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Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we show that members of the LUC7 protein family differentially regulate two major classes of 5′ splice sites (5′SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5′SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5′SS with stronger consensus matching on the intron side of the near invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5′SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5′SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies evolved before the split between animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess similar specificity to their human counterparts, indicating that 5′SS regulation by LUC7 proteins is highly conserved. Non-coding introns are excised from RNA transcripts of most eukaryotic genes by splicing. Here, Kenny et al. show that LUC7 proteins promote the splicing of different subsets of introns with distinct 5′ splice site sequences, and that these preferences are conserved from plants to human.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation (“birthdate”) in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an “inside-out” layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.

Creutzfeldt–Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP , the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in , the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in may underlie sCJD, we performed deep DNA sequencing of in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed Parallel RNA and DNA analysis after Deep-sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables the first-ever simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation (\"birthdate\") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an \"inside-out\" layer formation of excitatory neurons as seen in other species. In addition our analysis allows the first estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition. Competing Interest Statement The authors have declared no competing interest.

Human LUC7 family proteins associate with the U1 small nuclear ribonucleoprotein (snRNP) complex. Mutation or deletion of LUC7L2 is associated with myeloid neoplasms, and depletion of LUC7L2 alters cellular metabolism. Here, we describe distinctive 5' splice site (5'SS) features of exons impacted by each of the three human LUC7s. We find that LUC7L2 and LUC7L enhance splicing of 'right-handed' 5'SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 preferentially enhances splicing of 'left-handed' 5'SS with stronger consensus matching on the exon side of the splice junction. Specificity for right- or left-handed 5'SS is conferred by the distinct structured N-terminal domains of LUC7L2 and LUC7L3. Evolutionary analysis shows that divergence of LUC7L3 and LUC7L2 subfamilies occurred prior to the divergence of plants from animals/fungi, and suggests that loss of the LUC7L3 subfamily from the fungal lineage contributed to the predominance of right-handed 5'SS in fungi.Competing Interest StatementThe authors have declared no competing interest.

Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we uncover that members of the LUC7 protein family differentially regulate two major classes of 5’ splice sites (5’SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5’SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5’SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5’SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5’SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies diverged before the divergence of animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess specificity similar to their human counterparts, indicating that 5’SS regulation by LUC7 proteins is deeply conserved.

A comprehensive dataset of microphysical and radiative properties of aerosols and clouds in the boundary layer in the vicinity of Barrow, Alaska, was collected in April 2008 during the Indirect and Semi-Direct Aerosol Campaign (ISDAC). ISDAC's primary aim was to examine the effects of aerosols, including those generated by Asian wildfires, on clouds that contain both liquid and ice. ISDAC utilized the Atmospheric Radiation Measurement Pro- gram's permanent observational facilities at Barrow and specially deployed instruments measuring aerosol, ice fog, precipitation, and radiation. The National Research Council of Canada Convair-580 flew 27 sorties and collected data using an unprecedented 41 stateof- the-art cloud and aerosol instruments for more than 100 h on 12 different days. Aerosol compositions, including fresh and processed sea salt, biomassburning particles, organics, and sulfates mixed with organics, varied between flights. Observations in a dense arctic haze on 19 April and above, within, and below the single-layer stratocumulus on 8 and 26 April are enabling a process-oriented understanding of how aerosols affect arctic clouds. Inhomogeneities in reflectivity, a close coupling of upward and downward Doppler motion, and a nearly constant ice profile in the single-layer stratocumulus suggests that vertical mixing is responsible for its longevity observed during ISDAC. Data acquired in cirrus on flights between Barrow and Fairbanks, Alaska, are improving the understanding of the performance of cloud probes in ice. Ultimately, ISDAC data will improve the representation of cloud and aerosol processes in models covering a variety of spatial and temporal scales, and determine the extent to which surface measurements can provide retrievals of aerosols, clouds, precipitation, and radiative heating.

Background: The current coronavirus 2019 (COVID-19) pandemic has prompted a multitude of public health response measures including social distancing, school cancellations, and cessation of organized sports. Purpose: To examine the impact of COVID-19 and corresponding public health measures on the characteristics of common pediatric musculoskeletal injuries associated with sports. Study Design: Cohort study; Level of evidence, 3. Methods: This was a multicenter retrospective cohort study comparing patients with sports injuries presenting to 3 geographically diverse level I pediatric trauma hospitals and outpatient orthopaedic surgery clinics in the United States during the COVID-19 pandemic and a prepandemic period at the same institutions. Patients were included if they presented for care between February 15 and July 15 in 2020 (pandemic cohort) or between March 15 and April 15 in 2018 and 2019 (prepandemic cohort). Results: Included were 1455 patients with an average age of 12.1 ± 4.5 years. When comparing patients presenting in 2018 and 2019 with those presenting in 2020, we observed a decrease in mean age during the pandemic (12.6 ± 4.0 vs 11.0 ± 5.2 years; P = .048). Additionally, a decrease in the proportion of injuries attributed to sports (48.8% vs 33.3%; P < .001) and those occurring at school (11.9% vs 4.0%; P = .001) was observed. The proportion of injuries attributable to clavicle fractures increased during the early stages of the pandemic (13.2% vs 34.7%; P < .001). There was no statistically significant delay to care in injuries presenting during the pandemic (41.5 ± 141.2 vs 19.23 ± 79.1 days; P = .175). Conclusion: Across 3 tertiary care institutions, patients were seen without significant delay during the pandemic. We observed a significant decline in pediatric musculoskeletal injuries associated with sports during the COVID-19 pandemic. This decrease has been accompanied by a shift in both injury type and mechanism.

The present study was designed to provide further information about the relevance of raised urinary levels of N-methylnicotinamide (NMN), and/or its metabolites N-methyl-4-pyridone-3-carboxamide (4PY) and N-methyl-2-pyridone-3-carboxamide (2PY), to peroxisome proliferation by dosing rats with known peroxisome proliferator-activated receptor alpha (PPARalpha) ligands [fenofibrate, diethylhexylphthalate (DEHP) and long-chain fatty acids (LCFA)] and other compounds believed to modulate lipid metabolism via PPARalpha-independent mechanisms (simvastatin, hydrazine and chlorpromazine). Urinary NMN was correlated with standard markers of peroxisome proliferation and serum lipid parameters with the aim of establishing whether urinary NMN could be used as a biomarker for peroxisome proliferation in the rat. Data from this study were also used to validate a previously constructed multivariate statistical model of peroxisome proliferation (PP) in the rat. The predictive model, based on 1H nuclear magnetic resonance (NMR) spectroscopy of urine, uses spectral patterns of NMN, 4PY and other endogenous metabolites to predict hepatocellular peroxisome count. Each treatment induced pharmacological (serum lipid) effects characteristic of their class, but only fenofibrate, DEHP and simvastatin increased peroxisome number and raised urinary NMN, 2PY and 4PY, with simvastatin having only a transient effect on the latter. These compounds also reduced mRNA expression for aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase, EC 4.1.1.45), the enzyme believed to be involved in modulating the flux of tryptophan through this pathway, with decreasing order of potency, fenofibrate (-10.39-fold) >DEHP (-3.09-fold) >simvastatin (-1.84-fold). Of the other treatments, only LCFA influenced mRNA expression of ACMSDase (-3.62-fold reduction) and quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) (-2.42-fold) without any change in urinary NMN excretion. Although there were no correlations between urinary NMN concentration and serum lipid parameters, NMN did correlate with peroxisome count (r2=0.63) and acyl-CoA oxidase activity (r2=0.61). These correlations were biased by the large response to fenofibrate compared to the other treatments; nevertheless the data do indicate a relationship between the tryptophan-NAD+ pathway and PPARalpha-dependent pathways, making this metabolite a potentially useful biomarker to detect PP. In order to strengthen the observed link between the metabolites associated with the tryptophan-NAD+ pathway and more accurately predict PP, other urinary metabolites were included in a predictive statistical model. This statistical model was found to predict the observed PP in 26/27 instances using a pre-determined threshold of 2-fold mean control peroxisome count. The model also predicted a time-dependent increase in peroxisome count for the fenofibrate group, which is important when considering the use of such modelling to predict the onset and progression of PP prior to its observation in samples taken at autopsy.