Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements.

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

Expanding the scope of problem-based-learning at Hackensack Meridian School of Medicine; integrating domain-general skills with domain-specific content.

The Master Adaptive Learner is a model used to develop students to become self-regulated and adaptable lifelong learners to practice medicine in a complex and ever-changing environment. The Hackensack Meridian School of Medicine (HMSOM) proposes a new course, Patient Presentation Problem-Based Learning Curriculum (PPPC), a dynamic and integrated course that goes beyond the scope of traditional Problem-Based-Learning (PBL). PPPC allows students to build domain-general skills in tandem with domain-specific content learned during a pre-clerkship curriculum. An integrated case provides weekly scaffolding, such that the course takes place throughout the week and is not isolated from the rest of the curriculum. Students receive iterative feedback and structured assignments which allows development of self-directed learning skills along with integration and consolidation of weekly curricular content. A layered analysis approach was used to outline the philosophies, principles and techniques that link to our course objectives. Techniques used could easily be translated to other pre-clerkship curriculum to promote development of self-directed learning and clinical reasoning skills, as well as promote more meaningful learning of basic, clinical, and health system science content.

Introducing Life-long Learning Skills in a Patient Presentation Problem-Based Curriculum: The Case for Librarian Integration.

Medical students must learn how to find, critically appraise, and apply high-quality information to support their clinical decisions. To reinforce these skills, the Hackensack Meridian School of Medicine embedded medical librarians into a longitudinal case-based, problem-based learning curriculum, where they provide individualized feedback on student's skills in this area.

Understanding Carotenoid Dynamics via the Vibronic Energy Relaxation Approach.

Carotenoids are an integral part of natural photosynthetic complexes, with tasks ranging from light harvesting to photoprotection. Their underlying energy deactivation network of optically dark and bright excited states is extremely efficient: after excitation of light with up to 2.5 eV of photon energy, the system relaxes back to ground state on a time scale of a few picoseconds. In this article, we summarize how a model based on the vibrational energy relaxation approach (VERA) explains the main characteristics of relaxation dynamics after one-photon excitation with special emphasis on the so-called S* state. Lineshapes after two-photon excitation are beyond the current model of VERA. We outline this future line of research in our article. In terms of experimental method development, we discuss which techniques are needed to better describe energy dissipation effects in carotenoids and within the first solvation shell.

Ultrafast energy transfer between lipid-linked chromophores and plant light-harvesting complex II.

Light-Harvesting Complex II (LHCII) is a membrane protein found in plant chloroplasts that has the crucial role of absorbing solar energy and subsequently performing excitation energy transfer to the reaction centre subunits of Photosystem II. LHCII provides strong absorption of blue and red light, however, it has minimal absorption in the green spectral region where solar irradiance is maximal. In a recent proof-of-principle study, we enhanced the absorption in this spectral range by developing a biohybrid system where LHCII proteins together with lipid-linked Texas Red (TR) chromophores were assembled into lipid membrane vesicles. The utility of these systems was limited by significant LHCII quenching due to protein-protein interactions and heterogeneous lipid structures. Here, we organise TR and LHCII into a lipid nanodisc, which provides a homogeneous, well-controlled platform to study the interactions between TR molecules and single LHCII complexes. Fluorescence spectroscopy determined that TR-to-LHCII energy transfer has an efficiency of at least 60%, resulting in a 262% enhancement of LHCII fluorescence in the 525-625 nm range, two-fold greater than in the previous system. Ultrafast transient absorption spectroscopy revealed two time constants of 3.7 and 128 ps for TR-to-LHCII energy transfer. Structural modelling and theoretical calculations indicate that these timescales correspond to TR-lipids that are loosely- or tightly-associated with the protein, respectively, with estimated TR-to-LHCII separations of ∼3.5 nm and ∼1 nm. Overall, we demonstrate that a nanodisc-based biohybrid system provides an idealised platform to explore the photophysical interactions between extrinsic chromophores and membrane proteins with potential applications in understanding more complex natural or artificial photosynthetic systems.

Medical Students on the Virtual Front Line: A Literature Review Elective to Provide COVID-19 Clinical Teams With Essential Information.

PROBLEM: At the Hackensack Meridian School of Medicine (HMSOM) in New Jersey, clinical activities for students were suspended on March 15, 2020, due to the COVID-19 pandemic. Clinical teams at Hackensack Meridian Health (HMH) needed resources for identifying and assimilating the medical literature regarding COVID-19, which was expanding and evolving daily. HMH leaders reached out to HMSOM leaders for assistance. The HMSOM leadership and faculty quickly organized a literature review elective. APPROACH: Eight second-year medical students participated in a literature review elective course to research and synthesize the COVID-19 clinical literature to provide synopses of best practices for various clinical teams. By March 23, students were searching the literature and writing reports independently, mentored by a senior dean (an infectious diseases specialist) and supported by the associate dean of libraries and library team. The library team updated and categorized student reports daily on a website dedicated to the elective. OUTCOMES: During the 6-week elective, 8 students produced 70 reports synthesizing the emerging COVID-19 literature to help answer practitioners' clinical questions in real time. One student report was posted on the American Academy of Ophthalmology website. All 70 were published online in Elsevier's health education faculty hub. On course evaluations, students expressed regret about not being directly involved in patient care but articulated their gratitude to be able to contribute to the clinical teams. NEXT STEPS: In June 2020, the students returned to their clinical clerkships as COVID-19 clinical volumes declined and personal protective equipment became more available. Students continued to be available to the clinical teams to assist with COVID-19 questions. This literature review elective can serve as a model for other medical schools to use to deploy students to help synthesize the evolving literature on COVID-19 or other rapidly emerging research topics.

Trivial Excitation Energy Transfer to Carotenoids Is an Unlikely Mechanism for Non-photochemical Quenching in LHCII.

Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favors excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S 1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S 1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD) trajectories about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely its linear absorption (LA), transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII free energy surface. We show that trivial, Coulomb-mediated energy transfer to S 1 is an unlikely quenching mechanism, with pigment movements insufficiently pronounced to switch the system between quenched and unquenched states. Modulation of S 1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is possibly an artifact of quantum chemical over-estimation of S 1 oscillator strength and the real mechanism likely involves short-range interaction and/or non-trivial inter-molecular states.

A Protein Environment-Modulated Energy Dissipation Channel in LHCII Antenna Complex.

The major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (non-photochemical quenching, NPQ). In this work, we locked single LHCII trimers in a quenched conformation after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.

Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions.

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

Excitation quenching in chlorophyll-carotenoid antenna systems: 'coherent' or 'incoherent'.

Plants possess an essential ability to rapidly down-regulate light-harvesting in response to high light. This photoprotective process involves the formation of energy-quenching interactions between the chlorophyll and carotenoid pigments within the antenna of Photosystem II (PSII). The nature of these interactions is currently debated, with, among others, 'incoherent' or 'coherent' quenching models (or a combination of the two) suggested by a range of time-resolved spectroscopic measurements. In 'incoherent quenching', energy is transferred from a chlorophyll to a carotenoid and is dissipated due to the intrinsically short excitation lifetime of the latter. 'Coherent quenching' would arise from the quantum mechanical mixing of chlorophyll and carotenoid excited state properties, leading to a reduction in chlorophyll excitation lifetime. The key parameters are the energy gap, [Formula: see text] and the resonance coupling, J, between the two excited states. Coherent quenching will be the dominant process when [Formula: see text] i.e., when the two molecules are resonant, while the quenching will be largely incoherent when [Formula: see text] One would expect quenching to be energetically unfavorable for [Formula: see text] The actual dynamics of quenching lie somewhere between these limiting regimes and have non-trivial dependencies of both J and [Formula: see text] Using the Hierarchical Equation of Motion (HEOM) formalism we present a detailed theoretical examination of these excitation dynamics and their dependence on slow variations in J and [Formula: see text] We first consider an isolated chlorophyll-carotenoid dimer before embedding it within a PSII antenna sub-unit (LHCII). We show that neither energy transfer, nor the mixing of excited state lifetimes represent unique or necessary pathways for quenching and in fact discussing them as distinct quenching mechanisms is misleading. However, we do show that quenching cannot be switched 'on' and 'off' by fine tuning of [Formula: see text] around the resonance point, [Formula: see text] Due to the large reorganization energy of the carotenoid excited state, we find that the presence (or absence) of coherent interactions have almost no impact of the dynamics of quenching. Counter-intuitively significant quenching is present even when the carotenoid excited state lies above that of the chlorophyll. We also show that, above a rather small threshold value of [Formula: see text]quenching becomes less and less sensitive to J (since in the window [Formula: see text] the overall lifetime is independent of it). The requirement for quenching appear to be only that [Formula: see text] Although the coherent/incoherent character of the quenching can vary, the overall kinetics are likely robust with respect to fluctuations in J and [Formula: see text] This may be the basis for previous observations of NPQ with both coherent and incoherent features.

How carotenoid distortions may determine optical properties: lessons from the Orange Carotenoid Protein.

Carotenoids in photosynthetic proteins carry out the dual function of harvesting light and defending against photo-damage by quenching excess energy. The latter involves the low-lying, dark, excited state labelled S1. Here "dark" means optically-forbidden, a property that is often attributed to molecular symmetry, which leads to speculation that its optical properties may be strongly-perturbed by structural distortions. This has been both explicitly and implicitly proposed as an important feature of photo-protective energy quenching. Here we present a theoretical analysis of the relationship between structural distortions and S1 optical properties. We outline how S1 is dark not because of overall geometric symmetry but because of a topological symmetry related to bond length alternation in the conjugated backbone. Taking the carotenoid echinenone as an example and using a combination of molecular dynamics, quantum chemistry, and the theory of spectral lineshapes, we show that distortions that break this symmetry are extremely stiff. They are therefore absent in solution and only marginally present in even a very highly-distorted protein binding pocket such as in the Orange Carotenoid Protein (OCP). S1 remains resolutely optically-forbidden despite any breaking of bulk molecular symmetry by the protein environment. However, rotations of partially conjugated end-rings can result in fine tuning of the S1 transition density which may exert some influence on interactions with neighbouring chromophores.

Structural Basis for Allosteric Regulation in the Major Antenna Trimer of Photosystem II.

The allosteric regulation of protein function proves important in many life-sustaining processes. In plant photosynthesis, LHCII, the major antenna complex of Photosystem II, employs a delicate switch between light harvesting and photoprotective modes. The switch is triggered by an enlarged pH gradient (ΔpH) across the thylakoid membranes. Using molecular simulations and quantum calculations, we show that ΔpH can tune the light-harvesting potential of the antenna via allosteric regulation of the excitonic coupling in chlorophyll-carotenoid pairs. To this end, we propose how the LHCII excited state lifetime is coupled to the environmental conditions. In line with experimental findings, our theoretical model provides crucial evidence toward the elucidation of the photoprotective switch of higher plants at an all-atom resolution.

Research questions to facilitate the future development of European long-term ecosystem research infrastructures: A horizon scanning exercise.

Distributed environmental research infrastructures are important to support assessments of the effects of global change on landscapes, ecosystems and society. These infrastructures need to provide continuity to address long-term change, yet be flexible enough to respond to rapid societal and technological developments that modify research priorities. We used a horizon scanning exercise to identify and prioritize emerging research questions for the future development of ecosystem and socio-ecological research infrastructures in Europe. Twenty research questions covered topics related to (i) ecosystem structures and processes, (ii) the impacts of anthropogenic drivers on ecosystems, (iii) ecosystem services and socio-ecological systems and (iv), methods and research infrastructures. Several key priorities for the development of research infrastructures emerged. Addressing complex environmental issues requires the adoption of a whole-system approach, achieved through integration of biotic, abiotic and socio-economic measurements. Interoperability among different research infrastructures needs to be improved by developing standard measurements, harmonizing methods, and establishing capacities and tools for data integration, processing, storage and analysis. Future research infrastructures should support a range of methodological approaches including observation, experiments and modelling. They should also have flexibility to respond to new requirements, for example by adjusting the spatio-temporal design of measurements. When new methods are introduced, compatibility with important long-term data series must be ensured. Finally, indicators, tools, and transdisciplinary approaches to identify, quantify and value ecosystem services across spatial scales and domains need to be advanced.

The full dynamics of energy relaxation in large organic molecules: from photo-excitation to solvent heating.

In some molecular systems, such as nucleobases, polyenes or the active ingredients of sunscreens, substantial amounts of photo-excitation energy are dissipated on a sub-picosecond time scale, raising questions such as: where does this energy go or among which degrees of freedom it is being distributed at such early times? Here we use transient absorption spectroscopy to track excitation energy dispersing from the optically accessible vibronic subsystem into the remaining vibrational subsystem of the solute and solvent. Monitoring the flow of energy during vibrational redistribution enables quantification of local molecular heating. Subsequent heat dissipation away from the solute molecule is characterized by classical thermodynamics and molecular dynamics simulations. Hence, we present a holistic approach that tracks the internal temperature and vibronic distribution from the act of photo-excitation to the restoration of the global equilibrium. Within this framework internal vibrational redistribution and vibrational cooling are emergent phenomena. We demonstrate the validity of the framework by examining a highly controversial example, carotenoids. We show that correctly accounting for the local temperature unambiguously explains their energetically and temporally congested spectral dynamics without the ad hoc postulation of additional 'dark' states. An immediate further application of this approach would be to monitor the excitation and thermal dynamics of pigment-protein systems.

A possible molecular basis for photoprotection in the minor antenna proteins of plants.

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals.

Fine control of chlorophyll-carotenoid interactions defines the functionality of light-harvesting proteins in plants.

Photosynthetic antenna proteins can be thought of as "programmed solvents", which bind pigments at specific mutual orientations, thus tuning the overall energetic landscape and ensuring highly efficient light-harvesting. While positioning of chlorophyll cofactors is well understood and rationalized by the principle of an "energy funnel", the carotenoids still pose many open questions. Particularly, their short excited state lifetime (<25 ps) renders them potential energy sinks able to compete with the reaction centers and drastically undermine light-harvesting efficiency. Exploration of the orientational phase-space revealed that the placement of central carotenoids minimizes their interaction with the nearest chlorophylls in the plant antenna complexes LHCII, CP26, CP29 and LHCI. At the same time we show that this interaction is highly sensitive to structural perturbations, which has a profound effect on the overall lifetime of the complex. This links the protein dynamics to the light-harvesting regulation in plants by the carotenoids.

New Paths to Professional Nursing: Using Encouragement to Prepare a Minority Workforce to Enter the Nursing Profession.

The current professional nursing workforce in the United States is predominantly White and female, even though minorities compose 33% of the national population (Travers, Smaldone, & Cohn, 2015). Minority patients are more effectively cared for when their particular cultural milieu is taken into consideration as part of their health care plan (Sullivan, 2004). According to the Agency for Healthcare Research and Quality (AHRQ), health care quality remains suboptimal for diverse populations in the United States because some individuals do not receive quality care or do not believe their values are honored or respected (AHRQ, 2016). Minority professional nurses are necessary to address the racial and ethnic disparities in health care. Robert Wood Johnson Barnabas Health (RWJBH), in collaboration with Rutgers University School of Nursing (RUSON), implemented New Paths to Professional Nursing (NPPN) to increase the number of minority professional nurses in practice at RWJBH. The program provided financial resources as well as infrastructural, group, and personal support for RWJBH minority employees who desired to complete prerequisites to enter RUSON. The academic success of the employees who participated in NPPN was attributed to a unique combination of financial assistance and support and encouragement. The purpose of this article is to describe in detail the development of the program and the effective encouragement strategies that have led to success for NPPN minority student/employees. This article examines, defines, and illustrates particular types of effective encouragement and suggests that this encouragement was the key to success for the NPPN minority students.

The carotenoid pathway: what is important for excitation quenching in plant antenna complexes?

Plant light-harvesting is regulated by the Non-Photochemical Quenching (NPQ) mechanism involving the reversible formation of excitation quenching sites in the Photosystem II (PSII) antenna in response to high light. While the major antenna complex, LHCII, is known to be a site of NPQ, the precise mechanism of excitation quenching is not clearly understood. A preliminary model of the quenched crystal structure of LHCII implied that quenching arises from slow energy capture by Car pigments. It predicted a thoroughly quenched system but offered little insight into the defining aspects of this quenching. In this work, we present a thorough theoretical investigation of this quenching, addressing the factors defining the quenching pathway and possible mechanism for its (de)activation. We show that quenching in LHCII crystals is the result of slow energy transfer from chlorophyll to the centrally-bound lutein Cars, predominantly the Lut620 associated with the chlorophyll 'terminal emitter', one of the proposed in vivo pathways. We show that this quenching is rather independent of the particular species of Car and excitation 'site' energy. The defining parameter is the resonant coupling between the pigment co-factors. Lastly, we show that these interactions must be severely suppressed for a light-harvesting state to be recovered.