems.

The use of organic-based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic-based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limit their effectiveness and longevity both in vitro and in vivo. As an alternative, lanthanide-doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and FRET sensing; they exhibit high photo- and chemical stability and utilise near infrared (nIR) excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd2SO4:Yb:Er (PTIR545) and Gd2SO4:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

Photochemical Spin Dynamics of the Vitamin B12 Derivative, Methylcobalamin.

Derivatives of vitamin B12 are six-coordinate cobalt corrinoids found in humans, other animals, and microorganisms. By acting as enzymatic cofactors and photoreceptor chromophores, they serve vital metabolic and photoprotective functions. Depending on the context, the chemical mechanisms of the biologically active derivatives of B12-methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl)-can be very different from one another. The extent to which this chemistry is tuned by the upper axial ligand, however, is not yet clear. Here, we have used a combination of time-resolved Fourier transform-electron paramagnetic resonance (FT-EPR), magnetic field effect experiments, and spin dynamic simulations to reveal that the upper axial ligand alone only results in relatively minor changes to the photochemical spin dynamics of B12. By studying the photolysis of MeCbl, we find that, similar to AdoCbl, the initial (or "geminate") radical pairs (RPs) are born predominantly in the singlet spin state and thus originate from singlet excited-state precursors. This is in contrast to the triplet RPs and precursors proposed previously. Unlike AdoCbl, the extent of geminate recombination is limited following MeCbl photolysis, resulting in significant distortions to the FT-EPR signal caused by polarization from spin-correlated methyl-methyl radical "f-pairs" formed following rapid diffusion. Despite the photophysical mechanism that precedes photolysis of MeCbl showing wavelength dependence, the subsequent spin dynamics appear to be largely independent of excitation wavelength, again similar to AdoCbl. Our data finally provide clarity to what in the literature to date has been a confused and contradictory picture. We conclude that, although the upper axial position of MeCbl and AdoCbl does impact their reactivity to some extent, the remarkable biochemical diversity of these fascinating molecules is most likely a result of tuning by their protein environment.

Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor.

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a ß-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.