Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Twelve-month Natural History Study of Centrosomal Protein 290 (CEP290)-associated Inherited Retinal Degeneration.

Purpose: To define the clinical characteristics of centrosomal protein 290 (CEP290)-associated inherited retinal degeneration (IRD) and determine which assessments may provide reliable endpoints in future interventional trials. Design: Participants in this natural history study were enrolled into 2 best-corrected visual acuity (BCVA) cohorts: light perception to > 1.0 logarithm of the minimum angle of resolution (logMAR) and 1.0 logMAR to 0.4 logMAR. Each comprised 4 age cohorts (3-5, 6-11, 12-17, and ≥ 18 years). Participants: Patients with CEP290-associated IRD caused by the intron 26 c.2991+1655A>G mutation and BCVA ranging from light perception to 0.4 logMAR. Methods: Best-corrected visual acuity, full-field stimulus threshold (FST) sensitivity, Ora-Visual Navigation Challenge (Ora-VNC) composite score, and OCT-outer nuclear layer (OCT-ONL) average thickness were assessed at screening, baseline, 3 months, 6 months, and 12 months. Main Outcome Measures: Best-corrected visual acuity, FST sensitivity, Ora-VNC composite score, and OCT-ONL average thickness. Results: Twenty-six participants were included in this analysis. Nineteen were female. All participants were White and 4 reported Hispanic ethnicity. At screening, 13 of 16 adult and 9 of 10 pediatric participants had BCVA > 1.0 logMAR. Baseline BCVA was variable (median [range] = 2.0 [0.5, 3.9] logMAR) and was uncorrelated with age, as were VNC composite score, FST sensitivity, and OCT-ONL average thickness. Mean (95% confidence interval [CI]) test-retest variability was -0.04 (-0.09, 0.01) logMAR for BCVA (n = 25); 0.6 (-0.1, 1.3) for VNC composite score (n = 18); and 0.10 (-0.07, 0.27) log cd.s/m2 for red FST (n = 14). A greater than expected test-retest variability (5 [0, 10] µm, n = 14) was observed for OCT-ONL average thickness as nystagmus impacted ability to repeat measures at the same retinal location. Functional assessments were stable over 12 months. Mean (95% CI) change from baseline was 0.06 (-0.17, 0.29) logMAR for BCVA (n = 23); -0.1 (-1.2, 1.0) for VNC composite score (n = 21); and -0.15 (-0.43, 0.14) log cd.s/m2 for red FST (n = 16). Conclusions: Vision was stable over 12 months. Best-corrected visual acuity, FST, and VNC composite score are potentially viable endpoints for future studies in CEP290-associated IRD. Repeatability of OCT measures poses challenges for quantifying anatomical changes in this population. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

Structure-based network analysis predicts pathogenic variants in human proteins associated with inherited retinal disease.

Advances in gene sequencing technologies have accelerated the identification of genetic variants, but better tools are needed to understand which are causal of disease. This would be particularly useful in fields where gene therapy is a potential therapeutic modality for a disease-causing variant such as inherited retinal disease (IRD). Here, we apply structure-based network analysis (SBNA), which has been successfully utilized to identify variant-constrained amino acid residues in viral proteins, to identify residues that may cause IRD if subject to missense mutation. SBNA is based entirely on structural first principles and is not fit to specific outcome data, which makes it distinct from other contemporary missense prediction tools. In 4 well-studied human disease-associated proteins (BRCA1, HRAS, PTEN, and ERK2) with high-quality structural data, we find that SBNA scores correlate strongly with deep mutagenesis data. When applied to 47 IRD genes with available high-quality crystal structure data, SBNA scores reliably identified disease-causing variants according to phenotype definitions from the ClinVar database. Finally, we applied this approach to 63 patients at Massachusetts Eye and Ear (MEE) with IRD but for whom no genetic cause had been identified. Untrained models built using SBNA scores and BLOSUM62 scores for IRD-associated genes successfully predicted the pathogenicity of novel variants (AUC = 0.851), allowing us to identify likely causative disease variants in 40 IRD patients. Model performance was further augmented by incorporating orthogonal data from EVE scores (AUC = 0.927), which are based on evolutionary multiple sequence alignments. In conclusion, SBNA can used to successfully identify variants as causal of disease in human proteins and may help predict variants causative of IRD in an unbiased fashion.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Measuring pulsatile cortical blood flow and volume during carotid endarterectomy.

Carotid endarterectomy (CEA) involves removal of plaque in the carotid artery to reduce the risk of stroke and improve cerebral perfusion. This study aimed to investigate the utility of assessing pulsatile blood volume and flow during CEA. Using a combined near-infrared spectroscopy/diffuse correlation spectroscopy instrument, pulsatile hemodynamics were assessed in 12 patients undergoing CEA. Alterations to pulsatile amplitude, pulse transit time, and beat morphology were observed in measurements ipsilateral to the surgical side. The additional information provided through analysis of pulsatile hemodynamic signals has the potential to enable the discovery of non-invasive biomarkers related to cortical perfusion.

Coding and non-coding variants in the ciliopathy gene CFAP410 cause early-onset non-syndromic retinal degeneration.

Inherited retinal degenerations are blinding genetic disorders characterized by high genetic and phenotypic heterogeneity. The implementation of next-generation sequencing in routine diagnostics, together with advanced clinical phenotyping including multimodal retinal imaging, have contributed to the increase of reports describing novel genotype-phenotype associations and phenotypic expansions. In this study, we describe sixteen families with early-onset non-syndromic retinal degenerations in which affected probands carried rare bi-allelic variants in CFAP410, a ciliary gene previously associated with syndromic recessive Jeune syndrome. The most common retinal phenotypes were cone-rod and rod-cone dystrophies, but the clinical presentations were unified by their early onset as well as the severe impact on central visual function. Twelve variants were detected (three pathogenic, seven likely pathogenic, two of uncertain significance), eight of which were novel. One deep intronic change, c.373+91A>G, led to the creation of a cryptic splice acceptor site in intron four, followed by the inclusion of a 200- base pair pseudoexon and subsequent premature stop codon formation. To our knowledge this is the first likely pathogenic deep-intronic variant identified in this gene. Meta-analysis of all published and novel CFAP410 variants revealed no clear correlation between the severity of the CFAP410-associated phenotypes and the identified causal variants. This is supported by the fact that the frequently encountered missense variant p.(Arg73Pro), often found in syndromic cases, was also associated with non-syndromic retinal degeneration. This study expands the current knowledge of CFAP410-associated ciliopathy by enriching its mutational landscape and supports its association with non-syndromic retinal degeneration.

Generation of a human induced pluripotent stem cell line (OGIi001) from peripheral blood mononuclear cells of a healthy male donor.

We have successfully derived a novel human induced pluripotent stem cell (hiPSC) line using non-integrative Sendai virus. This hiPSC line was generated from a healthy male adult donor, aged 55, and subjected to thorough characterization and extensive quality control. The analysis confirmed the expression of undifferentiated stem cell markers, demonstrated the ability to differentiate into the three germ layers, and revealed the absence of any chromosomal abnormalities.

Sulfur speciation in Sphagnum peat moss modified by mutualistic interactions with cyanobacteria.

Peat moss (Sphagnum spp.) develops mutualistic interactions with cyanobacteria by providing carbohydrates and S compounds in exchange for N-rich compounds, potentially facilitating N inputs into peatlands. Here, we evaluate how colonization of Sphagnum angustifolium hyaline cells by Nostoc muscorum modifies S abundance and speciation at the scales of individual cells and across whole leaves. For the first time, S K-edge X-ray Absorption Spectroscopy was used to identify bulk and micron-scale S speciation across isolated cyanobacteria colonies, and in colonized and uncolonized leaves. Uncolonized leaves contained primarily reduced organic S and oxidized sulfonate- and sulfate-containing compounds. Increasing Nostoc colonization resulted in an enrichment of S and changes in speciation, with increases in sulfate relative to reduced S and sulfonate. At the scale of individual hyaline cells, colonized cells exhibited localized enrichment of reduced S surrounded by diffuse sulfonate, similar to observations of cyanobacteria colonies cultured in the absence of leaves. We infer that colonization stimulates plant S uptake and the production of sulfate-containing metabolites that are concentrated in stem tissues. Sulfate compounds that are produced in response to colonization become depleted in colonized cells where they may be converted into reduced S metabolites by cyanobacteria.

Exome copy number variant detection, analysis and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and with new innovative methods can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the GREGoR consortium. Each family's CNV data was analyzed using the seqr platform and candidate CNVs classified using the 2020 ACMG/ClinGen CNV interpretation standards. We developed additional evidence criteria to address situations not covered by the current standards. The addition of CNV calling to exome analysis identified causal CNVs for 173 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb with estimates that 44% would not have been detected by standard chromosomal microarrays. The causal CNVs consisted of 141 deletions, 15 duplications, 4 suspected complex structural variants (SVs), 3 insertions and 10 complex SVs, the latter two groups being identified by orthogonal validation methods. We interpreted 153 CNVs as likely pathogenic/pathogenic and 20 CNVs as high interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Early prediction of response to palliative chemotherapy in patients with stage-IV gastric and esophageal cancer.

BACKGROUND: The goal of therapy for many patients with advanced stage malignancies, including those with metastatic gastric and esophageal cancers, is to extend overall survival while also maintaining quality of life. After weighing the risks and benefits of treatment with palliative chemotherapy (PC) with non-curative intent, many patients decide to pursue treatment. It is known that a subset of patients who are treated with PC experience significant side effects without clinically significant survival benefits from PC. METHODS: We use data from 150 patients with stage-IV gastric and esophageal cancers to train machine learning models that predict whether a patient with stage-IV gastric or esophageal cancers would benefit from PC, in terms of increased survival duration, at very early stages of the treatment. RESULTS: Our findings show that machine learning can predict with high accuracy whether a patient will benefit from PC at the time of diagnosis. More accurate predictions can be obtained after only two cycles of PC (i.e., about 4 weeks after diagnosis). The results from this study are promising with regard to potential improvements in quality of life for patients near the end of life and a potential overall survival benefit by optimizing systemic therapy earlier in the treatment course of patients.

Dissolution Potential of Elemental Mercury in the Presence of Bisulfide and Implications for Mobilization.

Liquid elemental mercury (Hg0L) pollution can remain in soils for decades and, over time, will undergo corrosion, a process in which the droplet surface oxidizes soil constituents to form more reactive phases, such as mercury oxide (HgO). While these reactive coatings may enhance Hg migration in the subsurface, little is known about the transformation potential of corroded Hg0L in the presence of reduced inorganic sulfur species to form sparingly soluble HgS particles, a process that enables the long-term sequestration of mercury in soils and generally reduces its mobility and bioavailability. In this study, we investigated the dissolution of corroded Hg0L in the presence of sulfide by quantifying rates of aqueous Hg release from corroded Hg0L droplets under different sulfide concentrations (expressed as the S:Hg molar ratio). For droplets corroded in ambient air, no differences in soluble Hg release were observed among all sulfide exposure levels (S:Hg mole ratios ranging from 10-4 to 10). However, for droplets oxidized in the presence of a more reactive oxidant (hydrogen peroxide, H2O2), we observed a 10- to 25-fold increase in dissolved Hg when the oxidized droplets were exposed to low sulfide concentrations (S:Hg ratios from 10-4 to 10-1) relative to droplets exposed to high sulfide concentrations. These results suggest two critical factors that dictate the release of soluble Hg from Hg0L in the presence of sulfide: the extent of surface corrosion of the Hg0L droplet and sufficient sulfide concentration for the formation of HgS solids. The mobilization of Hg0L in porous media, therefore, largely depends on aging conditions in the subsurface and chemical reactivity at the Hg0L droplet interface.

Extracellular Vesicle RNA Contents as Biomarkers for Ocular Diseases.

Extracellular vesicles (EVs) are small vesicles secreted from cells into extracellular space. EVs contain proteins, lipids, and nucleic acids of the cells from which they originate. For this reason, EVs are being studied for use as biomarkers as they can be surrogates for the status of the cell from which they are secreted. Moreover, EVs are found in numerous biofluids and can be taken up by other cells, which allows for transfer of functional cargo, like RNAs, and changes in gene regulation in the recipient cell. Several potential RNA biomarkers have been identified in many diseases, and there is great potential in the vision field for extracellular RNA biomarkers as a diagnostic tool as well as a measure for treatment efficacy.

Role of Nuclear NAD+ in Retinal Homeostasis.

The retina is one of the most metabolically active tissues and maintenance of metabolic homeostasis is critical for retinal function. Nicotinamide adenine dinucleotide (NAD+) is a cofactor that is required for key processes, including the electron transport chain, glycolysis, fatty acid oxidation, and redox reactions. NAD+ also acts as a co-substrate for enzymes involved in maintaining genomic DNA integrity and cellular homeostasis, including poly-ADP ribose polymerases (PARPs) and Sirtuins. This review highlights the importance of NAD+ in the retina, including the role of enzymes involved in NAD+ production in the retina and how NAD+-consuming enzymes may play a role in disease pathology. We also suggest a cell death pathway that may be common in multiple models of photoreceptor degeneration and highlight the role that NAD+ likely plays in this process. Finally, we explore future experimental approaches to enhance our understanding of the role of NAD+ in the retina.

Structure-based network analysis predicts mutations associated with inherited retinal disease.

With continued advances in gene sequencing technologies comes the need to develop better tools to understand which mutations cause disease. Here we validate structure-based network analysis (SBNA)1,2 in well-studied human proteins and report results of using SBNA to identify critical amino acids that may cause retinal disease if subject to missense mutation. We computed SBNA scores for genes with high-quality structural data, starting with validating the method using 4 well-studied human disease-associated proteins. We then analyzed 47 inherited retinal disease (IRD) genes. We compared SBNA scores to phenotype data from the ClinVar database and found a significant difference between benign and pathogenic mutations with respect to network score. Finally, we applied this approach to 65 patients at Massachusetts Eye and Ear (MEE) who were diagnosed with IRD but for whom no genetic cause was found. Multivariable logistic regression models built using SBNA scores for IRD-associated genes successfully predicted pathogenicity of novel mutations, allowing us to identify likely causative disease variants in 37 patients with IRD from our clinic. In conclusion, SBNA can be meaningfully applied to human proteins and may help predict mutations causative of IRD.

Natural history of retinitis pigmentosa based on genotype, vitamin A/E supplementation, and an electroretinogram biomarker.

BACKGROUNDA randomized clinical trial from 1984 to 1992 indicated that vitamin A supplementation had a beneficial effect on the progression of retinitis pigmentosa (RP), while vitamin E had an adverse effect.METHODSSequencing of banked DNA samples from that trial provided the opportunity to determine whether certain genotypes responded preferentially to vitamin supplementation.RESULTSThe genetic solution rate was 587 out of 765 (77%) of sequenced samples. Combining genetic solutions with electroretinogram outcomes showed that there were systematic differences in severity and progression seen among different genetic subtypes of RP, extending findings made for USH2A, RHO, RPGR, PRPF31, and EYS. Baseline electroretinogram 30-Hz flicker implicit time was an independent, strong predictor of progression rate. Using additional data and baseline implicit time as a predictor, the deleterious effect of vitamin E was still present. Surprisingly, the effect of vitamin A progression in the cohort as a whole was not detectable, with or without data from subsequent trials. Subgroup analyses are also discussed.CONCLUSIONOverall, genetic subtype and implicit time have significant predictive power for a patient's rate of progression, which is useful prognostically. While vitamin E supplementation should still be avoided, these data do not support a generalized neuroprotective effect of vitamin A for all types of RP.TRIAL REGISTRATIONClinicalTrials.gov NCT00000114, NCT00000116, and NCT00346333.FUNDINGFoundation Fighting Blindness and the National Eye Institute: R01 EY012910, R01 EY031036, R01 EY026904, and P30 EY014104.

Adsorption and intracellular uptake of mercuric mercury and methylmercury by methanotrophs and methylating bacteria.

The cell surface adsorption and intracellular uptake of mercuric mercury Hg(II) and methylmercury (MeHg) are important in determining the fate and transformation of Hg in the environment. However, current information is limited about their interactions with two important groups of microorganisms, i.e., methanotrophs and Hg(II)-methylating bacteria, in aquatic systems. This study investigated the adsorption and uptake dynamics of Hg(II) and MeHg by three strains of methanotrophs, Methylomonas sp. strain EFPC3, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath, and two Hg(II)-methylating bacteria, Pseudodesulfovibrio mercurii ND132 and Geobacter sulfurreducens PCA. Distinctive behaviors of these microorganisms towards Hg(II) and MeHg adsorption and intracellular uptake were observed. The methanotrophs took up 55-80% of inorganic Hg(II) inside cells after 24 h incubation, lower than methylating bacteria (>90%). Approximately 80-95% of MeHg was rapidly taken up by all the tested methanotrophs within 24 h. In contrast, after the same time, G. sulfurreducens PCA adsorbed 70% but took up <20% of MeHg, while P. mercurii ND132 adsorbed <20% but took up negligible amounts of MeHg. These results suggest that microbial surface adsorption and intracellular uptake of Hg(II) and MeHg depend on the specific types of microbes and appear to be related to microbial physiology that requires further detailed investigation. Despite being incapable of methylating Hg(II), methanotrophs play important roles in immobilizing both Hg(II) and MeHg, potentially influencing their bioavailability and trophic transfer. Therefore, methanotrophs are not only important sinks for methane but also for Hg(II) and MeHg and can influence the global cycling of C and Hg.

Expression of NMNAT1 in the photoreceptors is sufficient to prevent NMNAT1-associated retinal degeneration.

Nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a ubiquitously expressed enzyme involved in nuclear NAD+ production throughout the body. However, mutations in the NMNAT1 gene lead to retina-specific disease with few reports of systemic effects. We have previously demonstrated that AAV-mediated gene therapy using self-complementary AAV (scAAV) to ubiquitously express NMNAT1 throughout the retina prevents retinal degeneration in a mouse model of NMNAT1-associated disease. We aimed to develop a better understanding of the cell types in the retina that contribute to disease pathogenesis in NMNAT1-associated disease, and to identify the cell types that require NMNAT1 expression for therapeutic benefit. To achieve this goal, we treated Nmnat1V9M/V9M mice with scAAV using cell type-specific promoters to restrict NMNAT1 expression to distinct retinal cell types. We hypothesized that photoreceptors are uniquely vulnerable to NAD+ depletion due to mutations in NMNAT1. Consistent with this hypothesis, we identified that treatments that drove NMNAT1 expression in the photoreceptors led to preservation of retinal morphology. These findings suggest that gene therapies for NMNAT1-associated disease should aim to express NMNAT1 in the photoreceptor cells.

Sonochemical oxidation and stabilization of liquid elemental mercury in water and soil.

Over 3000 mercury (Hg)-contaminated sites worldwide contain liquid metallic Hg [Hg(0)l] representing a continuous source of elemental Hg(0) in the environment through volatilization and solubilization in water. Currently, there are few effective treatment technologies available to remove or sequester Hg(0)l in situ. We investigated sonochemical treatments coupled with complexing agents, polysulfide and sulfide, in oxidizing Hg(0)l and stabilizing Hg in water, soil and quartz sand. Results indicate that sonication is highly effective in breaking up and oxidizing liquid Hg(0)l beads via acoustic cavitation, particularly in the presence of polysulfide. Without complexing agents, sonication caused only minor oxidation of Hg(0)l but increased headspace gaseous Hg(0)g and dissolved Hg(0)aq in water. However, the presence of polysulfide essentially stopped Hg(0) volatilization and solubilization. As a charged polymer, polysulfide was more effective than sulfide in oxidizing Hg(0)l and subsequently stabilizing the precipitated metacinnabar (ß-HgS) nanocrystals. Sonochemical treatments with sulfide yielded incomplete oxidation of Hg(0)l, likely resulting from the formation of HgS coatings on the dispersed µm-size Hg(0)l bead surfaces. Sonication with polysulfide also resulted in rapid oxidation of Hg(0)l and precipitation of HgS in quartz sand and in the Hg(0)l-contaminated soil. This research indicates that sonochemical treatment with polysulfide could be an effective means in rapidly converting Hg(0)l to insoluble HgS precipitates in water and sediments, thereby preventing its further emission and release to the environment. We suggest that future studies are performed to confirm its technical feasibility and treatment efficacy for remediation applications.

Inhibition of Methylmercury and Methane Formation by Nitrous Oxide in Arctic Tundra Soil Microcosms.

Climate warming causes permafrost thaw predicted to increase toxic methylmercury (MeHg) and greenhouse gas [i.e., methane (CH4), carbon dioxide (CO2), and nitrous oxide (N2O)] formation. A microcosm incubation study with Arctic tundra soil over 145 days demonstrates that N2O at 0.1 and 1 mM markedly inhibited microbial MeHg formation, methanogenesis, and sulfate reduction, while it slightly promoted CO2 production. Microbial community analyses indicate that N2O decreased the relative abundances of methanogenic archaea and microbial clades implicated in sulfate reduction and MeHg formation. Following depletion of N2O, both MeHg formation and sulfate reduction rapidly resumed, whereas CH4 production remained low, suggesting that N2O affected susceptible microbial guilds differently. MeHg formation strongly coincided with sulfate reduction, supporting prior reports linking sulfate-reducing bacteria to MeHg formation in the Arctic soil. This research highlights complex biogeochemical interactions in governing MeHg and CH4 formation and lays the foundation for future mechanistic studies for improved predictive understanding of MeHg and greenhouse gas fluxes from thawing permafrost ecosystems.