Lysosome-related organelles contain an expansion compartment that mediates delivery of zinc transporters to promote homeostasis.

Zinc is an essential nutrient-it is stored during periods of excess to promote detoxification and released during periods of deficiency to sustain function. Lysosome-related organelles (LROs) are an evolutionarily conserved site of zinc storage, but mechanisms that control the directional zinc flow necessary for homeostasis are not well understood. In Caenorhabditis elegans intestinal cells, the CDF-2 transporter stores zinc in LROs during excess. Here, we identify ZIPT-2.3 as the transporter that releases zinc during deficiency; ZIPT-2.3 transports zinc, localizes to the membrane of LROs in intestinal cells, and is necessary for zinc release from LROs and survival during zinc deficiency. In zinc excess and deficiency, the expression levels of CDF-2 and ZIPT-2.3 are reciprocally regulated at the level of mRNA and protein, establishing a fundamental mechanism for directional flow to promote homeostasis. To elucidate how the ratio of CDF-2 and ZIPT-2.3 is altered, we used super-resolution microscopy to demonstrate that LROs are composed of a spherical acidified compartment and a hemispherical expansion compartment. The expansion compartment increases in volume during zinc excess and deficiency. These results identify the expansion compartment as an unexpected structural feature of LROs that facilitates rapid transitions in the composition of zinc transporters to mediate homeostasis, likely minimizing the disturbance to the acidified compartment.

HONO Measurement by Catalytic Conversion to NO on Nafion Surfaces.

A selective catalytic converter has been developed to quantify nitrous acid (HONO), a photochemical precursor to NO and OH radicals that drives the formation of ozone and other pollutants in the troposphere. The converter is made from a sulfonated tetrafluoroethylene-based fluoropolymer-copolymer (Nafion) that was found to convert HONO to NO with unity yield under specific conditions. When coupled to a commercially available NOx (=NO + NO2) chemiluminescence (CL) analyzer, the system measures HONO with a limit of detection as low as 64 parts-per-trillion (ppt) (1 min average) in addition to NOx. The converter is selective for HONO when tested against other common gas-phase reactive nitrogen species, although loss of O3 on Nafion is a potential interference. The sensitivity and selectivity of this method allow for accurate measurement of atmospherically relevant concentrations of HONO. This was demonstrated by good agreement between HONO measurements made with the Nafion-CL method and those made with chemical ionization mass spectrometry in a simulation chamber and in indoor air. The observed reactivity of HONO on Nafion also has significant implications for the accuracy of CL NOx analyzers that use Nafion to remove water from sampling lines.

de novo variant calling identifies cancer mutation signatures in the 1000 Genomes Project.

Detection of de novo variants (DNVs) is critical for studies of disease-related variation and mutation rates. To accelerate DNV calling, we developed a graphics processing units-based workflow. We applied our workflow to whole-genome sequencing data from three parent-child sequenced cohorts including the Simons Simplex Collection (SSC), Simons Foundation Powering Autism Research (SPARK), and the 1000 Genomes Project (1000G) that were sequenced using DNA from blood, saliva, and lymphoblastoid cell lines (LCLs), respectively. The SSC and SPARK DNV callsets were within expectations for number of DNVs, percent at CpG sites, phasing to the paternal chromosome of origin, and average allele balance. However, the 1000G DNV callset was not within expectations and contained excessive DNVs that are likely cell line artifacts. Mutation signature analysis revealed 30% of 1000G DNV signatures matched B-cell lymphoma. Furthermore, we found variants in DNA repair genes and at Clinvar pathogenic or likely-pathogenic sites and significant excess of protein-coding DNVs in IGLL5; a gene known to be involved in B-cell lymphomas. Our study provides a new rapid DNV caller for the field and elucidates important implications of using sequencing data from LCLs for reference building and disease-related projects.

Microbial mechanisms and ecosystem flux estimation for aerobic NOy emissions from deciduous forest soils.

Reactive nitrogen oxides (NOy; NOy = NO + NO2 + HONO) decrease air quality and impact radiative forcing, yet the factors responsible for their emission from nonpoint sources (i.e., soils) remain poorly understood. We investigated the factors that control the production of aerobic NOy in forest soils using molecular techniques, process-based assays, and inhibitor experiments. We subsequently used these data to identify hotspots for gas emissions across forests of the eastern United States. Here, we show that nitrogen oxide soil emissions are mediated by microbial community structure (e.g., ammonium oxidizer abundances), soil chemical characteristics (pH and C:N), and nitrogen (N) transformation rates (net nitrification). We find that, while nitrification rates are controlled primarily by chemoautotrophic ammonia-oxidizing archaea (AOA), the production of NOy is mediated in large part by chemoautotrophic ammonia-oxidizing bacteria (AOB). Variation in nitrification rates and nitrogen oxide emissions tracked variation in forest communities, as stands dominated by arbuscular mycorrhizal (AM) trees had greater N transformation rates and NOy fluxes than stands dominated by ectomycorrhizal (ECM) trees. Given mapped distributions of AM and ECM trees from 78,000 forest inventory plots, we estimate that broadleaf forests of the Midwest and the eastern United States as well as the Mississippi River corridor may be considered hotspots of biogenic NOy emissions. Together, our results greatly improve our understanding of NOy fluxes from forests, which should lead to improved predictions about the atmospheric consequences of tree species shifts owing to land management and climate change.

Nitrogen cycling microbiomes are structured by plant mycorrhizal associations with consequences for nitrogen oxide fluxes in forests.

Volatile nitrogen oxides (N2 O, NO, NO2 , HONO, …) can negatively impact climate, air quality, and human health. Using soils collected from temperate forests across the eastern United States, we show microbial communities involved in nitrogen (N) cycling are structured, in large part, by the composition of overstory trees, leading to predictable N-cycling syndromes, with consequences for emissions of volatile nitrogen oxides to air. Trees associating with arbuscular mycorrhizal (AM) fungi promote soil microbial communities with higher N-cycle potential and activity, relative to microbial communities in soils dominated by trees associating with ectomycorrhizal (ECM) fungi. Metagenomic analysis and gene expression studies reveal a 5 and 3.5 times greater estimated N-cycle gene and transcript copy numbers, respectively, in AM relative to ECM soil. Furthermore, we observe a 60% linear decrease in volatile reactive nitrogen gas flux (NOy  ≡ NO, NO2 , HONO) as ECM tree abundance increases. Compared to oxic conditions, gas flux potential of N2 O and NO increase significantly under anoxic conditions for AM soil (30- and 120-fold increase), but not ECM soil-likely owing to small concentrations of available substrate ( NO 3 - ) in ECM soil. Linear mixed effects modeling shows that ECM tree abundance, microbial process rates, and geographic location are primarily responsible for variation in peak potential NOy flux. Given that nearly all tree species associate with either AM or ECM fungi, our results indicate that the consequences of tree species shifts associated with global change may have predictable consequences for soil N cycling.

Oxidation of Volatile Organic Compounds as the Major Source of Formic Acid in a Mixed Forest Canopy.

Formic acid (HCOOH) is among the most abundant carboxylic acids in the atmosphere, but its budget is poorly understood. We present eddy flux, vertical gradient, and soil chamber measurements from a mixed forest and apply the data to better constrain HCOOH source/sink pathways. While the cumulative above-canopy flux was downward, HCOOH exchange was bidirectional, with extended periods of net upward and downward flux. Net above-canopy fluxes were mostly upward during warmer/drier periods. The implied gross canopy HCOOH source corresponds to 3% and 38% of observed isoprene and monoterpene carbon emissions and is 15× underestimated in a state-of-science atmospheric model (GEOS-Chem). Gradient and soil chamber measurements identify the canopy layer as the controlling source of HCOOH or its precursors to the forest environment; below-canopy sources were minor. A correlation analysis using an ensemble of marker volatile organic compounds suggests that secondary formation, not direct emission, is the major source driving ambient HCOOH.

Analysis of the northern pitcher plant (Sarracenia purpurea L.) phytotelm bacteriome throughout a temperate region growing season.

The insectivorous Northern Pitcher Plant, Sarracenia purpurea, recruits a dynamic biotic community in the rainwater collected by its pitcher-shaped leaves. Insect capture and degradation within the pitcher fluid (phytotelma) has been well documented as a mechanism for supplementing the plant's nitrogen, phosphorous, and micronutrient requirements. Metagenomic studies have shown a diverse microbiome in this phytotelm environment, including taxa that contribute metabolically to prey digestion. In this investigation, we used high-throughput 16S rDNA sequencing and bioinformatics to analyze the S. purpurea phytotelm bacteriome as it changes through the growing season (May-September) in plants from the north-central region of the species' native range. Additionally, we used molecular techniques to detect and quantify bacterial nitrogenase genes (nifH) in all phytotelm samples to explore the hypothesis that diazotrophy is an additional mechanism of supplying biologically available nitrogen to S. purpurea. The results of this study indicate that while prokaryote diversity remains relatively stable in plants at different locations within our region, diversity changes significantly as the growing season progresses. Furthermore, nifH genes were detected at biologically significant concentrations in one hundred percent of samples, suggesting that nitrogen fixation may be an important contributor to the S. purpurea nutrient budget.

Proteome-Wide Assessment of Clustering of Missense Variants in Neurodevelopmental Disorders Versus Cancer.

Missense de novo variants (DNVs) and missense somatic variants contribute to neurodevelopmental disorders (NDDs) and cancer, respectively. Proteins with statistical enrichment based on analyses of these variants exhibit convergence in the differing NDD and cancer phenotypes. Herein, the question of why some of the same proteins are identified in both phenotypes is examined through investigation of clustering of missense variation at the protein level. Our hypothesis is that missense variation is present in different protein locations in the two phenotypes leading to the distinct phenotypic outcomes. We tested this hypothesis in 1D protein space using our software CLUMP. Furthermore, we newly developed 3D-CLUMP that uses 3D protein structures to spatially test clustering of missense variation for proteome-wide significance. We examined missense DNVs in 39,883 parent-child sequenced trios with NDDs and missense somatic variants from 10,543 sequenced tumors covering five TCGA cancer types and two COSMIC pan-cancer aggregates of tissue types. There were 57 proteins with proteome-wide significant missense variation clustering in NDDs when compared to cancers and 79 proteins with proteome-wide significant missense clustering in cancers compared to NDDs. While our main objective was to identify differences in patterns of missense variation, we also identified a novel NDD protein BLTP2. Overall, our study is innovative, provides new insights into differential missense variation in NDDs and cancer at the protein-level, and contributes necessary information toward building a framework for thinking about prognostic and therapeutic aspects of these proteins.

A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis.

Genomic assemblies of the unicellular green alga Chlamydomonas reinhardtii have provided important resources for researchers. However, assembly errors, large gaps, and unplaced scaffolds as well as strain-specific variants currently impede many types of analysis. By combining PacBio HiFi and Oxford Nanopore long-read technologies, we generated a de novo genome assembly for strain CC-5816, derived from crosses of strains CC-125 and CC-124. Multiple methods of evaluating genome completeness and base-pair error rate suggest that the final telomere-to-telomere assembly is highly accurate. The CC-5816 assembly enabled previously difficult analyses that include characterization of the 17 centromeres, rDNA arrays on three chromosomes, and 56 insertions of organellar DNA into the nuclear genome. Using Nanopore sequencing, we identified sites of cytosine (CpG) methylation, which are enriched at centromeres. We analyzed CRISPR-Cas9 insertional mutants in the PF23 gene. Two of the three alleles produced progeny that displayed patterns of meiotic inviability that suggested the presence of a chromosomal aberration. Mapping Nanopore reads from pf23-2 and pf23-3 onto the CC-5816 genome showed that these two strains each carry a translocation that was initiated at the PF23 gene locus on chromosome 11 and joined with chromosomes 5 or 3, respectively. The translocations were verified by demonstrating linkage between loci on the two translocated chromosomes in meiotic progeny. The three pf23 alleles display the expected short-cilia phenotype, and immunoblotting showed that pf23-2 lacks the PF23 protein. Our CC-5816 genome assembly will undoubtedly provide an important tool for the Chlamydomonas research community.

Pilot-scale in situ bioremediation of HMX and RDX in soil pore water in Hawaii.

A nine-month in situ bioremediation study was conducted in Makua Military Reservation (MMR) in Oahu, Hawaii (USA) to evaluate the potential of molasses to enhance biodegradation of royal demolition explosive (RDX) and high-melting explosive (HMX) contaminated soil below the root zone. MMR has been in operation since the 1940's resulting in subsurface contamination that in some locations exceeds USEPA preliminary remediation goals for these chemicals. A molasses-water mixture (1 : 40 dilution) was applied to a treatment plot and clean water was applied to a control plot via seven flood irrigation events. Pore water samples were collected from 12 lysimeters installed at different depths in 3 boreholes in each test plot. The difference in mean concentrations of RDX in pore water samples from the two test plots was very highly significant (p < 0.001). The concentrations differences with depth were also very highly significant (p < 0.001) and degradation was greatly enhanced at depths from 5 to 13.5 ft. biodegradation was modeled as first order and the rate constant was 0.063 per day at 5 ft and decreased to 0.023 per day at 11 ft to 13.5 ft depth. Enhanced biodegradation of HMX was also observed in molasses treated plot samples but only at a depth of 5 ft. The difference in mean TOC concentration (surrogate for molasses) was highly significant with depth (p = 0.003) and very highly significant with treatment (p < 0.001). Mean total nitrogen concentrations also differed significantly with treatment (p < 0.001) and depth (p = 0.059). The molasses water mixture had a similar infiltration rate to that of plain water (average 4.12 ft per day) and reached the deepest sensor (31 ft) within 5 days of application. Most of the molasses was consumed by soil microorganisms by about 13.5 feet below ground surface and treatment of deeper depths may require greater molasses concentrations and/or more frequent flood irrigation. Use of the bioremediation method described herein could allow the sustainable use of live fire training ranges by enhancing biodegradation of explosives in situ and preventing them from migrating to through the vadose zone to underlying ground water and off-site.