Spatial detection of mitochondrial DNA and RNA in tissues.

Background: Mitochondrial health has gained attention in a number of diseases, both as an indicator of disease state and as a potential therapeutic target. The quality and amount of mitochondrial DNA (mtDNA) and RNA (mtRNA) can be important indicators of mitochondrial and cell health, but are difficult to measure in complex tissues. Methods: mtDNA and mtRNA in zebrafish retina samples were fluorescently labeled using RNAscope™ in situ hybridization, then mitochondria were stained using immunohistochemistry. Pretreatment with RNase was used for validation. Confocal images were collected and analyzed, and relative amounts of mtDNA and mtRNA were reported. Findings regarding mtDNA were confirmed using qPCR. Results: Signals from probes detecting mtDNA and mtRNA were localized to mitochondria, and were differentially sensitive to RNase. This labeling strategy allows for quantification of relative mtDNA and mtRNA levels in individual cells. As a demonstration of the method in a complex tissue, single photoreceptors in zebrafish retina were analyzed for mtDNA and mtRNA content. An increase in mtRNA but not mtDNA coincides with proliferation of mitochondria at night in cones. A similar trend was measured in rods. Discussion: Mitochondrial gene expression is an important component of cell adaptations to disease, stress, or aging. This method enables the study of mtDNA and mtRNA in single cells of an intact, complex tissue. The protocol presented here uses commercially-available tools, and is adaptable to a range of species and tissue types.

Comparative analysis of in-silico tools in identifying pathogenic variants in dominant inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.

Selective transcriptomic dysregulation of metabolic pathways in liver and retina by short- and long-term dietary hyperglycemia.

A high glycemic index (HGI) diet induces hyperglycemia, a risk factor for diseases affecting multiple organ systems. Here, we evaluated tissue-specific adaptations in the liver and retina after feeding HGI diet to mice for 1 or 12 month. In the liver, genes associated with inflammation and fatty acid metabolism were altered within 1 month of HGI diet, whereas 12-month HGI diet-fed group showed dysregulated expression of cytochrome P450 genes and overexpression of lipogenic factors including Srebf1 and Elovl5. In contrast, retinal transcriptome exhibited HGI-related notable alterations in energy metabolism genes only after 12 months. Liver fatty acid profiles in HGI group revealed higher levels of monounsaturated and lower levels of saturated and polyunsaturated fatty acids. Additionally, HGI diet increased blood low-density lipoprotein, and diet-aging interactions affected expression of mitochondrial oxidative phosphorylation genes in the liver and disease-associated genes in retina. Thus, our findings provide new insights into retinal and hepatic adaptive mechanisms to dietary hyperglycemia.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Tracking widespread climate-driven change on temperate and tropical reefs.

Warming seas, marine heatwaves, and habitat degradation are increasingly widespread phenomena affecting marine biodiversity, yet our understanding of their broader impacts is largely derived from collective insights from independent localized studies. Insufficient systematic broadscale monitoring limits our understanding of the true extent of these impacts and our capacity to track these at scales relevant to national policies and international agreements. Using an extensive time series of co-located reef fish community structure and habitat data spanning 12 years and the entire Australian continent, we found that reef fish community responses to changing temperatures and habitats are dynamic and widespread but regionally patchy. Shifts in composition and abundance of the fish community often occurred within 2 years of environmental or habitat change, although the relative importance of these two mechanisms of climate impact tended to differ between tropical and temperate zones. The clearest of these changes on temperate and subtropical reefs were temperature related, with responses measured by the reef fish thermal index indicating reshuffling according to the thermal affinities of species present. On low latitude coral reefs, the community generalization index indicated shifting dominance of habitat generalist fishes through time, concurrent with changing coral cover. Our results emphasize the importance of maintaining local ecological detail when scaling up datasets to inform national policies and global biodiversity targets. Scaled-up ecological monitoring is needed to discriminate among increasingly diverse drivers of large-scale biodiversity change and better connect presently disjointed systems of biodiversity observation, indicator research, and governance.

A Health System's Response to the Ongoing Global Shortage of Iodinated Contrast Media.

A production facility shutdown related to containment measures during the COVID-19 pandemic has resulted in a global shortage of iodinated contrast media. This article describes the strategies implemented at one large U.S. health system to maintain care continuity during the ongoing shortage. The strategies have included attempts to procure additional stock, repackage existing stock for use in larger numbers of patients, use noncontrast CT or alternative imaging modalities in place of contrast-enhanced CT, and collaborate with specialties outside of radiology to participate in conservation efforts. In addition, individual CT protocols underwent tailored modifications to use dual-energy technique and/or lower tube voltages, to allow lower contrast media doses with maintained visualization of tissue enhancement. The experiences during this period provide insights to facilitate long-term reductions in contrast media doses and ongoing CT protocol optimization after supplies return to normal levels. Critical throughout the efforts to mitigate the impact of the shortage have been system-level action, operational flexibility, and close communication by the health system's radiologists, technologists, physicists, pharmacists, and ordering providers.

GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products.

BACKGROUND: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. RESULTS: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein-glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. CONCLUSIONS: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.

Daily mitochondrial dynamics in cone photoreceptors.

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.

Increasing Ca2+ in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress.

Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.

Assessing National Biodiversity Trends for Rocky and Coral Reefs through the Integration of Citizen Science and Scientific Monitoring Programs.

Reporting progress against targets for international biodiversity agreements is hindered by a shortage of suitable biodiversity data. We describe a cost-effective system involving Reef Life Survey citizen scientists in the systematic collection of quantitative data covering multiple phyla that can underpin numerous marine biodiversity indicators at high spatial and temporal resolution. We then summarize the findings of a continental- and decadal-scale State of the Environment assessment for rocky and coral reefs based on indicators of ecosystem state relating to fishing, ocean warming, and invasive species and describing the distribution of threatened species. Fishing impacts are widespread, whereas substantial warming-related change affected some regions between 2005 and 2015. Invasive species are concentrated near harbors in southeastern Australia, and the threatened-species index is highest for the Great Australian Bight and Tasman Sea. Our approach can be applied globally to improve reporting against biodiversity targets and enhance public and policymakers' understanding of marine biodiversity trends.

Corrigendum: Assessing National Biodiversity Trends for Rocky and Coral Reefs through the Integration of Citizen Science and Scientific Monitoring Programs.

[This corrects the article DOI: 10.1093/biosci/biw180.].