12,595 research outputs found

    An in situ hybridization approach distinguishes <i>M-opsin</i> and <i>L-opsin</i> mRNA.

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    (A) Alignment of human M-opsin and L-opsin mRNA. Green bar = nucleotide difference. Horizontal pink and blue lines = location of in situ hybridization probes. (B) Alignment of portions of exon 5 from M- and L-opsin. In situ hybridization probes target mRNA sequences, indicated by blue (M-opsin) and pink (L-opsin) boxes. Green arrowheads indicate 8 nucleotide differences. Dots indicate nucleotide alignment between the opsins. (C–H) HEK293 cells probed for M-opsin mRNA (blue) and L-opsin mRNA (pink). Insets = schematic of transfected plasmid. Cells that did not express M-opsin mRNA or L-opsin mRNA were not quantified. (C) Quantification of transfected HEK293 cells expressing M-opsin mRNA only, L-opsin mRNA only, or M-opsin mRNA and L-opsin mRNA for the conditions in (D–H). (D–H) Brightfield images of cells with: (D) No plasmid transfected. (E) Transfection of a plasmid driving M-opsin. (F) Transfection of a plasmid driving L-opsin. (G) Transfection of either a plasmid driving M-opsin or a plasmid driving L-opsin independently and then the cells were mixed. (H) Transfection of both a plasmid driving M-opsin and a plasmid driving L-opsin. (I) Visualization of M-opsin mRNA, L-opsin mRNA, and M-/L-opsin protein (black) in HEK293 cells transfected with both a plasmid driving M-opsin and a plasmid driving L-opsin. M-opsin (blue) and L-opsin (pink). Blue arrow indicates a cell expressing M-opsin mRNA only. Pink arrows indicate cells expressing L-opsin mRNA only. Purple arrow indicates a cell expressing both M-opsin mRNA and L-opsin mRNA. Black arrow indicates an untransfected cell. Cells were identified based on nuclear Hoechst staining (S1A Fig). With this combined RNA in situ hybridization/immunohistochemistry approach, the mRNA signal was reduced, when compared to the mRNA signal observed when RNA in situ hybridization was conducted alone (Fig 1H). (J) Quantification of M-/L-opsin mRNA and M/L-opsin protein expression in transfected HEK293 cells (I). Original data sets are in S1 Data. (K) Quantification of M-/L-opsin mRNA and M/L-opsin protein expression in adult human retina (L). Original data sets are in S1 Data. (L) Visualization of M-opsin mRNA, L-opsin mRNA, and M-/L-opsin protein in cone cells in an adult human retina. M-opsin (blue) and L-opsin (pink). No cones co-expressed M-opsin mRNA and L-opsin mRNA. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Cell boundaries were determined by identifying layers from a nuclear Hoechst stain (S1B Fig) and analyzing opsin protein immunohistochemistry signal from the ONL to the OPL.</p

    RA signaling induces <i>M-opsin</i> and inhibits <i>L-opsin</i> early in human retinal organoids.

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    (A–C) Expression of ALDH1A1, ALDH1A2, and ALDH1A3 in fetal human retinas by day of gestation and retinal region. CPM, log counts per million. Analyzed from [16]. Error bars for the 2 samples from fetal day 94 indicate SEM. Original data sets are in S3 Data. (A) Whole retina. (B) Central retina. (C) Periphery. (D) Black bars indicate temporal windows of 1.0 μm RA addition during retinal organoid culture. (E) Quantification of M and L cone ratios for RA treatments. For “No RA,” N = 3; for “RA to day 60,” N = 6; for “RA to day 130,” N = 3; and for “Late RA,” N = 5. One-way ANOVA with Dunnett’s multiple comparisons test: “No RA” L-opsin versus “RA to day 60” L-opsin, p L-opsin versus “RA to day 130” L-opsin p L-opsin versus “Late RA” L-opsin p = 0.9635. Error bars indicate SEM. * Indicates p p (F–I) M-opsin (blue) and L-opsin (pink) expression in organoids grown in different RA conditions (D), quantified in (E). White dotted outlines indicate M- or L-opsin-expressing cells. White lines indicate the edge of the organoid. (F) No RA. (G) RA to day 60. (H) RA to day 130. (I) Late RA.</p

    Comparing Order and Fluidity of Omega-3 Polyunsaturated Fatty Acid Membranes Using Molecular Dynamics Simulations: An Epidemiologic Approach

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    In contemporary Western society, chronic diseases such as cardiovascular disease, mental health disorders, and eye disorders pose significant socioeconomic burdens. Despite extensive research on mitigating these issues, the role of omega-3 polyunsaturated fatty acids (PUFAs), essential nutrients obtained through dietary sources, remains a topic of ongoing debate concerning their efficacy in treating these diseases. This study utilized molecular dynamics to delve into the molecular effects of omega-3 PUFAs on cellular membranes, focusing on their impact on cell membrane fluidity and the surrounding molecular environment. Five molecular dynamics systems were created and simulated to investigate the PUFA species docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and tetracosapentaenoic acid (TPA). Three systems excluded integrated proteins, while the other two integrated dark-state opsin or meta II state rhodopsin with DHA. The lipid composition included head groups like phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol. Various analyses were conducted to assess membrane fluidity, thickness, and lipid-protein interactions. Results indicated that DHA and EPA exhibited higher fluidity in polyunsaturated tails, particularly near phospholipid head groups. The membrane thickness varied, with EPA being the thinnest and TPA being the thickest. Cholesterol distribution studies revealed differences in head and tail group positioning, influenced by membrane thickness. The cholesterol tilt was consistent although DHA and TPA displayed slightly lower angles relative to the bilayer normal. Further analyses highlighted phospholipid acyl chain concentrations and PUFA-cholesterol interactions, revealing closer associations between saturated phospholipid tails and cholesterol relative to the unsaturated Sn-2 tails. Molecular dynamics snapshots illustrated differences in protein-phospholipid interactions among various states of rhodopsin. These realizations are crucial for understanding the public health implications of PUFA's effects on membrane structures. Analyzing fluidity patterns and the distribution of atoms and molecules provides a clearer picture, allowing inference of the role of omega-3 PUFAs in disease prevention strategies targeting cardiovascular, mental, eye, and other diseases, influenced by cell signaling and membrane dynamics. Considering the evolutionary perspective of Western dietary patterns, the results prompt reflection on these shifts and advocate for holistic nutrition interventions to promote long-term public health

    BaseScope in situ probes.

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    Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.</div

    Expression of <i>M-opsin</i> mRNA and <i>L-opsin</i> mRNA in fetal and adult human retinas.

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    (A) 20 μm sections of 122-day-old and 130-day-old human fetal retinas. M-opsin (blue) and L-opsin (pink). White arrow indicates L-opsin-expressing cell. (B) Quantification of % M and L cones in 122-day-old and 130-day-old human fetal retinas. (C–F) M- and L-opsin mRNA expression in developing human fetal retinas and adult retinas. Values indicate total pileup count, normalized to total read count. Each data point indicates detection of M- or L-opsin mRNAs, based an individual nucleotide difference. N = 1 for each time point, except 94–2 where N = 2. 52/54 = exact date is unclear. Analyzed from [16,17]. Original data sets are in S2 Data. (C) M-opsin mRNA in fetal retinas. (D) L-opsin mRNA in fetal retinas. (E) M-opsin mRNA in adult retinas. (F) L-opsin mRNA in adult retinas. (G) Schematic of adult human retina with regions isolated using a 5 mm biopsy punch. White circle = optic nerve. Red lines = blood vessels. Yellow circle = macular pigment. (H–J) 20 μm sections were probed for M-opsin (blue) and L-opsin (pink) mRNA. (K) Average ratios of M and L cones as percent of M/L total cones across 3 individuals. One-way ANOVA with Tukey’s multiple comparisons test: Center L versus Middle L = no significance; Center L versus Periphery L p p p S2 Data.</p

    Organoid replicates and cell lines.

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    Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.</div

    Data that underlies Fig 1J, 1K.

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    Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.</div

    Opsin cDNA plasmids.

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    Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.</div

    Neuromodulatory effects on early visual signal processing

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    Understanding how the brain processes information and generates simple to complex behavior constitutes one of the core objectives in systems neuroscience. However, when studying different neural circuits, their dynamics and interactions researchers often assume fixed connectivity, overlooking a crucial factor - the effect of neuromodulators. Neuromodulators can modulate circuit activity depending on several aspects, such as different brain states or sensory contexts. Therefore, considering the modulatory effects of neuromodulators on the functionality of neural circuits is an indispensable step towards a more complete picture of the brain’s ability to process information. Generally, this issue affects all neural systems; hence this thesis tries to address this with an experimental and computational approach to resolve neuromodulatory effects on cell type-level in a well-define system, the mouse retina. In the first study, we established and applied a machine-learning-based classification algorithm to identify individual functional retinal ganglion cell types, which enabled detailed cell type-resolved analyses. We applied the classifier to newly acquired data of light-evoked retinal ganglion cell responses and successfully identified their functional types. Here, the cell type-resolved analysis revealed that a particular principle of efficient coding applies to all types in a similar way. In a second study, we focused on the issue of inter-experimental variability that can occur during the process of pooling datasets. As a result, further downstream analyses may be complicated by the subtle variations between the individual datasets. To tackle this, we proposed a theoretical framework based on an adversarial autoencoder with the objective to remove inter-experimental variability from the pooled dataset, while preserving the underlying biological signal of interest. In the last study of this thesis, we investigated the functional effects of the neuromodulator nitric oxide on the retinal output signal. To this end, we used our previously developed retinal ganglion cell type classifier to unravel type-specific effects and established a paired recording protocol to account for type-specific time-dependent effects. We found that certain retinal ganglion cell types showed adaptational type-specific changes and that nitric oxide had a distinct modulation of a particular group of retinal ganglion cells. In summary, I first present several experimental and computational methods that allow to study functional neuromodulatory effects on the retinal output signal in a cell type-resolved manner and, second, use these tools to demonstrate their feasibility to study the neuromodulator nitric oxide

    The IUPHAR/BPS Guide to PHARMACOLOGY in 2024

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    The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb; https://www.guidetopharmacology.org) is an open-access, expert-curated, online database that provides succinct overviews and key references for pharmacological targets and their recommended experimental ligands. It includes over 3039 protein targets and 12 163 ligand molecules, including approved drugs, small molecules, peptides and antibodies. Here, we report recent developments to the resource and describe expansion in content over the six database releases made during the last two years. The database update section of this paper focuses on two areas relating to important global health challenges. The first, SARS-CoV-2 COVID-19, remains a major concern and we describe our efforts to expand the database to include a new family of coronavirus proteins. The second area is antimicrobial resistance, for which we have extended our coverage of antibacterials in partnership with AntibioticDB, a collaboration that has continued through support from GARDP. We discuss other areas of curation and also focus on our external links to resources such as PubChem that bring important synergies to the resources. [Abstract copyright: © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
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