Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design ...of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.
A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with ...schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10⁻⁶. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10⁻⁶) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10⁻⁸) postsynaptic signalling complexes. In an analysis of 18 492 subjects (7907 cases and 10 585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.
Human breast tumours are diverse in their natural history and in their
responsiveness to treatments. Variation in transcriptional
programs accounts for much of the biological diversity of human cells ...and
tumours. In each cell, signal transduction and regulatory systems transduce
information from the cell's identity to its environmental status, thereby
controlling the level of expression of every gene in the genome. Here we have
characterized variation in gene expression patterns in a set of 65 surgical
specimens of human breast tumours from 42 different individuals, using complementary
DNA microarrays representing 8,102 human genes. These patterns provided a
distinctive molecular portrait of each tumour. Twenty of the tumours were
sampled twice, before and after a 16-week course of doxorubicin chemotherapy,
and two tumours were paired with a lymph node metastasis from the same patient.
Gene expression patterns in two tumour samples from the same individual were
almost always more similar to each other than either was to any other sample.
Sets of co-expressed genes were identified for which variation in messenger
RNA levels could be related to specific features of physiological variation.
The tumours could be classified into subtypes distinguished by pervasive differences
in their gene expression patterns.
Soil/atmosphere exchange fluxes of nitrous oxide were monitored for a 3-year period at 10 grassland sites in eight European countries (Denmark, France, Hungary, Ireland, Italy, The Netherlands, ...Switzerland and United Kingdom), spanning a wide range of climatic, environmental and soil conditions. Most study sites investigated the influence of one or several management practices on N
2O exchange, such as nitrogen fertilization and grazing intensity. Fluxes were measured using non-steady state chambers at most sites, and alternative measurement techniques such as eddy covariance and fast-box using tunable diode laser spectroscopy were implemented at some sites. The overall uncertainty in annual flux estimates derived from chamber measurements may be as high as 50% due to the temporal and spatial variability in fluxes, which warrants the future use of continuous measurements, if possible at the field scale. Annual emission rates were higher from intensive than from extensive grasslands, by a factor 4 if grazed (1.77
versus 0.48
kg
N
2O-N
ha
−1
year
−1) and by a factor 3 if ungrazed (0.95
versus 0.32
kg
N
2O-N
ha
−1
year
−1). Annual emission factors for fertilized systems were highly variable, ranging from 0.01% to 3.56%, but the mean emission factor across all sites (0.75%) was substantially lower than the IPCC default value of 1.25%. Emission factors for individual fertilization events increased with soil temperature and were generally higher for water-filled pore space values in the range 60–90%, though precipitation onto dry soils was also shown to lead to high losses of N
2O-N from applied fertilizer. An empirical, multiple regression model to predict N
2O emission factors on the basis of soil temperature, moisture and rainfall is developed, explaining half of the variability in observed emission factors.
BackgroundIn 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset ...lymphoproliferation and autoimmunity.ObjectiveThis pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants.MethodsWe identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3.ResultsOverall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD4−CD8−) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate.Conclusion: STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome.
Abstract Objective To determine the effects of a policy of “use acupuncture” on headache, health status, days off sick, and use of resources in patients with chronic headache compared with a policy ...of “avoid acupuncture.” Design Randomised, controlled trial. Setting General practices in England and Wales. Participants 401 patients with chronic headache, predominantly migraine. Interventions Patients were randomly allocated to receive up to 12 acupuncture treatments over three months or to a control intervention offering usual care. Main outcome measures Headache score, SF-36 health status, and use of medication were assessed at baseline, three, and 12 months. Use of resources was assessed every three months. Results Headache score at 12 months, the primary end point, was lower in the acupuncture group (16.2, SD 13.7, n = 161, 34% reduction from baseline) than in controls (22.3, SD 17.0, n = 140, 16% reduction from baseline). The adjusted difference between means is 4.6 (95% confidence interval 2.2 to 7.0; P = 0.0002). This result is robust to sensitivity analysis incorporating imputation for missing data. Patients in the acupuncture group experienced the equivalent of 22 fewer days of headache per year (8 to 38). SF-36 data favoured acupuncture, although differences reached significance only for physical role functioning, energy, and change in health. Compared with controls, patients randomised to acupuncture used 15% less medication (P = 0.02), made 25% fewer visits to general practitioners (P = 0.10), and took 15% fewer days off sick (P = 0.2). Conclusions Acupuncture leads to persisting, clinically relevant benefits for primary care patients with chronic headache, particularly migraine. Expansion of NHS acupuncture services should be considered.
Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been ...mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content.
Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over ...half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.
While children approaching end-stage kidney disease (ESKD) are considered at risk of uremic anorexia and underweight they are also exposed to the global obesity epidemic. We sought to investigate the ...variation of nutritional status in children undergoing chronic peritoneal dialysis (CPD) around the globe. The distribution and course of body mass index (BMI) standard deviation score over time was examined prospectively in 1001 children and adolescents from 35 countries starting CPD who were followed in the International Pediatric PD Network (IPPN) Registry. The overall prevalence of underweight, and overweight/obesity at start of CPD was 8.9% and 19.7%, respectively. Underweight was most prevalent in South and Southeast Asia (20%), Central Europe (16.7%) and Turkey (15.2%), whereas overweight and obesity were most common in the Middle East (40%) and the US (33%). BMI SDS at PD initiation was associated positively with current eGFR and gastrostomy feeding prior to PD start. Over the course of PD BMI SDS tended to increase on CPD in underweight and normal weight children, whereas it decreased in initially overweight patients. In infancy, mortality risk was amplified by obesity, whereas in older children mortality was markedly increased in association with underweight. Both underweight and overweight are prevalent in pediatric ESKD, with the prevalence varying across the globe. Late dialysis start is associated with underweight, while enteral feeding can lead to obesity. Nutritional abnormalities tend to attenuate with time on dialysis. Mortality risk appears increased with obesity in infants and with underweight in older children.
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