Rhodopsin Gene Research Articles

Flotillin-associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes.

Microbial rhodopsins are found more than once in a single genome (paralogs) often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59, and the Gammaproteobacteria Pseudothioglobus SAGs. These genomes always had a bona fide proteorhodopsin and a separate cluster of genes containing a second rhodopsin associated with a predicted flotillin coding gene and have thus been named flotillin-associated rhodopsins (FArhodopsins). Although they are members of the proteorhodopsin protein family, they form a separate clade within that family and are quite divergent from known proton-pumping proteorhodopsins. They contain either DTT, DTL, or DNI motifs in their key functional amino acids. FArhodopsins are mainly associated with the lower layers of the epipelagic zone. All marine FArhodopsins had the retinal binding lysine, but we found relatives in freshwater metagenomes lacking this key amino acid. AlphaFold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less. Freshwater FArhodopsins were more diverse than marine ones, but we could not determine if there were other rhodopsins in the genome due to the lack of SAGs or isolates. Although the function of FArhodopsins could not be established, their conserved genomic context indicated involvement in the formation of membrane microdomains. The conservation of FArhodopsins in diverse and globally abundant microorganisms suggests that they may be important in the adaptation to the twilight zone of aquatic environments. IMPORTANCE Rhodopsins have been shown to play a key role in the ecology of aquatic microbes. Here, we describe a group of widespread rhodopsins in aquatic microbes associated with dim light conditions. Their characteristic genomic context found in both marine and freshwater environments indicates a novel potential involvement in membrane microstructure that could be important for the function of the coexisting proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to a drastically different physiological role.

Rhodopsin-mediated nutrient uptake by cultivated photoheterotrophic Verrucomicrobiota

Rhodopsin photosystems convert light energy into electrochemical gradients used by the cell to produce ATP, or for other energy-demanding processes. While these photosystems are widespread in the ocean and have been identified in diverse microbial taxonomic groups, their physiological role in vivo has only been studied in few marine bacterial strains. Recent metagenomic studies revealed the presence of rhodopsin genes in the understudied Verrucomicrobiota phylum, yet their distribution within different Verrucomicrobiota lineages, their diversity, and function remain unknown. In this study, we show that more than 7% of Verrucomicrobiota genomes (n = 2916) harbor rhodopsins of different types. Furthermore, we describe the first two cultivated rhodopsin-containing strains, one harboring a proteorhodopsin gene and the other a xanthorhodopsin gene, allowing us to characterize their physiology under laboratory-controlled conditions. The strains were isolated in a previous study from the Eastern Mediterranean Sea and read mapping of 16S rRNA gene amplicons showed the highest abundances of these strains at the deep chlorophyll maximum (source of their inoculum) in winter and spring, with a substantial decrease in summer. Genomic analysis of the isolates suggests that motility and degradation of organic material, both energy demanding functions, may be supported by rhodopsin phototrophy in Verrucomicrobiota. Under culture conditions, we show that rhodopsin phototrophy occurs under carbon starvation, with light-mediated energy generation supporting sugar transport into the cells. Overall, this study suggests that photoheterotrophic Verrucomicrobiota may occupy an ecological niche where energy harvested from light enables bacterial motility toward organic matter and supports nutrient uptake.

Gene augmentation for autosomal dominant retinitis pigmentosa using rhodopsin genomic loci nanoparticles in the P23H+/- knock-in murine model.

Gene therapy for autosomal dominant retinitis pigmentosa (adRP) is challenged by the dominant inheritance of the mutant genes, which would seemingly require a combination of mutant suppression and wild-type replacement of the appropriate gene. We explore the possibility that delivery of a nanoparticle (NP)-mediated full-length mouse genomic rhodopsin (gRho) or human genomic rhodopsin (gRHO) locus can overcome the dominant negative effects of the mutant rhodopsin in the clinically relevant P23H+/--knock-in heterozygous mouse model. Our results demonstrate that mice in both gRho and gRHO NP-treated groups exhibit significant structural and functional recovery of the rod photoreceptors, which lasted for 3 months post-injection, indicating a promising reduction in photoreceptor degeneration. We performed miRNA transcriptome analysis using next generation sequencing and detected differentially expressed miRNAs as a first step towards identifying miRNAs that could potentially be used as rhodopsin gene expression enhancers or suppressors for sustained photoreceptor rescue. Our results indicate that delivering an intact genomic locus as a transgene has a greater chance of success compared to the use of the cDNA for treatment of this model of adRP, emphasizing the importance of gene augmentation using a gDNA that includes regulatory elements.

Gap junction Delta-2b (gjd2b/Cx35.1) depletion causes hyperopia and visual-motor deficiencies in the zebrafish.

The zebrafish is a powerful model to investigate the developmental roles of electrical synapses because many signaling pathways that regulate the development of the nervous system are highly conserved from fish to humans. Here, we provide evidence linking the mammalian connexin-36 (Cx36) ortholog gjd2b/Cx35.1, a major component of electrical synapses in the zebrafish, with a refractive error in the context of morphological, molecular, and behavioral changes of zebrafish larvae. Two abnormalities were identified. The optical coherence tomography analysis of the adult retina confirmed changes to the refractive properties caused by eye axial length reduction, leading to hyperopic shifts. The gjd2b/Cx35.1 depletion was also correlated with morphological changes to the head and body ratios in larvae. The differential expression of Wnt/ß-catenin signaling genes, connexins, and dopamine receptors suggested a contribution to the observed phenotypic differences. The alteration of visual-motor behavioral responses to abrupt light transitions was aggravated in larvae, providing evidence that cone photoreceptor cell activity was enhanced when gjd2b/Cx35.1 was depleted. The visual disturbances were reversed under low light conditions in gjd2b -/- /Cx35.1-/- larvae. Since qRT-PCR data demonstrated that two rhodopsin genes were downregulated, we speculated that rod photoreceptor cells in gjd2b/Cx35.1-/- larvae were less sensitive to bright light transitions, thus providing additional evidence that a cone-mediated process caused the VMR light-ON hyperactivity after losing Cx35.1 expression. Together, this study provides evidence for the role of gjd2b/Cx35.1 in the development of the visual system and visually guided behaviors.

Colour Selection and Olfactory Responses of Papilio demoleus during Foraging and Courtship.

Colours and odours are the most important cues for butterflies to localise food and mating partners. We studied the visual and olfactory responses of the widely distributed butterfly Papilio demoleus Linnaeus during foraging and courtship. P. demoleus visited odourless flowers with six colours except green and black, with red as the favourite colour (650-780 nm). Males and females differed in behaviour while visiting flowers. Males were more active than females during foraging. The application of honey water resulted in a significant increase in flower visits by both females and males, and they scarcely visited the apetalous branches with odours. Under natural conditions, four patterns were observed: males chasing males (42.28%), males chasing females (30.56%), females chasing females (13.73%), and females chasing males (13.43%). Males chasing males was the most frequent, probably because males drive away competing con-specific males. When butterflies visited odourless mimics, males chasing females (70.73%) and males chasing males (29.27%) also occurred, indicating that males could accurately distinguish mates using colours only without any chemical cues, and females need chemical cues. The behavioural responses of P. demoleus to floral visits and courtship suggest that colour is the dominant factor during foraging and courtship. We verified the presence of P. demoleus rhodopsin genes, including Rh2, Rh3, Rh4, and Rh5, for long wavelength, blue, and ultraviolet (UV) spectrum recognition, which is consistent with the colour recognition of flowers and wings during visiting flowers and courtship.

New insights into the diversity of cryptobenthic Cirripectes blennies in the Mascarene Archipelago sampled using Autonomous Reef Monitoring Structures (ARMS).

Autonomous Reef Monitoring Structures (ARMS) are artificial mini-reefs designed for standardized sampling of sessile and small motile cryptobenthic organisms. ARMS are also effective for collecting small cryptobenthic fishes, such as the combtooth blennies of the genus Cirripectes. Recent studies discovered several Cirripectes species endemic to islands or archipelagos, in spite of the generally broad distributions of tropical and subtropical blennies. Thus, to evaluate the diversity and distribution of Cirripectes species in the Mascarene Archipelago, a little-studied region but an important biodiversity hotspot, complete mitochondrial genomes, and nuclear rhodopsin genes were sequenced for 39 specimens collected with ARMS deployed on outer reef slopes at Reunion and Rodrigues islands. Mitochondrial COI sequences were analyzed to integrate these specimens within the largest dataset of publicly available sequences. Three species were found in the Mascarene Archipelago, Cirripectes castaneus, Cirripectes randalli, and Cirripectes stigmaticus. C.castaneus and C.stigmaticus both have an Indo-Pacific distribution with several haplotypes shared among distant localities. In agreement with the literature, C.randalli shows a small-range endemism restricted to the Mascarenes. We confirmed the presence of C.castaneus, C.randalli, and C.stigmaticus in Rodrigues, and the presence of C.stigmaticus in Reunion. This study contributes to filling the gaps in taxonomic and molecular knowledge of the reef cryptobiome in the South-West Indian Ocean, and provides the first complete mitogenomes for the genus, a crucial step for future molecular-based inventories (e.g., eDNA).

Binocular benefit following monocular subretinal AAV injection in a mouse model of autosomal dominant retinitis pigmentosa (adRP)

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Pharmacochaperones as a novel approach to rescue misfolding rod opsin mutants associated with retinitis pigmentosa.

The correct trafficking of folded, functional rhodopsin (Rho) is critical to sustaining vision. Inherited destabilizing mutations in the rhodopsin (RHO) gene lead to a spectrum of blinding diseases called retinitis pigmentosa (RP) with currently no cure available. Therapeutic approaches to correct these impartments focus on the development of pharmacological chaperones that stabilize rod opsin. The native ligand, 11-cis-retinal, improves protein stability, folding, and trafficking of some mutated Rho variants. However, its chemical instability, toxicity, and poor pharmacokinetics limit its use as a therapeutic. In our laboratory, we focus our attention on finding chaperones with natural-like product scaffolds due to their broad chemical diversity. Furthermore, ∼50% of marketed small molecule drugs are derived from natural products scaffold, and some of them target GPCRs. Thus, we integrated computational, biochemical, biophysical, and in vivo analysis to evaluate non-retinoid ligands with diverse chemical scaffolds as potential chaperones of misfolding Rho variants. Our study led to the discovery of a novel chromenone-containing small molecule with favorable pharmacological properties that through direct binding to the orthosteric pocket enhanced rod opsin stability. This compound improved the membrane trafficking of multiple rod opsin mutants in vitro. Importantly, our studies unraveled the pharmacological potential of this new chromenone rod opsin ligand to slow down the progression of photoreceptor degeneration in the P23H Rho knock-in mice, a model of RP. Together, our studies encourage further search and evaluation of novel non-retinoid compounds that could mitigate the misfolding and mistrafficking of the Rho mutants to prolong the survival of photoreceptors in the retina, and thus delaying vision loss in RP.

The genome of sheep ked (Melophagus ovinus) reveals potential mechanisms underlying reproduction and narrower ecological niches

BackgroundMelophagus ovinus is considered to be of great veterinary health significance. However, little is known about the information on genetic mechanisms of the specific biological characteristics and novel methods for controlling M. ovinus.ResultsIn total, the de novo genome assembly of M. ovinus was 188.421 Mb in size (330 scaffolds, N50 Length: 10.666 Mb), with a mean GC content of 27.74%. A total of 13,372 protein-coding genes were functionally annotated. Phylogenetic analysis indicated that the diversification of M. ovinus and Glossina fuscipes took place 72.76 Mya within the Late Cretaceous. Gene family expansion and contraction analysis revealed that M. ovinus has 65 rapidly-evolving families (26 expansion and 39 contractions) mainly involved DNA metabolic activity, transposases activity, odorant receptor 59a/67d-like, IMD domain-containing protein, and cuticle protein, etc. The universal and tightly conserved list of milk protein orthologues has been assembled from the genome of M. ovinus. Contractions and losses of sensory receptors and vision-associated Rhodopsin genes were significant in M. ovinus, which indicate that the M. ovinus has narrower ecological niches.ConclusionsWe sequenced, assembled, and annotated the whole genome sequence of M. ovinus, and launches into the preliminary genetic mechanisms analysis of the adaptive evolution characteristics of M. ovinus. These resources will provide insights to understand the biological underpinnings of this parasite and the disease control strategies.

Galanin receptor 3 − A new pharmacological target in retina degeneration

The neuropeptide galanin receptor 3 (GALR3) is a class A G protein-coupled receptor (GPCR) broadly expressed in the nervous system, including the retina. GALR3 is involved in the modulation of immune and inflammatory responses. Tight control of these processes is critical for maintaining homeostasis in the retina and is required to sustain vision. Here, we investigated the role of GALR3 in retina pathologies triggered by bright light and P23H mutation in the rhodopsin (RHO) gene, associated with the activation of oxidative stress and inflammatory responses. We used a multiphase approach involving pharmacological inhibition of GALR3 with its antagonist SNAP-37889 and genetic depletion of GALR3 to modulate the GALR3 signaling. Our in vitro experiments in the retinal pigment epithelium-derived cells (ARPE19) susceptible to all-trans-retinal toxicity indicated that GALR3 could be involved in the cellular stress response to this phototoxic product. Indeed, blocking the GALR3 signaling in Abca4-/-/Rdh8-/- and wild-type Balb/cJ mice, sensitive to bright light-induced retina damage, protected retina health in these mice exposed to light. The retina morphology and function were substantially improved, and stress response processes were reduced in these mouse models compared to the controls. Furthermore, in P23H Rho knock-in mice, a model of retinitis pigmentosa (RP), both pharmacological inhibition and genetic ablation of GALR3 prolonged the survival of photoreceptors. These results indicate that GALR3 signaling contributes to acute light-induced and chronic RP-linked retinopathies. Together, this work provides the pharmacological knowledge base to evaluate GALR3 as a potential target for developing novel therapies to combat retinal degeneration.

Overexpression of Rhodopsin or Its Mutants Leads to Energy Metabolism Dysfunction in 661w Cells.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders characterized by photoreceptor degeneration. The rhodopsin gene (RHO) is the most frequent cause of autosomal dominant RP (ADRP), yet it remains unclear how RHO mutations cause heterogeneous phenotypes. Energy failure is a main cause of the secondary cone death during RP progression; however, its role in primary rod death induced by ADRP RHO mutants is unknown. Three RHO missense mutations were chosen from different clinical classes. Wild-type (WT) RHO and its mutants, P23H (class B1), R135L (class A), and G188R (class B2), were overexpressed in 661w cells, a mouse photoreceptor cell line, and their effects on oxidative phosphorylation (OXPHOS) and aerobic glycolysis were compared separately. Here, we report that energy failure is an early event in the cell death caused by overexpression of WT RHO and its mutants. RHO overexpression leads to OXPHOS deficiency, which might be a result of mitochondrial loss. Nonetheless, only in WT RHO and P23H groups, energy stress triggers AMP-activated protein kinase activation and metabolic reprogramming to increase glycolysis. Metabolic reprogramming impairment in R135L and G188R groups might be the reason why energy failure and cell injury are much more severe in those groups. Our results imply that overexpression of RHO missense mutants have distinct impacts on the two energy metabolic pathways, which might be related to their heterogeneous phenotypes.

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.

Identification of the Channelrhodopsin Genes in the Green and Cryptophytic Algae from the White and Black Seas.

Due to the unique capability of modulating cell membrane potential upon photoactivation, channelrhodopsins of green (Chlorophyta) and cryptophytic (Cryptophyta) algae are widely employed in optogenetics, a modern method of light-dependent regulation of biological processes. To enable the search for new genes perspective for optogenetics, we have developed the PCR tests for the presence of genes of the cation and anion channelrhodopsins. Six isolates of green algae Haematococcus and Bracteacoccus from the White Sea region and 2 specimens of Rhodomonas sp. (Cryptophyta) from the regions of White and Black Seas were analyzed. Using our PCR test we have demonstrated the known Haematococcus rhodopsin genes and have discovered novel rhodopsin genes in the genus of Bracteacoccus. Two distantly homologous genes of anion channelrhodopsins were also identified in the cryptophytic Rhodomonas sp. from the White and Black Seas. These results indicate that the developed PCR tests might be useful tool for a broad-range screening of the Chlorophyta and Cryptophyta algae to identify unique channelrhodopsin genes.

Gene Therapy for Rhodopsin Mutations.

Mutations in RHO, the gene for rhodopsin, account for a large fraction of autosomal-dominant retinitis pigmentosa (adRP). Patients fall into two clinical classes, those with early onset, pan retinal photoreceptor degeneration, and those who experience slowly progressive disease. The latter class of patients are candidates for photoreceptor-directed gene therapy, while former may be candidates for delivery of light-responsive proteins to interneurons or retinal ganglion cells. Gene therapy for RHO adRP may be targeted to the mutant gene at the DNA or RNA level, while other therapies preserve the viability of photoreceptors without addressing the underlying mutation. Correcting the RHO gene and replacing the mutant RNA show promise in animal models, while sustaining viable photoreceptors has the potential to delay the loss of central vision and may preserve photoreceptors for gene-directed treatments.

CRISPR DNA Base Editing Strategies for Treating Retinitis Pigmentosa Caused by Mutations in Rhodopsin.

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerations and pathogenic variants in the Rhodopsin (RHO) gene are major cause for autosomal dominant RP (adRP). Despite extensive attempts to treat RHO-associated adRP, standardized curative treatment is still lacking. Recently developed base editors offer an exciting opportunity to correct pathogenic single nucleotide variants and are currently able to correct all transition variants and some transversion variants. In this study, we analyzed previously reported pathogenic RHO variants (n = 247) for suitable PAM sites for currently available base editors utilizing the Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) or the KKH variant of SaCas9 (KKH-SaCas9) to assess DNA base editing as a treatment option for RHO-associated adRP. As a result, 55% of all the analyzed variants could, in theory, be corrected with base editors, however, PAM sites were available for only 32% of them and unwanted bystander edits were predicted for the majority of the designed guide RNAs. As a conclusion, base editing offers exciting possibilities to treat RHO-associated adRP in the future, but further research is needed to develop base editing constructs that will provide available PAM sites for more variants and that will not introduce potentially harmful bystander edits.

Morphometric, meristic and genetic characteristics of Atlantic bonito (Sarda sarda) in the Black Sea

The Atlantic bonito (Sarda sarda) is a commercially important and popular species in the world as well as in Turkish fisheries sector. In this study, the morphologic and meristic features and genetic characteristics of Atlantic bonito in the Black Sea were examined. We found that most of the morphometric measurements in females were greater than males. Besides, 10 haplotypes were found for COI gene region, 3 haplotypes for 16s gene region and 4 haplotypes for the rhodopsin gene region. Genetic closure was determined in Tirebolu population for the first time.

Molecular basis for variations in the sensitivity of pathogenic rhodopsin variants to 9-cis-retinal

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Ablation of pigment epithelium‐derived factor receptor, PEDF‐R, causes photoreceptor degeneration

Photoreceptor cells produce a receptor protein for pigment epithelium‐derived factor (PEDF‐R), which is encoded by the PNPLA2 gene of the patatin‐like phospholipase domain‐containing 2 family. PEDF‐R is a membrane‐linked lipase detected in the inner segments of photoreceptors. We aim to reveal the physiological role of PEDF‐R in photoreceptors by characterizing CRISPR Pnpla2 knock‐out mouse lines.CRISPR‐derived Pnpla2 knock‐out mouse lines were generated in a C57BL/6 and rd8 free background. RT‐PCR was performed from dissected retinas. Mice were evaluated for fundoscopy, electroretinography and angiography. Organs of the thorax were harvested and imaged using a LEICA microscope. Plasma was collected for lipid analyses. Eyes were enucleated and processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. Pnpla2‐/‐ mice had undetectable retinal Pnpla2 expression levels, enlarged whiter hearts, and lower plasma free fatty acids and triglycerides levels than their Pnpla2+/+ littermate controls. Their retinal pigment epithelial cells accumulated lipid droplets and their photoreceptors were deformed. Fundi of Pnpla2+/‐ eyes had white spots. The optic nerve area of Pnpla2‐/‐ and Pnpla2+/‐ mice had an excess of pigment. The a‐wave in Pnpla2‐/‐and Pnpla2+/‐ mice, and b‐wave in Pnpla2+/‐ mice were attenuated relative to controls. Retinal rhodopsin and opsin gene expression of Pnpla2‐/‐and Pnpla2+/‐ were lower than in controls. The immunofluorescent intensities of rhodopsin and opsin antibodies decreased in Pnpla2+/‐and Pnpla2‐/‐ photoreceptors relative to controls. The immunofluorescence of PKCα and synaptophysin antibodies (markers for bipolar cells and ribbon synapse) in the Pnpla2+/‐ retinas were lower than in controls. Ablation of Pnpla2 in mice causes malformation of photoreceptors and affects photoreceptor performance, identifying PEDF‐R as an important component for photoreceptor structure and function. These findings suggest that the absence of PEDF‐R in photoreceptor cells interrupts PEDF neurotrophic action, thereby heightening degeneration in the mice.

3-Indoleacrylic acid from canola straw as a promising antialgal agent - Inhibition effect and mechanism on bloom-forming Prorocentrum donghaiense

Harmful algal blooms (HABs) have induced severe damage worldwide. A novel high-efficient antialgal natural chemical, 3-indoleacrylic acid (3-IDC) with a 5-day half-maximal inhibitory concentration (IC50, 5d), was discovered from canola straw, and its algal inhibition mechanism was investigated. Adverse effects were observed on the growth of P. donghaiense with 3-IDC addition, following an increase in reactive oxygen species (ROS) production. 3-IDC also hindered the photosynthetic mechanism of P. donghaiense cells. Transcriptional results showed 3-IDC inhibiting the functions of all the nutrient assimilating genes, down-regulated ribulose-1,5-bisphosphate carboxylase/oxygenase II, and cytochrome f genes. The expression of heat shock protein (HSP) 70 and 90 and rhodopsin genes were also suppressed. The binding affinity of investigated receptors was observed. The conformational changes induced by the spatial microstructural alteration through 3-IDC may further contribute to the perturbation of those enzyme catalytic activities. The present results provide new insights on controlling HABs using 3-IDC.