Models of Photoreceptor Degeneration in Adult Zebrafish.

Zebrafish maintain a remarkable ability to regenerate their neural retina following rapid and extensive loss of retinal neurons. This is mediated by Müller glial cells (MG), which re-enter the cell cycle to produce amplifying progenitor cells that eventually differentiate into the lost retinal neurons. For example, exposing adult albino zebrafish to intense light destroys large numbers of rod and cone photoreceptors, which are then restored by MG-mediated regeneration. Here, we describe an updated method for performing these acute phototoxic lesions to adult zebrafish retinas. Next, we contrast this method to a chronic, low light lesion model that results in a more muted and sustained damage to photoreceptors and does not trigger a MG-mediated regeneration response. Thus, these two methods can be used to compare and contrast the genetic and morphological changes associated with acute and chronic methods of photoreceptor degeneration.

Efficient photodegradation of perfluoroalkyl substances under visible light by hexagonal ZnIn2S4 nanosheets.

Perfluoroalkyl substances (PFASs) are typical persistent organic pollutants, and their removal is urgently required but challenging. Photocatalysis has shown potential in PFASs degradation due to the redox capabilities of photoinduced charge carriers in photocatalysts. Herein, hexagonal ZnIn2S4 (ZIS) nanosheets were synthesized by a one-pot oil bath method and were well characterized by a series of techniques. In the degradation of sodium p-perfluorous nonenoxybenzenesulfonate (OBS), one kind of representative PFASs, the as-synthesized ZIS showed activity superior to P25 TiO2 under both simulated sunlight and visible-light irradiation. The good photocatalytic performance was attributed to the enhanced light absorption and facilitated charge separation. The pH conditions were found crucial in the photocatalytic process by influencing the OBS adsorption on the ZIS surface. Photogenerated e- and h+ were the main active species involved in OBS degradation in the ZIS system. This work confirmed the feasibility and could provide mechanistic insights into the degradation and defluorination of PFASs by visible-light photocatalysis.

Red light-induced localized release of carbon monoxide for alleviating postoperative cognitive dysfunction.

Inflammation within the central nervous system (CNS), which may be triggered by surgical trauma, has been implicated as a significant factor contributing to postoperative cognitive dysfunction (POCD). The relationship between mitigating inflammation at peripheral surgical sites and its potential to attenuate the CNS inflammatory response, thereby easing POCD symptoms, remains uncertain. Notably, carbon monoxide (CO), a gasotransmitter, exhibits pronounced anti-inflammatory effects. Herein, we have developed carbon monoxide-releasing micelles (CORMs), a nanoparticle that safely and locally liberates CO upon exposure to 650 nm light irradiation. In a POCD mouse model, treatment with CORMs activated by light (CORMs + hv) markedly reduced the concentrations of interleukin (IL)-6, IL-1ß, and tumor necrosis factor-alpha (TNF-α) in both the peripheral blood and the hippocampus, alongside a decrease in ionized calcium-binding adapter molecule 1 in the hippocampal CA1 region. Furthermore, CORMs + hv treatment diminished Evans blue extravasation, augmented the expression of tight junction proteins zonula occludens-1 and occludin, enhanced neurocognitive functions, and fostered fracture healing. Bioinformatics analysis and experimental validation has identified Htr1b and Trhr as potential key regulators in the neuroactive ligand-receptor interaction signaling pathway implicated in POCD. This work offers new perspectives on the mechanisms driving POCD and avenues for therapeutic intervention.

Tumor microenvironment-modulated nanoparticles with cascade energy transfer as internal light sources for photodynamic therapy of deep-seated tumors.

Photodynamic therapy (PDT) is an appealing modality for cancer treatments. However, the limited tissue penetration depth of external-excitation light makes PDT impossible in treating deep-seated tumors. Meanwhile, tumor hypoxia and intracellular reductive microenvironment restrain the generation of reactive oxygen species (ROS). To overcome these limitations, a tumor-targeted self-illuminating supramolecular nanoparticle T-NPCe6-L-N is proposed by integrating photosensitizer Ce6 with luminol and nitric oxide (NO) for chemiluminescence resonance energy transfer (CRET)-activated PDT. The high H2O2 level in tumor can trigger chemiluminescence of luminol to realize CRET-activated PDT without exposure of external light. Meanwhile, the released NO significantly relieves tumor hypoxia via vascular normalization and reduces intracellular reductive GSH level, further enhancing ROS abundance. Importantly, due to the different ROS levels between cancer cells and normal cells, T-NPCe6-L-N can selectively trigger PDT in cancer cells while sparing normal cells, which ensured low side effect. The combination of CRET-based photosensitizer-activation and tumor microenvironment modulation overcomes the innate challenges of conventional PDT, demonstrating efficient inhibition of orthotopic and metastatic tumors on mice. It also provoked potent immunogenic cell death to ensure long-term suppression effects. The proof-of-concept research proved as a new strategy to solve the dilemma of PDT in treatment of deep-seated tumors.

The effects of emotional arousal on pupil size depend on luminance.

Pupillometry is widely used to measure arousal states. The primary functional role of the pupil, however, is to respond to the luminance of visual inputs. We previously demonstrated that cognitive effort-related arousal interacted multiplicatively with luminance, with the strongest pupillary effects of arousal occurring at low-to-mid luminances (< 37 cd/m2), implying a narrow range of conditions ideal for assessing cognitive arousal-driven pupillary differences. Does this generalize to other forms of arousal? To answer this, we assessed luminance-driven pupillary response functions while manipulating emotional arousal, using well-established visual and auditory stimulus sets. At the group level, emotional arousal interacted with the pupillary light response differently from cognitive arousal: the effects occurred primarily at much lower luminances (< 20 cd/m2). Analyses at the individual-participant level revealed qualitatively distinct patterns of modulation, with a sizable number of individuals displaying no arousal response to the visual or auditory stimuli, regardless of luminance. Together, our results suggest that effects of arousal on pupil size are not monolithic: different forms of arousal exert different patterns of effects. More practically, our findings suggest that lower luminances create better conditions for measuring pupil-linked arousal, and when selecting ambient luminance levels, consideration of the arousal manipulation and individual differences is critical.

Spectral transmission of commercially available high-energy visible light-filtering spectacle lenses.

SIGNIFICANCE: High-energy visible (HEV) light-filtering spectacle lenses are being widely promoted despite limited evidence to support their use. The spectral transmission properties of commercially available lenses varied, particularly in the HEV light region, and they do not perform in the same way for transmission of ultraviolet (UV)-A and HEV light. PURPOSE: Although HEV light is important for several visual and nonvisual functions, there are concerns over the potential adverse effects of increased HEV light exposure. High-energy visible light-filtering spectacle lenses are being widely marketed to promote ocular health and improve sleep by reducing exposure to HEV light. This study reports on the spectral transmission characteristics of commercially available HEV light-filtering spectacle lenses based on the recommendations of the Spectral Bands Task Force technical report. METHODS: The spectral transmission of light through nine afocal plastic lenses, including eight commercially available HEV light-filtering lenses and one clear uncoated control lens, was evaluated using a Cary 5000 UV-Vis-NIR spectrophotometer (Agilent Technologies, Johannesburg, South Africa) for wavelengths 250 to 780 nm. The percentage transmission values are reported for UV radiation and visible light, with emphasis for HEV light (380 to 500 nm) and the three subbands therein. RESULTS: All lenses blocked UV-C and UV-B radiation (250 to 315 nm). For UV-A radiation (315 to 380 nm), six lenses showed optimal 100% absorption, whereas three lenses allowed ≤12%. The transmission values for the HEV light-filtering lenses ranged from 55 to 90% and 75 to 95% for HEV light of wavelengths 400 to 455 nm and 455 to 500 nm, respectively. In contrast, the control lens showed 92 (400 to 455 nm) and 93% (455 to 500 nm) transmission. CONCLUSIONS: The HEV light-filtering spectacle lenses varied in their transmission properties and do not necessarily perform in the same way for transmission of UV-A and HEV light. Optometric personnel should consider these transmission properties, particularly for HEV light, when making recommendations to patients about HEV light-filtering spectacle lenses.

Exploring effects of light intensity on sustainability indicators in finishing pig production.

With an ongoing transition towards the use of Light Emitting Diodes, more knowledge is needed on which light settings optimise sustainability parameters in pig production. We studied the effects of four light intensities on social, environmental and economic sustainability indicators, including ammonia emissions, space use, pen fouling, weight gain, carcass quality, perception of the stockkeeper, costs of the light system, and use of drinking water, electricity and medicines. Light treatments included a low (45 lux), medium (198 lux) and high (968 lux) uniform intensity, and a spatial gradient treatment ranging from 71 lux in the front to 330 lux in the back of each pen. The latter treatment aimed to improve the space use of functional areas. A total of 448 growing-finishing pigs were studied on a commercial farm using two consecutive batches of four rooms containing eight pens with seven pigs. Light intensity influenced some aspects of space use and pen fouling. For example, the proportion of pigs lying in the resting area was higher in the high and medium light intensity treatment than in the low intensity and gradient treatment. Moreover, the high-intensity treatment resulted in more fouling with faeces in the feeding area compared with the low-intensity and the gradient treatment. Ammonia emissions were higher in the gradient than in the low intensity treatment (not measured in medium and high intensity treatment). Furthermore, light intensity did not affect weight gain, carcass quality, water use and medicine use. The stockkeeper was content to work in all light conditions, but slightly preferred the medium intensity due to optimal visibility. Concerning economic performance, the costs of the light system and electricity use increased in the following order: low intensity, gradient, medium intensity, and high intensity. In conclusion, contrary to expectation the spatial gradient did not notably improve space use or reduce pen fouling, but rather increased ammonia emissions in comparison with uniform light. This is likely because the gradient could not be applied in an optimal way in the existing housing conditions. Among the other sustainability indicators, mainly electricity use and costs of the light system differed per treatment. These aspects can be improved by further optimising the number of light sources needed per pen to achieve the targeted intensities.

Photosensitized Oxidative Damage from a New Perspective: The Influence of Before-Light and After-Light Reaction Conditions.

Photooxidative damage is heavily influenced by the presence of bioactive agents. Conversely, bioactive agents influence the local environment, which in turn is perturbed by photooxidative damage. These sorts of processes give rise to a version of the "chicken-and-egg" quandary. In this Perspective, we probe this issue by referring to photooxidative damage in one direction as the light-dark (L-D) sequence and in a second direction as the dark-light (D-L) sequence with a reversed cause and effect. The L-D sequence can lead to the downstream production of reactive molecular species (RMS) in the dark, whereas the D-L sequence can be a pre-irradiation period, such as an additive to limit cellular iron levels to enhance biosynthesized amounts of a protoporphyrin sensitizer. A third direction comes from L-D or D-L sequences, or both simultaneously, which can also be useful for optimizing photodynamics. Photodynamic optimization will benefit from understanding and quantitating unidirectional L-D and D-L pathways, and bidirectional L-D/D-L pathways, for improved control over photooxidative damage. Photooxidative damage, which occurs during anticancer photodynamic therapy (PDT), will be shown to involve RMS. Such RMS include persulfoxides (R2S+OO-), NO2•, peroxynitrate (O2NOO-), OOSCN-, SO3•-, selenocyanogen [(SeCN)2], the triselenocyanate anion [(SeCN)3-], I•, I2•-, I3-, and HOOI, as well as additives to destabilize membranes (e.g., caspofungin and saponin A16), inhibit DNA synthesis (5-fluorouracil), or sequester iron (desferrioxamine). In view of the success that additive natural products and repurposed drugs have had in PDT, a Perspective of additive types is expected to reveal mechanistic details for enhanced photooxidation reactions in general. Indeed, strategies for how to potentiate photooxidations with additives remain highly underexplored.

Elucidating light and temperature-dependent signalling pathways from shoot to root in rice plants: Implications for stress responses.

The main aim of this work was to better understand how the low temperature signal from the leaves may affect the stress responses in the roots, and how the light conditions modify certain stress acclimation processes in rice plants. Rice plants grown at 27°C were exposed to low temperatures (12°C) with different light intensities, and in the case of some groups of plants, only the leaves received the cold, while the roots remained at control temperature. RNA sequencing focusing on the roots of plants grown under normal growth light conditions found 525 differentially expressed genes in different comparisons. Exposure to low temperature led to more down-regulated than up-regulated genes. Comparison between roots of the leaf-stressed plants and whole cold-treated or control plants revealed that nitrogen metabolism and nitric oxide-related signalling, as well as the phenylpropanoid-related processes, were specifically affected. Real-time PCR results focusing on the COLD1 and polyamine oxidase genes, as well as metabolomics targeting hormonal changes and phenolic compounds also showed that not only cold exposure of the leaves, either alone or together with the roots, but also the light conditions may influence certain stress responses in the roots of rice plants.

Blackthorn fruit peel polyphenol extracts and photodynamic effect under blue light against Listeria monocytogenes.

Photodynamic inactivation is an emerging antimicrobial treatment that can be enhanced by employing exogenous photosensitizers to eradicate foodborne pathogens. This study investigated a novel combinatory strategy to eradicate Listeria monocytogenes using blackthorn fruit peel (BFP) and blue light (BL). Extracts of BFP were characterized in terms of polyphenolic content, individual constituents, and antioxidant and antimicrobial activity. The concentration of phenolic compounds and antioxidant activity were both found to be determinants of antimicrobial activity. It was further speculated that flavonols, predominantly quercetin and rutin, were responsible for the activity of BFP against L. monocytogenes. A combination of BFP and BL resulted in a rapid inactivation of the pathogen by up to 4 log CFU/mL at 58.5 J/cm2, corresponding to 15 min BL illumination. Flow cytometry analysis revealed that the bacterial cells lost activity and suffered extensive membrane damage, exceeding 90% of the population. After photosensitizing L. monocytogenes with the BFP constituents quercetin and rutin, a 1.3-log reduction was observed. When applied together, these compounds could inflict the same damaging effect on cells as they did individually when effects were added. Therefore, the results indicate that BFP represents a natural source of (pro-)photosensitizers, which act additively to create inactivation effects. This study may help identify more effective plant-based photosensitizers to control L. monocytogenes in food-related applications.

Photosynthetic light requirement near the theoretical minimum detected in Arctic microalgae.

Photosynthesis is one of the most important biological processes on Earth, providing the main source of bioavailable energy, carbon, and oxygen via the use of sunlight. Despite this importance, the minimum light level sustaining photosynthesis and net growth of primary producers in the global ocean is still unknown. Here, we present measurements from the MOSAiC field campaign in the central Arctic Ocean that reveal the resumption of photosynthetic growth and algal biomass buildup under the ice pack at a daily average irradiance of not more than 0.04 ± 0.02 µmol photons m-2 s-1 in late March. This is at least one order of magnitude lower than previous estimates (0.3-5 µmol photons m-2 s-1) and near the theoretical minimum light requirement of photosynthesis (0.01 µmol photons m-2 s-1). Our findings are based on measurements of the temporal development of the under-ice light field and concurrent measurements of both chlorophyll a concentrations and potential net primary production underneath the sea ice at 86 °N. Such low light requirements suggest that euphotic zones where photosynthesis can occur in the world's oceans may extend further in depth and time, with major implications for global productivity estimates.

Wirelessly Powered Visible Light-Emitting Implant for Surgical Guidance during Lumpectomy.

Achieving negative surgical margins, defined as no tumor found on the edges of the resected tissue, during lumpectomy for breast cancer is critical for mitigating the risk of local recurrence. To identify nonpalpable tumors that cannot be felt, pre-operative placements of wire and wire-free localization devices are typically employed. Wire-free localization approaches have significant practical advantages over wired techniques. In this study, we introduce an innovative localization system comprising a light-emitting diode (LED)-based implantable device and handheld system. The device, which is needle injectable and wire free, utilizes multiple wirelessly powered LEDs to provide direct visual guidance for lumpectomy. Two distinct colors, red and blue, provide a clear indication of tissue depth: blue light is absorbed strongly in tissue, visible within a close range of <1 cm, while red light remains visible through several centimeters of tissue. The LEDs, integrated with an impedance-matching circuit and receiver coil, are encapsulated in biocompatible epoxy for injection with a 12 G needle. Our findings demonstrate that the implant exhibits clearly perceivable depth-dependent color changes and remains visible through >2 cm of ex vivo chicken breast and bovine muscle tissue using less than 4 W of transmitted power from a handheld antenna. These miniaturized needle-injectable localization devices show promise for improving surgical guidance of nonpalpable breast tumors.

Multicellularity and increasing Reynolds number impact on the evolutionary shift in flash-induced ciliary response in Volvocales.

BACKGROUND: Volvocales in green algae have evolved by multicellularity of Chlamydomonas-like unicellular ancestor. Those with various cell numbers exist, such as unicellular Chlamydomonas, four-celled Tetrabaena, and Volvox species with different cell numbers (~1,000, ~5,000, and ~10,000). Each cell of these organisms shares two cilia and an eyespot, which are used for swimming and photosensing. They are all freshwater microalgae but inhabit different fluid environments: unicellular species live in low Reynolds-number (Re) environments where viscous forces dominate, whereas multicellular species live in relatively higher Re where inertial forces become non-negligible. Despite significant changes in the physical environment, during the evolution of multicellularity, they maintained photobehaviors (i.e., photoshock and phototactic responses), which allows them to survive under changing light conditions. RESULTS: In this study, we utilized high-speed imaging to observe flash-induced changes in the ciliary beating manner of 27 Volvocales strains. We classified flash-induced ciliary responses in Volvocales into four patterns: "1: temporal waveform conversion", "2: no obvious response", "3: pause in ciliary beating", and "4: temporal changes in ciliary beating directions". We found that which species exhibit which pattern depends on Re, which is associated with the individual size of each species rather than phylogenetic relationships. CONCLUSIONS: These results suggest that only organisms that acquired different patterns of ciliary responses survived the evolutionary transition to multicellularity with a greater number of cells while maintaining photobehaviors. This study highlights the significance of the Re as a selection pressure in evolution and offers insights for designing propulsion systems in biomimetic micromachines.

Fabrication and properties of temperature-responsive imprinted sensors based on fluorescently labeled yeast cells via MVL ATRP.

Temperature-responsive yeast cell-imprinted sensors (CIPs/AuNPs/Ti3C2Tx/AuNPs/Au) were prepared based on fluorescein isothiocyanate labeled yeast cells (FITC-yeast) via metal-free visible-light-induced atom transfer radical polymerization (MVL ATRP). Here, N-isopropyl acrylamide (NIPAM) was used as a temperature-responsive functional monomer, α-methacrylic acid (MAA) was chosen as an auxiliary functional monomer, N,N'-methylene bisacrylamide (MBA) was used as a cross-linker, and FITC-yeast was selected as both a template and photocatalyst. Under the optimal conditions, the detection range of the yeast cell-imprinted sensor toward yeast cells was 1.0 × 102 to 1.0 × 109 cells per mL, and the detection limit was 11 cells per mL (S/N = 3), with a linear equation of ΔI (µA) = 8.44 log[C (cells per mL)] + 7.62 (R2 = 0.993). The sensor showed good selective recognition in the presence of interfering substances such as autolyzed yeast cells (AY), dead yeast cells (DY), human mammary epithelial cells (MCF-10A), human breast cancer cells (MCF-7) and Escherichia coli (EC). The sensor also had good consistency and reproducibility. Finally, spiked recovery experiments were performed to investigate the recognition of yeast cells in the actual sample using the yeast cell-imprinted sensor. The spiked recoveries were all in the range of 98.5-108.0%, and the RSD values were all less than 4%, indicating that the sensor had good application prospects.

Organic-Inorganic Interfacial Dipole Induced by Energy Level Alignment for Efficient Photocatalytic Sterilization.

Photocatalytic molecules are considered to be one of the most promising substitutions of antibiotics against multidrug-resistant bacterial infections. However, the strong excitonic effect greatly restricts their efficiency in antibacterial performance. Inspired by the interfacial dipole effect, a Ti3C2 MXene modified photocatalytic molecule (MTTTPyB) is designed and synthesized to enhance the yield of photogenerated carriers under light irradiation. The alignment of the energy level between Ti3C2 and MTTTPyB results in the formation of an interfacial dipole, which can provide an impetus for the separation of carriers. Under the role of a dipole electric field, these photogenerated electrons can rapidly migrate to the side of Ti3C2 for improving the separation efficiency of photogenerated electrons and holes. Thus, more electrons can be utilized to produce reactive oxygen species (ROS) under light irradiation. As a result, over 97.04% killing efficiency can be reached for Staphylococcus aureus (S. aureus) when the concentration of MTTTPyB/Ti3C2 was 50 ppm under 660 nm irradiation for 15 min. A microneedle (MN) patch made from MTTTPyB/Ti3C2 was used to treat the subcutaneous bacterial infection. This design of an organic-inorganic interface provides an effective method to minimize the excitonic effect of molecules, further expanding the platform of inorganic/organic hybrid materials for efficient phototherapy.

Development of an affordable light emitting diode spectrophotometer paired with a Python program for calibration and linearity testing and the measurement of uranium(VI).

Uranium (U) is a radiologically and chemically toxic element that occurs naturally in water, soil, and rock at generally low levels. However, anthropogenic uranium can also leach into groundwater sources due to mining, ore refining, and improper nuclear waste management. Over the last few decades, various methods for measuring uranium have emerged; however, most of these techniques require skilled scientists to run samples on expensive instrumentation for detection or require the pretreatment of samples in complex procedures. In this work, a Schiff base ligand (P1) is used to develop a simple spectrophotometric method for measuring the concentration of uranium (VI) with an accurate and affordable light-emitting diode (LED) spectrophotometer. A test for a higher-order polynomial relationship was used to objectively determine the calibration data's linearity. This test was done with a Python program on a Raspberry Pi computer that captured the spectrophotometer's calibration and sample measurement data.

Anticancer activity and mechanism studies of photoactivated iridium(III) complexes toward lung cancer A549 cells.

Cyclometalated iridium(III) compounds have been widely explored due to their outstanding photo-physical properties and multiple anticancer activities. In this paper, three cyclometalated iridium(III) compounds [Ir(ppy)2(DBDIP)]PF6 (5a), [Ir(bzq)2(DBDIP)]PF6 (5b), and [Ir(piq)2(DBDIP)]PF6 (5c) (ppy: 2-phenylpyridine; bzq: benzo[h]quinoline; piq: 1-phenylisoquinoline, and DBDIP: 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and the mechanism of antitumor activity was investigated. Compounds photoactivated by visible light show strong cytotoxicity against tumor cells, especially toward A549 cells. Biological experiments such as migration, cellular localization, mitochondrial membrane potential and permeability, reactive oxygen species (ROS) and calcium ion level detection were performed, and they demonstrated that the compounds induced the apoptosis of A549 cells through a mitochondrial pathway. At the same time, oxidative stress caused by ROS production increases the release of damage-related molecules and the expression of porogen gasdermin D (GSDMD), and the content of LDH released from damaged cell membranes also increased. Besides, the content of the lipid peroxidation product, malondialdehyde (MDA), increased and the expression of GPX4 decreased. These indicate that the compounds promote cell death by combining ferroptosis and pyroptosis. The results reveal that cyclometalated iridium(III) compounds 5a-5c may be a potential chemotherapeutic agent for photodynamic therapy of cancers.

Light-Activated Molecular Purification (LAMP) of Recombinant Proteins.

The production of recombinant proteins has become a focal point in biotechnology, with potential applications in catalysis, therapeutics, and diagnostics. Before their application, these proteins undergo cumbersome downstream processing, including multiple resin-based chromatography steps (ion exchange or affinity-based) to isolate the protein of interest from host cell proteins, which are more abundant. These methods often involve (1) nonspecific binding of host cell proteins onto the resin, (2) a trial and error approach in determining elution conditions for the protein of interest, and (3) complex functionalization of the resin. These processes are also further supplemented with additional processing steps including buffer exchange through dialysis or desalting. Despite the prevalence and need for proteins, challenges persist in optimizing elution conditions and minimizing downstream processing steps, which contribute to the overall cost, impeding their translation into the market. To address these challenges, there has been a growing interest in stimuli-responsive purification systems, which allow for precise control and modulation of the purification process for protein recovery by altering their properties or behavior in response to specific external conditions, such as heat, light, or chemicals. We have developed a light-activated molecular purification (LAMP) system, a stimuli-responsive chromatography technique where the purification of recombinant proteins is triggered by light. We employed a photocleavable protein (PhoCl1) that binds specifically to Ni-NTA resin through a hexa-histidine tag at its N-terminus. We harnessed the ability of PhoCl1 to undergo photocleavage into two fragments for the development of LAMP. To demonstrate LAMP, the protein of interest (POI) is genetically fused to the C-terminus of PhoCl1. The exposure to 405 nm light (1.5 mW cm-2 for 12 h) results in the release of POI into the supernatant. We showcased the potential of LAMP by purifying highly charged green fluorescent proteins and an enzyme, riboflavin kinase. Our custom-built violet light LED setup achieved more than 50% light-induced photocleavage of the fusion constructs, resulting in the release of more than 30% of the POI into the supernatant, with the remainder retained within the resin. All the proteins purified using LAMP were more than 90% pure. Moreover, the comparison of the riboflavin kinase purified through LAMP and the traditional chromatography (Ni-NTA affinity method) revealed no significant changes in the activity levels. These highlight the broad potential of LAMP in providing a facile, yet robust stimuli-responsive protein purification technique, which leverages the potential of light to purify the proteins and overcome the limitations of current conventional chromatography systems.

Reversible Control of Native GluN2B-Containing NMDA Receptors with Visible Light.

NMDA receptors (NMDARs) are glutamate-gated ion channels playing a central role in synaptic transmission and plasticity. NMDAR dysregulation is linked to various neuropsychiatric disorders. This is particularly true for GluN2B-containing NMDARs (GluN2B-NMDARs), which have major pro-cognitive, but also pro-excitotoxic roles, although their exact involvement in these processes remains debated. Traditional GluN2B-selective antagonists suffer from slow and irreversible effects, limiting their use in native tissues. We therefore developed OptoNAM-3, a photoswitchable negative allosteric modulator selective for GluN2B-NMDARs. OptoNAM-3 provided light-induced reversible inhibition of GluN2B-NMDAR activity with precise temporal control both in vitro and in vivo on the behavior of freely moving Xenopus tadpoles. When bound to GluN2B-NMDARs, OptoNAM-3 displayed remarkable red-shifting of its photoswitching properties allowing the use of blue light instead of UV light to turn-off its activity, which we attributed to geometric constraints imposed by the binding site onto the azobenzene moiety of the ligand. This study therefore highlights the importance of the binding site in shaping the photochemical properties of azobenzene-based photoswitches. In addition, by enabling selective, fast, and reversible photocontrol of native GluN2B-NMDARs with in vivo compatible photochemical properties (visible light), OptoNAM-3 should be a useful tool for the investigation of the GluN2B-NMDAR physiology in native tissues.

The retinal chromophore environment in an inward light-driven proton pump studied by solid-state NMR and hydrogen-bond network analysis.

Inward proton pumping is a relatively new function for microbial rhodopsins, retinal-binding light-driven membrane proteins. So far, it has been demonstrated for two unrelated subgroups of microbial rhodopsins, xenorhodopsins and schizorhodopsins. A number of recent studies suggest unique retinal-protein interactions as being responsible for the reversed direction of proton transport in the latter group. Here, we use solid-state NMR to analyze the retinal chromophore environment and configuration in an inward proton-pumping Antarctic schizorhodopsin. Using fully 13C-labeled retinal, we have assigned chemical shifts for every carbon atom and, assisted by structure modelling and molecular dynamics simulations, made a comparison with well-studied outward proton pumps, identifying locations of the unique protein-chromophore interactions for this functional subclass of microbial rhodopsins. Both the NMR results and molecular dynamics simulations point to the distinctive polar environment in the proximal part of the retinal, which may result in a hydration pattern dramatically different from that of the outward proton pumps, causing the reversed proton transport.