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Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies., Summary: Combining different domains of human visual opsins and melanopsin creates functionally unique chimeric opsins with potential optogenetic applications.

There is no consensus on the best optogenetic tool for neuronal inhibition. Lamprey parapinopsin (‘Lamplight’) is a Gi/o-coupled bistable animal opsin that can be activated and deactivated by short and long wavelength light, respectively. Since native mechanisms of neuronal inhibition frequently employ Gi/o signalling, we asked here whether Lamplight could be used for optogenetic silencing. We show that short (405nm) and long (525nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, and that combining these wavelengths can be used to achieve intermediate levels of activity. We demonstrate that these properties can be applied to produce switchable and scalable neuronal hyperpolarisation, and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. We show that expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, and that 405 and 525nm stimuli can produce responses of opposite sign in output neurons of the retina. Lamplight-driven responses to both activating (405nm) and deactivating (525nm) light can occur within 500ms and be elicited by intensities at least 10x below threshold for available inhibitory optogenetic tools. We conclude that Lamplight can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and rapidly reversible.

An animal's temporal niche – the time of day at which it is active – is known to drive a variety of adaptations in the visual system. These include variations in the topography, spectral sensitivity and density of retinal photoreceptors, and changes in the eye's gross anatomy and spectral transmission characteristics. We have characterised visual spectral sensitivity in the murid rodent Rhabdomys pumilio (the four-striped grass mouse), which is in the same family as (nocturnal) mice and rats but exhibits a strong diurnal niche. As is common in diurnal species, the R. pumilio lens acts as a long-pass spectral filter, providing limited transmission of light, Summary: The visual spectral sensitivity of a diurnal rodent, Rhabdomys pumilio, is biased against UV-A wavelengths thanks to lens filtering, but not cone spectral sensitivity.

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure–function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities. Electronic supplementary material The online version of this article (10.1007/s00018-018-2813-0) contains supplementary material, which is available to authorized users.

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼492nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

Background Infection and disease from human cytomegalovirus (CMV) is a major complicating factor for both solid organ and hematopoietic stem cell transplant recipients. Antiviral therapy is often used to control CMV infections, but presents problems of toxicity, antiviral resistance and excessive costs. Currently, treating physicians are limited in the information and data available to assess a patient’s ability to control a potential CMV infection post-transplant. Recent studies have shown that measuring a patient’s CMV specific T cell mediated immunity may provide valuable information to physicians for monitoring CMV infection/disease in transplant patients and may aid in determining which patients need antiviral therapy. Methods For this purpose, a flow cytometry assay was developed to determine the percentages of CD4+ and CD8+ T cells that respond to stimulation with CMV antigen. Assessment of CMV specific response is based upon the cellular activation surface marker CD69 in conjunction with IFNg, TNFα, and IL-2 cytokine production. Three CMV antigens were used to assess patient immunity; a whole viral lysate, a peptide pool of pp65, and a peptide pool of IE-1. Results Our data indicate that CD8 T cells respond primarily to the pp65 and/or IE-1 peptide pools while the CD4 T cells respond primarily to the viral lysate. Detection of both CD4 and CD8 responding populations at levels above background, ≥ 0.2% of the parent population, indicates that a patient’s immune system has previously been exposed to CMV. Validation of 23 CMV seropositive samples demonstrated immune responses for all 23 samples above 0.2% for at least one of the three intra-cellular cytokines and at least one of the three CMV antigens. Validation of five CMV seronegative samples demonstrated immune responses below 0.2% (when excluding underlying, unrelated immune responses). Included for each sample is a positive (Staphylococcal Enterotoxin type B) control to assess patient’s overall ability to mount an immune response and negative (media) control to capture the presence of an underlying immune response. Conclusion This assay evaluates a patient’s pre-existing CMV specific T cell immunity and their global T cell function. Disclosures C. B. Lutgen, Viracor Eurofins Clinical Diagnostics: Employee, Salary; L. Flebbe-Rehwaldt, Viracor Eurofins Clinical Diagnostics: Employee, Salary; S. Kleiboeker, Viracor Eurofins Laboratories: Employee, Salary; S. Fausett, Viracor Eurofins Clinical Diagnostics: Employee, Salary; C. Cordes, Viracor Eurofins Clinical Diagnostics: Employee, Salary; J. Rodgers, Viracor Eurofins Clinical Diagnostics: Employee, Salary; K. Steffens, Viracor Eurofins Clinical Diagnostics: Employee, Salary; M. Altrich, Viracor Eurofins Laboratories: Employee, Salary