A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

Global mercury concentrations in biota: their use as a basis for a global biomonitoring framework.

An important provision of the Minamata Convention on Mercury is to monitor and evaluate the effectiveness of the adopted measures and its implementation. Here, we describe for the first time currently available biotic mercury (Hg) data on a global scale to improve the understanding of global efforts to reduce the impact of Hg pollution on people and the environment. Data from the peer-reviewed literature were compiled in the Global Biotic Mercury Synthesis (GBMS) database (>550,000 data points). These data provide a foundation for establishing a biomonitoring framework needed to track Hg concentrations in biota globally. We describe Hg exposure in the taxa identified by the Minamata Convention: fish, sea turtles, birds, and marine mammals. Based on the GBMS database, Hg concentrations are presented at relevant geographic scales for continents and oceanic basins. We identify some effective regional templates for monitoring methylmercury (MeHg) availability in the environment, but overall illustrate that there is a general lack of regional biomonitoring initiatives around the world, especially in Africa, Australia, Indo-Pacific, Middle East, and South Atlantic and Pacific Oceans. Temporal trend data for Hg in biota are generally limited. Ecologically sensitive sites (where biota have above average MeHg tissue concentrations) have been identified throughout the world. Efforts to model and quantify ecosystem sensitivity locally, regionally, and globally could help establish effective and efficient biomonitoring programs. We present a framework for a global Hg biomonitoring network that includes a three-step continental and oceanic approach to integrate existing biomonitoring efforts and prioritize filling regional data gaps linked with key Hg sources. We describe a standardized approach that builds on an evidence-based evaluation to assess the Minamata Convention's progress to reduce the impact of global Hg pollution on people and the environment.

Ectomycorrhizal fungi integrate nitrogen mobilisation and mineral weathering in boreal forest soil.

Tree growth in boreal forests is driven by ectomycorrhizal fungal mobilisation of organic nitrogen and mineral nutrients in soils with discrete organic and mineral horizons. However, there are no studies of how ectomycorrhizal mineral weathering and organic nitrogen mobilisation processes are integrated across the soil profile. We studied effects of organic matter (OM) availability on ectomycorrhizal functioning by altering the proportions of natural organic and mineral soil in reconstructed podzol profiles containing Pinus sylvestris plants, using 13 CO2 pulse labelling, patterns of naturally occurring stable isotopes (26 Mg and 15 N) and high-throughput DNA sequencing of fungal amplicons. Reduction in OM resulted in nitrogen limitation of plant growth and decreased allocation of photosynthetically derived carbon and mycelial growth in mineral horizons. Fractionation patterns of 26 Mg indicated that magnesium mobilisation and uptake occurred primarily in the deeper mineral horizon and was driven by carbon allocation to ectomycorrhizal mycelium. In this horizon, relative abundance of ectomycorrhizal fungi, carbon allocation and base cation mobilisation all increased with increased OM availability. Allocation of carbon through ectomycorrhizal fungi integrates organic nitrogen mobilisation and mineral weathering across soil horizons, improving the efficiency of plant nutrient acquisition. Our findings have fundamental implications for sustainable forest management and belowground carbon sequestration.

Investigating the diet source influence on freshwater fish mercury bioaccumulation and fatty acids-Experiences from Swedish lakes and Chinese reservoirs.

Dietary uptake is key for transferring potentially toxic contaminants, such as mercury (Hg) and essential dietary nutrients, such as polyunsaturated fatty acids (PUFA), to consumers at higher trophic levels of aquatic food webs. We evaluated the role of diet sources for Hg bioaccumulation and PUFA retention in fish across lake food webs in seven Swedish lakes and two Chinese reservoirs. Fish total Hg (THg) and methyl-Hg (MeHg) differed greatly between the two countries: the Chinese fish contained less than 300 ng g-1 dry weight (d.w.) THg with less than 50% as MeHg, versus the Swedish fishes which contained approximately 2000 ng g-1 d.w. THg and nearly 100% as MeHg. Fatty acids enrichment of linoleic acids (LIN) were more prevalent in the Chinese fishes regardless of size (p < 0.05). Here we examined food web length, fish growth rates, and fatty acids patterns in relation to the quality of fish as a food source for both Hg and FA. Contrary to the expectation that biodilution of Hg throughout the food chain would explain these differences, a more complex picture emerged with high levels of Hg at the base of the food web in the Chinese reservoirs, a decoupling of fatty acid and Hg bioaccumulation, and a major role for both fish stocking and fish feed. It is hoped that this work will provide a nuanced picture of fish quality as a food source in different ecosystems.

The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.

ROESY and 13C NMR to distinguish between D- and L-rhamnose in the α-D-Manp-(1 → 4)-ß-Rhap-(1 → 3) repeating motif.

Many children suffering from autism spectrum disorder (ASD) experience gastrointestinal (GI) conditions. Enterocloster bolteae has been regularly detected in the stool of individuals suffering from GI symptoms and autism. Literature has suggested that E. bolteae strains WAL 16351 and WAL 14578 produce an immunogenic capsular polysaccharide (CPS) comprised of disaccharide repeating units: α-D-Man-(1 → 4)-ß-Rha-(1 → 3) that could be used for the development of an immunotherapeutic vaccine. Ambiguity in the configuration of rhamnose led to the synthesis of tri- and disaccharide analogues containing D-rhamnose and L-rhamnose, respectively. ROESY-NMR spectra showed that CH3-6 of rhamnose and H-2 of mannose in the L-Rha containing disaccharide gave correlation. No such correlation was seen between the CH3-6 of rhamnose and the H-2 of mannose in the D-Rha containing trisaccharide. Molecular dynamics studies on hexasaccharide containing L-Rha or D-Rha confirmed that these structures adopt conformations resulting in different distances between the C6-rhamnose and the H-2 mannose of the preceding residue. We also demonstrate that assignment of the absolute configuration of the rhamnosyl residue in the ß-Rhap-(1 → 3)-D-Man linkage can be determined using the 13C chemical shift of C-2 in of D-Mannose. While ß-D-Rha will lead to an upfield shift of C-2 due to γ-gauche interaction between H-1 Rha and H-2 Man, ß-L-Rha will not. Our results provide insights to distinguish between D- and L-rhamnose in the α-D-Manp-(1 → 4)-ß-Rhap-(1 → 3) repeating motif.

Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish.

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.

Fast Ion-Conducting Thioboracite with a Perovskite Topology and Argyrodite-like Lithium Substructure.

We report a new fast ion-conducting lithium thioborate halide, Li6B7S13I, that crystallizes in either a cubic or tetragonal thioboracite structure, which is unprecedented in boron-sulfur chemistry. The cubic phase exhibits a perovskite topology and an argyrodite-like lithium substructure that leads to superionic conduction with a theoretical Li-ion conductivity of 5.2 mS cm-1 calculated from ab initio molecular dynamics (AIMD). Combined single-crystal X-ray diffraction, neutron powder diffraction, and AIMD simulations elucidate the Li+-ion conduction pathways through 3D intra- and intercage connections and Li-ion site disorder, which are all essential for high lithium mobility. Furthermore, we demonstrate that Li+ ordering in the tetragonal polymorph impedes lithium-ion conduction, thus highlighting the importance of the lithium substructure and lattice symmetry in dictating transport properties.

Splicing factor DHX15 affects tp53 and mdm2 expression via alternate splicing and promoter usage.

DHX15, a DEAH box containing RNA helicase, is a splicing factor required for the last step of splicing. Recent studies identified a recurrent mutational hotspot, R222G, in DHX15 in ∼ 6% of acute myeloid leukemia (AML) patients that carry the fusion protein RUNX1-RUNX1T1 produced by t (8;21) (q22;q22). Studies using yeast mutants showed that substitution of G for the residue equivalent to R222 leads to loss of its helicase function, suggesting that it is a loss-of-function mutation. To elucidate the role of DHX15 during development, we established the first vertebrate knockout model with CRISPR/Cas9 in zebrafish. Our data showed that dhx15 expression is enriched in the brain, eyes, pectoral fin primordia, liver and intestinal bulb during embryonic development. Dhx15 deficiency leads to pleiotropic morphological phenotypes in homozygous mutant embryos starting at 3 days post fertilization (dpf) that result in lethality by 7 dpf, revealing an essential role during embryonic development. RNA-seq analysis suggested important roles of Dhx15 in chromatin and nucleosome assembly and regulation of the Mdm2-p53 pathway. Interestingly, exons corresponding to the alternate transcriptional start sites for tp53 and mdm2 were preferentially expressed in the mutant embryos, leading to significant upregulation of their alternate isoforms, Δ113p53 (orthologous to Δ133p53 isoform in human) and mdm2-P2 (isoform using distal promoter P2), respectively. We speculate that these alterations in the Mdm2-p53 pathway contribute to the development of AML in patients with t(8;21) and somatically mutated DHX15.

Compound heterozygous KCTD7 variants in progressive myoclonus epilepsy.

KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients' variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

Monitoring and assessment of environmental resources in the changing landscape of Ethiopia: a focus on forests and water.

Monitoring and Assessment (M&A) of environmental resources aims to support the formulation of policies and follow up on outcomes of their implementation. In this study, the state of M&A is explored for Ethiopia with a focus on forests and water resources. The study is intended to serve as recommendations for future M&A applications in Ethiopia, as well as fulfillment of SDGs and other national and international commitments. Expert meetings, key informant interviews, and selected document analysis served as sources of information. The findings were summarized using qualitative grading and institutional mapping. Basic results of the study are that monitoring data on climate and streamflow are standardized in forms that can be communicated to policymakers. Scantier and less standardized are environmental data on soils, sediment transport, forests, biodiversity, and air quality. Water quality, soil moisture, groundwater level, forest biomass, and soil carbon are rarely monitored and can only be found in reports or studies for the fulfillment of academic degree requirements. Resources like nutrient fluxes have rarely been documented, not at all in some cases. There is considerable scope for tapping both technological advances and experiences of citizen science and local participation in environmental governance to rapidly expand and improve monitoring from local to regional and national scales. The study showed that there is a need for establishing a coordinated national system for monitoring and assessing the status of the environment, including the use of natural resources. Communicating such data to the scientific and wider public will support evidence-based planning and policy-making towards national development.

Methanogenesis Is an Important Process in Controlling MeHg Concentration in Rice Paddy Soils Affected by Mining Activities.

Rice paddies are agricultural sites of special concern because the potent toxin methylmercury (MeHg), produced in rice paddy soils, accumulates in rice grains. MeHg cycling is mostly controlled by microbes but their importance in MeHg production and degradation in paddy soils and across a Hg concentration gradient remains unclear. Here we used surface and rhizosphere soil samples in a series of incubation experiments in combination with stable isotope tracers to investigate the relative importance of different microbial groups on MeHg production and degradation across a Hg contamination gradient. We showed that sulfate reduction was the main driver of MeHg formation and concentration at control sites, and that methanogenesis had an important and complex role in MeHg cycling as Hg concentrations increased. The inhibition of methanogenesis at the mining sites led to an increase in MeHg production up to 16.6-fold and a decrease in MeHg degradation by up to 77%, suggesting that methanogenesis is associated with MeHg degradation as Hg concentrations increased. This study broadens our understanding of the roles of microbes in MeHg cycling and highlights methanogenesis as a key control of MeHg concentrations in rice paddies, offering the potential for mitigation of Hg contamination and for the safe production of rice in Hg-contaminated areas.

Correction: ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

[This corrects the article DOI: 10.1371/journal.pgen.1006481.].

ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease.

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors.

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Quantum mechanical free energy profiles with post-quantization restraints: Binding free energy of the water dimer over a broad range of temperatures.

Free energy calculations are a crucial part of understanding chemical systems but are often computationally expensive for all but the simplest of systems. Various enhanced sampling techniques have been developed to improve the efficiency of these calculations in numerical simulations. However, the majority of these approaches have been applied using classical molecular dynamics. There are many situations where nuclear quantum effects impact the system of interest and a classical description fails to capture these details. In this work, path integral molecular dynamics has been used in conjunction with umbrella sampling, and it has been observed that correct results are only obtained when the umbrella sampling potential is applied to a single path integral bead post quantization. This method has been validated against a Lennard-Jones benchmark system before being applied to the more complicated water dimer system over a broad range of temperatures. Free energy profiles are obtained, and these are utilized in the calculation of the second virial coefficient as well as the change in free energy from the separated water monomers to the dimer. Comparisons to experimental and ground state calculation values from the literature are made for the second virial coefficient at higher temperature and the dissociation energy of the dimer in the ground state.