UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in  UBE3A gene dysregulation is associated with neurodevelopmental disorders, but predicting the function of UBE3A variants remains difficult. The authors use a high-throughput assay to categorize variants by functional activity, and show that UBE3A hyperactivity increases the risk of neurodevelopmental disease. However, the clinical significance for the vast majority of known variants remains undetermined3, representing an immensely understudied area of human genetics.The identification of genes linked to human disorders traditionally focuses on lesions that can be interpreted easily as reducing gene function. Although this method has been effective in establishing causality, a simple loss-of-function model is insufficient to account for the broad phenotypic heterogeneity observed in many neurodevelopmental disorders. This is especially important for the millions of uncharacterized coding variants identified in the human genomeUBE3A encodes an E3 ubiquitin ligase that promotes the proteasomal degradation of proteins4. Abnormal changes in UBE3A activity are associated with various human conditions including human papilloma virus-mediated cancer4 and neurodevelopmental disorders. In neurons, UBE3A expression is epigenetically modified such that transcription of paternal UBE3A is silenced and only the maternally-inherited copy is expressed7. It is well established that loss of maternal UBE3A causes Angelman syndrome (AS), a severe form of intellectual disability characterized by epilepsy, motor deficits, dysmorphic facial features, and a unique happy demeanor10. In contrast, excessive UBE3A activity resulting from duplication of maternal chromosome 15q11-13, the region where UBE3A resides, is linked to an autistic disorder known as Dup15q syndrome11. However, there are only two known examples that specifically link UBE3A gain-of-function to neurodevelopmental disease. This includes one de novo hyperactivating missense mutation identified in a child with autism13, and a microduplication of UBE3A that segregates with neuropsychiatric phenotypes in one family14. These limited observations have raised questions about the extent to which excessive UBE3A activity contributes to neurodevelopmental pathology.15. They all possess the eponymous HECT domain which houses the biochemical machinery necessary to accept activated ubiquitin from E2 enzyme, and transfers it to substrate proteins. In previous work, we and others found that UBE3A activates WNT reporter gene expression in HEK293T cells in a manner that is dependent on its ubiquitin ligase activity19.UBE3A belongs to the HECT (Homologous to E6-AP C-terminus) domain superfamily of E3 ubiquitin ligases which are implicated in an array of human conditionsUBE3A variants identified in individuals. We discovered many mutations that impact UBE3A activity, including 18 hyperactivating mutations. By generating a knock-in mouse model, we show that hyperactivation of maternal, but not paternal UBE3A is sufficient to perturb neonatal motor behaviors and communication, and using patient data, we show that UBE3A hyperactivity produces phenotypes that are distinct from AS. Finally, using the rich structure–function data derived from our screen, we uncovered a conserved regulatory mechanism in UBE3A that is commonly perturbed by both loss and gain-of-function mutations. Together, our results provide strong evidence that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggest that detailed functional variant analysis can provide a widely-applicable method to identify mechanistic sub-classes of genetic disorders.Here, we used this insight to quantitatively assess the functional significance of 152 UBE3A missense variants contained in the Genome Aggregation (gnomAD v2.1.1)2 and ClinVar1 databases. Most variants are exceedingly rare  and nearly two-thirds of variants in ClinVar have conflicting interpretations or are classified as a variant of uncertain significance  UBE3A E3A Fig. , to deteE3A Fig. . WhereasUBE3A missense variants present in the ClinVar database and assessed their effect on UBE3A activity  finger known as the AZUL domain  that dictates UBE3A localization31. Six variants were located within the AZUL domain including a loss-of-function mutation that disrupts a Zn2+ chelating residue  and the adjacent amino acid  and T787, two positions that contained the strongest hyperactivating mutations in our screen , every hyperactivating mutation tested in our assay decreased RING1B protein levels. Indeed, R516Q (67.48% ± 3.68), R516W (60.16% ± 4.63), A521T (55.16% ± 4.71), Q588E (60.58% ± 4.44), L781H (52.31% ± 4.30), T787A (52.96% ± 6.31), and T787M (52.37% ± 4.91) all reduced RING1B abundance to a fraction of WT UBE3A levels  in NEDD4 and isoleucine 44 (I44) in ubiquitin, and another hydrophobic contact formed by two tyrosines (Y605 and Y634) that interact with leucine 73 (L73) near the C-terminus of ubiquitin  to perform constrained rigid-body docking with the Rosetta molecular modeling program44. Modeling showed that F665 in UBE3A contacts I44 of ubiquitin, and in functional experiments, we found that mutation of F665 to alanine (F665A) caused a near-complete loss of UBE3A activity in our BAR assay , proline (Q588P), and arginine (Q588R), all of which altered the overall charge in the ubiquitin-binding pocket of UBE3A  of 65.7 ± 28.5 μM  animals, we found that mQ588E pups accomplished this task just as effectively as their respective WT littermates  whereas no effect was seen in pQ588E animals  to determine whether early-life communication is affected in mQ588E animals. We found that on average, the number of USVs produced by WT animals increased gradually from 68.11 ± 12.67 calls at P5, to 80.81 ± 15.53 at P7, and 95.03 ± 19.10 at P9. In contrast, mQ588E animals produced dramatically fewer calls at P5 (12.57 ± 3.32) and at P9  of loss-of-function mutations identified in our screen were deemed pathogenic whereas most gain-of-function mutations were classified as VUS (84%) with a few designated as benign (16%) and none designated as pathogenic  and displayed autistic behaviors and was non-verbal until the age of 4 Fig. . It was ADAR1 variant that was inherited from the mother. However, as ADAR1 mutation causes an autosomal recessive disorder, this variant was not determined to be causative of disease phenotypes54. Individual 5 had three siblings. One adolescent sibling , possessed the same L726Δ variant and exhibited intellectual disability and ADHD. The cognitive ability of Individual 6 was estimated at 4 years of developmental age and required special resources at school  and raised UBE3A activity 180.75% ± 9.9 above WT levels. The individual presented with global developmental delay, microcephaly, and intractable epilepsy requiring vagal nerve stimulation placement. Delayed motor and language milestones were evident in early childhood and the proband was later diagnosed with quadriplegic cerebral palsy and severe intellectual disability. Individual 5 is currently G-tube dependent for feeding, possesses generalized muscle atrophy and spasticity in the lower extremities, pectus carinatum deformity, severe thoracolumbar scoliosis and thoracic kyphosis. Osteoporosis in the femoral head and neck was also evident. Individual 5 was found to be heterozygous for a pathogenic ool Fig. . IndividUBE3A revealed that ~12% of variants cause a gain of enzyme function. We found that hyperactivating mutations segregate with affected phenotypes in two unrelated families, and consistent with the neuronal imprinting of UBE3A, our mouse modeling and patient data both strongly suggest that hyperactivating mutations confer an increased risk for pathogenicity when present on the maternally-inherited allele.Historically, the need to infer the impact of mutations strictly from sequence changes has biased the discovery of disease-causing genes toward lesions that produce loss-of-function alleles. Our functional analyses of missense variants in 39, thereby implicating its dysfunction in UBE3A-dependent disorders. In general, the exosite is thought to facilitate ubiquitin chain elongation by binding the distal ubiquitin on a growing chain and stabilizing ubiquitinated target proteins to the E3 enzyme46. However, this mechanism appears context-dependent as ubiquitin binding to the exosite can also inhibit chain elongation40 and ubiquitin variants that bind the exosite with high affinity can alter enzyme activity in complex ways36. We also found that additional HECT domain enzymes possess VUS or pathogenic/likely pathogenic variants within their exosites  at a position that is analogous to Q588 in UBE3A and is associated with an X-linked form of intellectual disability57 , produced a loss-of-function response in our BAR assay , which allowed us to control for transfection efficiency and loading in our in vitro assays. All constructs were verified by sequencing. Disease-linked UBE3A missense mutations used in this study were identified in previous publications30, and are listed in the ClinVar1 and gnomAD databases2. Cloning for C21Y, R39H, T106P, T106K, C117R, I130T, A178T, N243K, E269G, L273F, I329T, S349P, R477P, M478I, R482P, T485A, L502P, R506C, E550K, and E550L variants were described previously12. For most constructs, variants were introduced by PCR using site-specific primers (Integrated DNA Technologies). A complete list of oligonucleotide sequences used in this study are presented in Supplementary Table 70. Primers matching this extended sequence were designed, annealed, and ligated using an internal SphI site and XmaI. To generate FLAG-tagged RING1B, we placed the epitope tag on the N-terminus of RING1B by polymerase chain reaction using a plasmid encoding a catalytically inactive human RING1B (RING1B I53S)71 and cloned into pCIG2 using SacI and XmaI sites. The vector to express the pGEX GST-TEV-Cys-ubiquitin used in the METRIS assay was acquired as a gift from Dr. Brenda Schulman (Max Planck) and described in a previous study72. pGL3-BAR and TK-Renilla constructs were described previously12. The pCDNA3.1 3× FLAG-tagged ubiquitin was a gift from Dr. Mark Zylka (University of North Carolina) and used in a previous study12. For UBE3A protein purification, an N-terminal 6×-His tag was added to each UBE3A missense variant by polymerase chain reaction and cloned into the pET-45b(+) plasmid (EMD Millipore) using SacI and NotI restriction sites. Primary antibodies used were mouse anti-Myc , mouse anti-UBE3A , mouse anti-GFP (Santa Cruz Biotechnology #sc-9996), and mouse anti-FLAG . All secondary antibodies were obtained from LI-COR Biosciences: donkey anti-rabbit 800CW (926-32213), donkey anti-rabbit 680RD (925-68073), donkey anti-mouse 800CW (926-32212), and donkey anti-mouse 680RD (926-68072); all used at a dilution of 1:10,000.The nomenclature used in this study was based on the amino acid sequence from human UBE3A isoform I (NCBI accession: NP_001361390.1). Myc epitope tags were placed on the N-terminus of UBE3A by polymerase chain reaction and all UBE3A constructs were cloned into pCIG2 plasmid DNA2 humidified incubator in DMEM containing 25 mM glucose, 0.4 mM glutamine, and 1 mM Sodium Pyruvate (Gibco #10569044), supplemented with 10% fetal bovine serum  and 1× antibiotic–antimycotic containing penicillin, streptomycin, and amphotericin B (ThermoFisher #15240062). For biochemical analyses, transfections were performed using FuGENE (Promega) according to the manufacturer’s instructions. HEK293T cells were lysed in 2× sample buffer (Fisher NuPAGE™ LDS Sample Buffer #NP0008) supplemented with 5 mM DTT and 1× EDTA-free Pierce protease inhibitor mini tablets (ThermoFisher #A32955). Lysates were boiled and proteins were resolved by 4–20% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 1× fish gelatin blocking agent (Biotium #22010) and probed with the appropriate primary antibodies overnight at 4 °C or 2 h at RT. Protein bands were visualized using the Odyssey CLx infrared imaging system and Image Studio v5.2 software (LI-Cor Biosciences) and western blots analyzed using ImageJ v2.1.0/1.53c.HEK293T cells (ATCC) were maintained in a 5% CORenilla, 30 ng of BAR-pGL3, and 30 ng of the indicated constructs using the FuGENE (Promega) transfection reagent, according to the manufacturer’s instructions. Forty-eight hours after transfection, reporter gene expression was assessed using the Dual-Luciferase reporter assay system (Promega) and measured on a Synergy HTX Multi-Mode Reader (BioTek) using Gen5 software v3.08. Luciferase activity was normalized against Renilla activity, and UBE3A missense variant signals were normalized to WT UBE3A.BAR assays were performed in 96-well plates for all experiments. HEK293T cells were plated at a density of 10,000/well. Cells were transiently transfected with 10 ng of pRL-TK-E. coli cells (Sigma-Aldrich #70954). The next day, a colony was picked and transferred into 1 mL of growth media , 0.5% glycerol, 100 µg/mL ampicillin, and 30 µg/mL chloramphenicol). Cultures were allowed to shake at 225 RPM at 37 °C for 2–5 h. Afterwards, 1 mL of culture was transferred into 50 mL of growth media and incubated overnight at 225 RPM at 25 °C. The next morning, 500 mL of induction media (inoculation media + 0.5% glucose) was inoculated to an OD600 of 0.4. The induction culture was shaken at 175 RPM at 37 °C until an O.D. of 0.7 was reached (~2 h). Five hundred microliters of 1 M IPTG was then added to induce UBE3A protein expression and the culture was allowed to shake at 175 RPM at 25 °C for 6 h. Cells were pelleted by centrifugation at 1425×g at 4 °C for 15 min. For protein purification, pellets were resuspended in 25 mL lysis buffer , 10 mM imidazole and lysed by sonication. After centrifugation at 15,000×g for 15 min at 4 °C, the resulting lysate was incubated with HisPur™ Ni-NTA Resin beads (ThermoFisher #88221) at 4 °C for 60 min. The beads were collected on elution columns and washed with wash buffer . UBE3A was eluted with 500 mM imidazole. UBE3A was further purified through size-exclusion chromatography and buffer exchange with 7 K MWCO Zeba™ Spin desalting columns (Thermo Scientific #89889) according to the manufacturer’s instructions .For protein expression, pET-45b(+) UBE3A constructs were transformed into chloramphenicol-resistant Rosetta™ BL21DE3 competent 12. In brief, a single reaction contained 1x E3 ligase reaction buffer, 0.1 µM UBE1, 0.5 µM UBE2L3 (UbcH7), 12.5 µM FLAG-ubiquitin, 2 µM ATP, and 4 µg of purified UBE3A. The reaction was allowed to proceed at room temperature for the indicated times. Reactions were stopped by adding 2× Laemmli buffer and boiling for 5 min. UBE3A ubiquitination was also assayed in HEK293T cells treated with 30 μM MG-132 for 1 h. The cells were then lysed in RIPA buffer containing 1% SDS and 30 μM MG-132. Cell lysates were boiled for 20 min and clarified by centrifugation at 15,000×g for 10 min. The resulting supernatant was diluted 1:10 (v/v) in an immunoprecipitation buffer . For UBE3A immunoprecipitation, an anti-Myc-conjugated affinity gel (Sigma #A7470) was used at 4 °C for 1 h. The final complex was washed three times with wash buffer (immunoprecipitation buffer containing 125 mM NaCl), resuspended in sample buffer, and subject to SDS-PAGE and immunoblot analysis.In vitro ubiquitination assays were performed using human His6 Ubiquitin-activating enzyme/UBE1 recombinant protein , 10× E3 ligase reaction buffer , UbcH7 recombinant protein , human FLAG-ubiquitin recombinant protein , and ATP. (Sigma). Assays were performed as described previously73. To build a starting structure we aligned UBE3A (PDB ID: 1C4Z)25 to NEDD4 in the NEDD4-ubiquitin co-complex (PDB ID: 4BBN)42, which has a complete ubiquitin tail. We used this starting structure with the relax application in Rosetta to generate 30,000 models of ubiquitin docked to UBE3A. To calculate the RMSD of the ubiquitin tail, we used ProFit with residues 71–76 of ubiquitin. The same protocol was used with WT UBE3A, and the Q588E and Q588R mutations which were generated using the Rosetta fixbb application74.To model ubiquitin bound to the exosite of UBE3A, we used the Rosetta Relax application through the Rosetta scripts interface3JVZ), HUWE1 (3H1D), WWP1 (6J1Y), and ITCH (3TUG).Sequence alignments of UBE3A from various species were performed using amino acid sequences with the following accession numbers: mouse (NP_001029134.1), frog (NP_001080693.1), fish (NP_001007319.1), and fly (NP_648452.1). Sequence alignments of UBE3A with human NEDD4 subfamily enzymes were performed using amino acid sequences from the following accession numbers: NEDD4 (NP_006145.2), NEDD4-2 (NP_001138439.1), HUWE1 (NP_113584.3), HECW1 (NP_055867.3), HECW2 (NP_001335697.1), SMURF1 (NP_001186776.1), SMURF2 (NP_073576.1), WWP1 (NP_008944.1), WWP2 (NP_001257383.1), and ITCH (NP_001311127.1). PDB IDs for additional crystal structures of HECT domains used in this study are as follows: NEDD4-2 . This experiment measures the translational displacement of magnetic particles, immobilized with ubiquitin, on a surface that contains immobilized UBE3A. Streptavidin-biotin was used to immobilize proteins to their respective material. To attach ubiquitin to the magnetic beads we used a commonly used ubiquitin variant with an N-terminal cysteine. Cysteine biotinylation was carried out using Poly(ethylene glycol) [N-carbamoyl]methyl ether 2-(biotinylamino)ethane (Sigma #757748) . Ubiquitin was singly biotinylated and purified over size exclusion chromatography, and dialyzed to remove excess biotin-maleamide.2. Ten liters of the bead slurry (1.0% w/v) was used to coat with biotinylated-ubiquitin at room temperature for at least 2 h. The amount of protein used was 50× the theoretical limit needed to coat each bead to that ensure all of the binding sites on the beads were occupied (1 mg of beads bound 0.16 nmoles of biotin). Biotinylated UBE3A was fixed to avidin-coated glass slides (Arrayit) with a ligand density of 1.1 × 1010 ligands per mm2. Microfluidic channels were created on this substrate using two pieces of double-sided tape (3 M). The amount of proteins inserted was enough to coat the channel surface 50× the theoretical limit to ensure that all of the sites on the substrate were coated. The substrate and solution were left in a sealed container for 2 h, then washed to remove excess unbound protein.The streptavidin coated ferromagnetic particles, provided by Spherotech were composed of a core of polystyrene and CrOCoated ferromagnetic beads were diluted approximately 2000-fold, to reduce the probability of bead aggregation. Beads were placed into channels on the coated slides and sealed. The sealed slide containing the magnetic particles and functionalized substrate was placed in a Helmholtz-inspired apparatus. A clockwise field was actuated at 1 Hz for 5 s, turned off for 5 s, then a counter-clockwise field was actuated at 1 Hz for 5 s, then turned off for 5 s. This actuation cycle was repeated 18 times. Video captured from the CMOS camera was converted into an.avi file using ArcSoft Media Converter 8 then converted into a sequence of jpeg images. Able Particle Tracker was used to track the motion of the beads and the data then imported into Mathematica for analysis.x is translational displacement, D is the diameter of the beads, τ is the actuation time, and ω is the frequency of the rotation of the magnetic field.To analyze the rolling of the particles, we used a dimensionless value called the rolling parameter (ζ) which is a ratio between the maximum possible rolling  and the observed rolling of the particles. The rolling parameter is given by the following equation:E. coli BL21 codon plus RIL (Agilent) cells, and expression was induced by adding 0.6 mM IPTG at 16 °C for 18 h. UBE3A was purified on a nickel affinity gravity column followed by TEV protease digestion and size exclusion purification on an AKTA FPLC . The purity of the fractions was confirmed by SDS-gel electrophoresis, combined, and concentrated to ~10 mg/mL for biophysical assays. N-terminal fluorescein-labeled ubiquitin (Anaspec) was prepared as described previously36. Mixtures of 1 µM fluorescein-labeled ubiquitin and varied concentrations of UBE3A were prepared in a 384-well plate. Milli-fluorescence polarization (mP) values were detected by a Tecan Infinite M1000Pro plate reader at 30 °C. Iterative nonlinear curve fitting using a one-site specific binding model was applied in ProFit to obtain binding affinities (KD).The HECT domain from WT UBE3A was cloned into pRSFDuet-1 (Novagen) with an Octa-histidine-GST double affinity tag fused to the N-terminus. A TEV protease cleavage sequence was introduced between His-GST and the HECT domain to excise the affinity tag. The UBE3A Q588E mutant was made by site-directed mutagenesis. Both wild type and UBE3A Q588E were transformed into All animal procedures were performed in accordance with guidelines set by the Washington University Institutional Animal Care and Use Committee. Mice were maintained in standard housing conditions, where food and water were provided ad libitum. Each generation was crossed to wildtype C57BL/J6 mice from Jackson labs.An analogous Q585E mutation in mouse UBE3A  was created by designing single guide RNAs (sgRNAs) to create a C<G substitution at Chr7:59,286,122 (GRCm38/mm10 assembly). The resulting mutation altered a CAG codon into GAG Fig. . The gRNFounders were crossed to WT C57/Bl6 mice (Jackson Laboratory #000664). Animals were backcrossed for at least six generations prior to use in the study. Mice were genotyped using either tail-tips or toe clippings. Tissue samples were incubated overnight at 55 °C in 400 µL tail lysis buffer . Proteinase K was inactivated by incubation at 95 °C for 10 min and genomic DNA purified by isopropanol precipitation. PCR was performed by amplifying a 407 bp fragment with ~200 bp flanking either side of the Q588 codon. The products were purified using a PCR purification kit (Qiagen) and submitted for Sanger sequencing (Genewiz). PCR was performed using GoTaq DNA polymerase (Promega #M3001). An initial denaturation step of 95 °C for 2 min was used followed by 35 cycles of 95 °C for 15 s, 43 °C for 15 s, and an extension step of 72 °C for 30 s.UBE3A were crossed with WT C57BL/6 males, and to generate pQ588E animals, heterozygous males possessing the Q588E mutation on the paternal copy were crossed with WT females. For all mQ588E tests used in this paper, a cohort of 45 mice in total was used: 20 female  and 25 male , and for all pQ588E tests, a cohort of 53 mice was used: 31 female (13 WT and 18 pQ588E) and 21 male (8 WT and 13 pQ588E). Although sex differences were assessed in all experiments, no significant differences observed and data from male and female mice was pooled for our analyses. All behavioral experiments were performed blind to genotype by a female experimenter and also analyzed blind to genotype.Mice were group housed in standard mouse cages with ad libitum access to food and water, and with a 12 h light/dark cycle in a temperature (~20–22 °C) and humidity-controlled (50% relative humidity) barrier facility. To generate mQ588E animals, heterozygous females possessing the Q588E mutation on the paternal copy of 50. Pups were habituated for at least 10 min in the testing room. At P10, grip strength, forelimb suspension, and negative geotaxis was assessed. On P14, grip strength, the surface righting reflex, and the grasping reflex were measured. Each task was performed by each mouse in triplicate and mice were allowed at least 1 min to rest between trials. The values of the three trials were averaged for analysis.Neonatal motor assessments were performed as described in refs. Negative geotaxis was determined by placing the pup head downward on a flat, padded 45° incline and holding it for five seconds to allow it to acclimate. The pup was released, and the time it took the pup to turn to face upward was measured (2 min max). Surface righting reflex was conducted by placing the pup on its back on a flat, padded surface for 5 s and recording the time until the pup positioned itself upright and touched all four paws on the surface. Grasping reflex was determined by holding the mouse vertically by the scruff of the neck, and gently stroking each paw, palm-to-fingertip, five times with the blunt, rounded side of a razor blade. A successful trial was recorded if the pup grasped the blade within five strokes. Forelimb suspension was measured by positioning pups in front of a stable wire strung horizontally across the opening of a wire cup with a padded bottom. Once the mouse grasped the wire with both forepaws, the mouse was released, and the time until the mouse dropped to the padded bottom of the cup was recorded. Grip strength was measured by placing the pup on a plastic mesh screen laid upon a flat, padded surface. Keeping one edge of the screen flat against the table, the screen was slowly rotated  until the screen was inverted (180°). A protractor was placed perpendicularly to the screen to measure the angle at which the pup fell from the screen.75 to assess early communicative behaviors. Pup vocalizations were recorded on postnatal days 5, 7, and 9 in the home colony room of the pups. For each recording session, the dam was placed in a separate cage, and the home cage containing the pups was placed in a warming box (Harvard Apparatus) set to 33 °C to ensure pup temperature in the nest remained between 34–37 °C. An individual pup was selected and its skin temperature taken from the lower dorsal region using a non-contact HDE Infrared Thermometer, as it is known that decreased body temperature results in increased number of USVs76. We did not observe a difference in body temperature between genotypes. The pup was then placed in an empty standard mouse cage which in turn was placed within an MDF sound-attenuating box (Med Associates) (36 × 64 × 60 cm). The doors of the sound-attenuating box were shut and USVs were detected using an Avisoft UltraSoundGate CM16 microphone positioned 5 cm above the bottom of the cage, along with the Avisoft UltraSoundGate 116H amplifier and the Avisoft v4.3.01 recorder software. Gain was set to 3 dB and the sampling rate set to 250 kHz. Pups were recorded for 3 min, after which their weight was taken and they were returned to their home cage within the warming box. MATLAB software was used to prepare sonograms of the recordings  using a previously published protocol75.USV recordings were performed as described in ref. https://www.ncbi.nlm.nih.gov/clinvar) is a freely accessible, public archive that catalogs relationships among human variants and phenotypes with supporting evidence. Information for this study was collected by searching for missense variants in UBE3A. For each variant, the resulting protein change, associated conditions, clinical significance, and accession number was recorded. In addition, de-identified evidence details were recorded if present in the database. For variants without evidence details, the submitting entity was contacted for additional information.The ClinVar database  with Benjamini–Hochberg multiple comparisons correction . BAR responses were normalized to WT UBE3A and tested for deviance from a theoretical mean value of 100. Individual p values are shown in Table p < 0.05, **p < 0.005, *p < 0.0005.Figure t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ***p < 0.0005.Figure t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ****p < 0.0005.Figure t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ****p < 0.0005.Figure t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ***p < 0.0005.Figure q = 8.580, p < 0.001, and between WT UBE3A × Q588R: q = 7.004, p < 0.001.Figure p < 0.05, **p < 0.005, ***p < 0.0005.Figure U-test, exact significance (two-tailed). *p < 0.05, **p < 0.005, ***p < 0.0005.Figure U-test, exact significance (two-tailed). *p < 0.05, **p < 0.005, ***p < 0.0005.Figure p < 0.05, **p < 0.005, ***p < 0.0005.Figure p < 0.05, **p < 0.005, ***p < 0.0005.Figure t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ***p < 0.0005.Supplementary Fig. t-test (two-tailed) with Bonferroni multiple comparisons correction . Mutant enzyme protein levels were normalized to WT UBE3A and tested for deviance from a theoretical mean abundance level of 100. *p < 0.05, **p < 0.005.Supplementary Fig. t-test (two-tailed) with Bonferroni multiple comparisons correction . RING1B protein levels for each UBE3A mutant-transfected sample was normalized to WT UBE3A and tested for deviance from a theoretical mean abundance level of 100. *p < 0.05, **p < 0.005.Supplementary Fig. t-test (two-tailed) with Benjamini–Hochberg multiple comparisons correction . *p < 0.05, **p < 0.005, ***p < 0.0005.Supplementary Fig. U-test, exact significance (two-tailed). *p < 0.05, **p < 0.005, ***p < 0.0005.Supplementary Fig. p < 0.05, **p < 0.005, ***p < 0.0005.Supplementary Fig. z-score histogram plot outputs. Mice that had a value greater than 3.29 standard deviations above the mean were deemed influential outliers and were removed from analysis. ANOVA assumption of equal variances was assessed with the Levene’s test. For all reported behavioral tests which were found to violate the assumptions of ANOVA, non-parametric tests were performed. Chi-square goodness of fit tests was used to assess associations between categorical variables. For tests performed across multiple ages, the linear mixed model was used to account for repeated measures.All behavioral statistical analyses were performed in SPSS (v.27). ANOVA assumption of normality was assessed by using the Shapiro–Wilks test and manual assessment of the Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Myospalax fontanieri), as a species to investigate (both in the laboratory and in the field) the possible factors responsible for burrow-sealing behavior. Our results showed that: (1) In the laboratory, the burrow-sealing frequency of plateau zokor in response to five factors were as follows: oxygen (52.63%) > light (34.58%) > temperature (20.24%) > gas flow (6.48%) > sound/control (0%). Except for light, the burrow-sealing frequency in response to other factors was significantly lower than that in response to oxygen (P < 0.05). (2) Burrow-sealing behavior in response to each treatment did not differ significantly between males and females in the laboratory experiment. (3) In the field, during the animal’s active periods in both the cold and warm season, the burrow-sealing frequency under the oxygen treatment was higher than that under the light and temperature treatments. Plateau zokors were found not to be sensitive to these treatments during their inactive periods during both the cold and warm season. (4) The latency to reseal the burrow showed no obvious differences between each treatment both in the laboratory and in the field. In conclusion, the main factor that influences the burrow-sealing behavior of plateau zokors is the variation in oxygen concentration, and this defensive behavior is related to their activity rhythm.Defensive behaviors are a response to immediate and potential threats in the environment, including abiotic and biotic threats. Subterranean rodents exhibit morphological and physiological adaptions for life underground, and they will seal with mounds and additional plugs when their burrow opened. However, little is known about the factors driving this defensive behavior. In this study, we selected a subterranean rodent, plateau zokor ( For the vast majority of species, these threat-response behaviors do not rely on specific experience, but instead are based on instinct. A defensive repertoire is constructed of such behaviors, each of which has proved successful in response to particular types of threats in particular situations2. Biotic threats, such as predators and conspecifics, may involve an intricate arms race that ultimately facilitates the development of elaborate patterns of species-specific defensive behaviors, whereas abiotic environmental threats, such as floods or fires, cause relatively few and simple defenses3.Defensive behaviors are a set of responses to threat stimuli and situations that have evolved on the basis of their adaptiveness in reducing harm to the threatened organism4. Across the globe, subterranean rodents, with at least 140 species belonging to 20 genera of 8 families, occur in all continents except Australia and Antarctica5. Compared to aboveground rodents, subterranean habitats are considered more stable and buffered than aboveground habitats. This shelter not only protects these animals from predators, but also from environmental fluctuations or extremes taking place above the ground6. However, sometimes, their tunnels are opened inadvertently because of trampling by livestock or researchers using open-hole assessments to monitor their population dynamics8. These opened tunnels, which constitute a threat to the occupants, will be sealed by the animals with additional plugs. Although most researchers have observed this defensive behavior in subterranean rodents, little is known about the factors driving this defensive behavior.Subterranean rodents are small mammals that live in underground tunnels and undergo most of their life activities below the soil surface9 concluded that wind stimulated the occurrence of such behavior in Gansu kozor . Han10 suggested that wind and light have a large influence on the burrow-sealing behavior in Transbaikal zokor . However, Chai et al.11 found that this behavior in Transbaikal zokor was unrelated to wind and light. In general, burrow systems are characterized by the absence of light, relatively invariant temperatures, low ratios of oxygen to carbon dioxide, and gas flow is generally reduced relative to the surface habitat12. In facing these challenges, the morphology of subterranean rodents has been molded by convergent evolution to the same extent as their physiology. For instance, the dark environment has resulted in small eye sizes that limit the image size on the retina, resulting in poor image quality and visual acuity, but they can still detect ambient light levels for photoperiodism13. In subterranean ecotypes, olfaction and audition play an important role in the lives of these animals that have successfully adapted to life underground. The role of olfaction is one not only of finding food, but also in recognizing familiar individuals and choosing mates15. However, the spread of predator odor over long distances in the subterranean environment is restricted14 and, besides, predators are generally unable to open tunnels to prey upon these species17. Compared to epigeic generalized rodents, the hearing range of subterranean rodents is not restricted, rather, it is shifted towards lower frequencies18. More importantly, poor ventilation in subterranean burrows leads to poor levels of oxygen and an abundance of carbon dioxide, and, as such, these species have evolved physiological strategies enabling their respiratory and cardiovascular systems to cope with hypoxia and hypercapnia20. For instance, they have a large oxygen carrying capacity facilitated by elevated hemoglobin concentrations, a high intrinsic affinity for oxygen, and large concentration of red blood cells21. Moreover, the critical O2 (pO2) partial pressure levels in these mammals are low and metabolic rates can be maintained at low pO222. Therefore, when a burrow is opened, the atmosphere inside the burrow will become richer in oxygen, which may result in harmful effects on these species. Clearly, all of these factors  need to be considered as possible determinants of burrow-sealing behavior, both in the laboratory and in field experiments.Previous studies have focused on the effect of wind and light on burrow-sealing behavior. Yu and QianMyospalax5, as our species of subterranean rodent to study. Most plateau zokors live in alpine meadows on the eastern Qinghai–Tibetan Plateau23, and like other subterranean rodents, they possess morphological, physiological, and behavioral adaptions for digging and living in the underground environment24. Plateau zokors construct complicated burrow systems that consist of one or two main nests, foraging and transportation tunnels, food store caches, and blind endings. When constructing tunnels, the animals move the loosened soil to the surface and deposit it in mounds23. In this study, we conducted laboratory experiments to determine the effects of oxygen, light, temperature, gas flow and sound on the burrow-sealing behavior of plateau zokors. In addition, field experiments were conducted during both the cold and warm season of 2016. The aims of this research were to (1) reveal the determinants of burrow-sealing behavior in plateau zokor, (2) determine whether the latency to reseal the burrow is the difference between the responses to different treatments, (3) determine whether burrow-sealing behavior differs between the sexes, and (4) determine whether activity rhythm has an influence on burrow-sealing behavior in the field.In the present study, we selected plateau zokors, belonging to the genus All experimental procedures were permitted by the Institutional Animal Care and Usage Committees of the Grassland Science College of Gansu Agricultural University (GSC-IACUC-2015-0011). Our experiments were conducted according to their guidelines, which are in accordance with the Guide for the Care and Use of Laboratory Animals . All experiments were performed in accordance with ARRIVE guidelines.25 set at fresh surface mounds. The individuals were then transported to the laboratory and housed in an acrylic box with a pipeline covered with soil. The box and pipeline were covered by black cloth to simulate the dark environment of plateau zokors. The temperature in the room was maintained between 20 and 25 °C. Food was supplied daily and consisted of potatoes, lettuce and carrot. After three days of acclimatization to the laboratory the animals were used in the different experiments. Our laboratory is located 2 km away from the field site. At the end of the experiments all animals were returned to the capture site in good health.Adults of both sexes  were captured in April 2015. Specifically, the animals were captured in Mayin Tan  using live trapsThe experimental setup for the laboratory experiments was as follows Fig. : A transA rubber stopper with seven holes was used for plugging one end of the tube Fig. . The oxyTo avoid the oxygen that was delivered into the tube causing the gas to flow too strongly, become drier, and create a sound, a steel oxygen cylinder and thin hose (0.3 cm in diameter) were selected, and one end of the hose was connected directly to the oxygen cylinder with a humidifier bottle, while the opposite end was inserted into the rubber stopper Fig. . Before The average light intensity—that is, 360 Lux from 8:00 am to 8:00 pm—was measured in the field. One end of a wire was connected to an LED light (1 Watt), and the other end to the power supply Fig. .The temperature in the burrow entrance in the field was about 3 °C warmer than that at a tunnel depth of 10 cm. As such, one end of a wire was connected to a heater strip and the other end to the electrical power supply Fig. . A thermWhen a burrow is opened, wind whistle can be produced around the burrow entrance. Accordingly, a voice recorder  was placed at the burrow entrance in the field to record the burrow-entrance sound, the duration of which was 30 min. In the laboratory, the two ends of a wire were connected to an AVOXIVY loudspeaker  and a voice recorder, respectively  connected the tube through a rubber stopper  and an alarm device. The alarm device was made up of a loudspeaker, two slide rails (15 cm long), two metal plates (approximately 7 cm in length and 3 cm in width), and three coiled metal springs . The three springs were joined to one of the metal plates, while the other metal plate was fixed on the slide rails. The two metal plates were touched by the plateau zokor when it was plugging, which triggered the alarm device, thus enabling us to know whether or not burrow-sealing behavior was occurring , and six zokors were caught in the cold season and warm season . We then fastened radio collars  to each captured individual to allow us to track the position in foraging tunnels of each zokor. Each zokor was used three times in the experiments under each treatment, and, after finishing each experiment, we changed the position of the foraging tunnel to ensure the test tunnel was not an abandoned tunnel. According to radio-tracking data, the straight-line distance between the test tunnel and the nest for each treatment was about 5 m. We conducted a control experiment that whether plateau zokor move to the test tunnel or not during the time between treatments. In the cold season, from 4 October 2015 to 2 November 2015, the burrow-sealing behavior of each zokor was tested under different treatments during their active time (12:00–18:00) and inactive time (09:00–11:00) for a total of 27 days , and significantly higher than in the other treatments , although the sensitivity of plateau zokor to low-frequency sounds was the worst of the subterranean rodents that have been studied. Interestingly, in the laboratory, our results from the gas flow treatment yielded both the shortest latency to reseal the burrow and the lowest frequency of burrow-sealing events, which seems contradictory. However, we thought that a stress reaction could be the trigger of rapid burrow-sealing behavior in plateau zokors. The shorter distance (about 1 m) between the end of tube and the zokor’s nest probably made the zokor feel uncomfortable because of the gas flow. Therefore, they would act to seal their burrow rapidly. However, the lowest frequency of burrow-sealing in this instance may be beneficial to plateau zokors in terms of conserving energy. Because gas flow did not produce a negative physiological response owing to the negative pressure drainage ball with plastic tubing used in the laboratory.In our study, we also found that light had a slight effect on burrow-sealing behavior after eliminating oxygen, temperature and other factors. Han24 and have their own home ranges. Thus, both males and females must rely on themselves to ensure all its own burrow systems are sealed. Furthermore, some researchers have found that both males and females have been captured when capturing zokors by using the opening–sealing burrow method34. Therefore, we confirm that opening the burrow entrance has the same influence on both males and females. However, we do not know whether different ages of individuals have similar responses to these treatments, which needs to be studied in future research.In our study, we found that the frequency of burrow-sealing and the latency to reseal the burrow under different treatments did not differ between males and females in the laboratory experiments. In other words, both males and females will seal the burrow when it is opened. Unlike social subterranean rodents, plateau zokors are solitary animals


Universal domain adaptation (UDA) is a crucial research topic for efficient deep learning model training using data from various imaging sensors. However, its development is affected by unlabeled target data. Moreover, the nonexistence of prior knowledge of the source and target domain makes it more challenging for UDA to train models. I hypothesize that the degradation of trained models in the target domain is caused by the lack of direct training loss to improve the discriminative power of the target domain data. As a result, the target data adapted to the source representations is biased toward the source domain. I found that the degradation was more pronounced when I used synthetic data for the source domain and real data for the target domain. In this paper, I propose a UDA method with target domain contrastive learning. The proposed method enables models to leverage synthetic data for the source domain and train the discriminativeness of target features in an unsupervised manner. In addition, the target domain feature extraction network is shared with the source domain classification task, preventing unnecessary computational growth. Extensive experimental results on VisDa-2017 and MNIST to SVHN demonstrated that the proposed method significantly outperforms the baseline by 2.7% and 5.1%, respectively. The application of deep learning has been rapidly improving performance in various fields of computer vision, such as object detection , human pAchieving significant improvement in deep learning relies on the abundance of labeled samples  in a supDomain adaptation (DA) ,14 is thCurrently, research is underway on how to adapt learned knowledge to different domains with no restrictions to the source and target domain label sets. Universal domain adaptation (UDA) ,26 incluEven though  demonstrIn this paper, I propose a novel universal domain adaptation method that improves the discriminative power of the target feature representation of . Here, dIn contrast to the existing universal domain adaptation methods ,26, I foIn this case, owing to the limitations of data synthesis, the source domain has different characteristics from the real data in the target domain. Thus, the target domain information should be fully utilized to learn the feature representation. I used contrastive learning  to extraFor contrastive learning, the target domain feature extraction network is not separately constructed. The source and target domains share a common feature extraction network, thus avoiding unnecessary computation surges.The experiments conducted on the VisDa-2017 dataset and MNIST to SVHN dataset indicate that the proposed method significantly outperforms baselines by 2.7% and 5.1%, respectively.The contributions are summarized as follows:The remainder of this paper is organized as follows: Closed-set domain adaptation, if Partial domain adaptation, if Open-set domain adaptation, if Let I here introduce recent domain adaptation methods.Haeusser et al.  producedCao et al.  introducBusto et al.  introducRecently, You et al.  introducRecent studies have shown that unsupervised feature representations for various downstream tasks can be learned in a contrastive manner. Oord et al.  capturedBased on the abovementioned success of contrastive learning, I extracted the target domain information in a contrastive manner for universal domain adaptation.i-th input sample and its corresponding true label, respectively. G, i.e., G are denoted by G is shared for both the source and target domains. Let k-th weight vector k-th class. The proposed method uses a memory bank Let the dataset for the source domain be In this study, domain adaptation aims to transfer the knowledge of labeled samples in the source domain to train a classification model for unlabeled target domain samples. However, the problem is made more challenging for universal domain adaptation by including unknown samples in the target domain. Furthermore, some classes in the source domain may not have samples in the target domain. Therefore, the feature extraction network should move target domain samples closer to known class samples from the source domain, simultaneously making it easier to distinguish between unknown and known samples. To achieve this goal, researchers in a previous work (DANCE)  trained To summarize, the proposed method consists of four loss functions as depicted in posed in . I sequeContrastive representation learning aims to learn a discriminative embedding space devoid of any true labels through self-supervised learning wherein similar pairs of samples are close to each other, and dissimilar pairs of samples are distant from each other. To achieve this, each image in a given dataset is considered its own class, i.e., instance-level classification .I used the SimCLR-based contrastive learning method  to learnGenerate different perspectives based on the same target sample:Randomly selected data augmentations G to generate feature representations Extract features from augmented target samples: The augmented target samples are fed into a feature extraction network e ResNet ,27, i.e.M, and contrastive learning loss is applied to them. I use a shallow multilayer perceptron  with the same random seeds. DANCE-A represents another trained version of DANCE by the same augmentation G because contrastive learning needs different views generated augmentations I denote the accuracies reported in  as DANCEI used VisDA-2017 , MNIST  and SVHNVisDA-2017  is a larAs expected, the results in DANCE-* in For another synthetic-to-real domain adaptation scenario, I used two-digit datasets: MNIST  and SVHNMNIST consists of 28 × 28 sized gray images containing handwritten numbers ranging from zero to nine. The standard training and test splits included 60,000 and 10,000 images, respectively. I resized it to 32 × 32 for the experiments. Examples of the images are shown in the first row of SVHN, the Street View House Numbers dataset, consists of 32 × 32 sized natural scene images of numbers acquired from Google Street View. The training and test splits contain 73,257 and 26,032 images, respectively. The images in the SVHN dataset are aligned at the center of the desired number, but the surroundings are cluttered with visual artifacts and distractors. Examples of the images are depicted in Images in the MNIST dataset are not synthetic but appear synthetic because they contain only black-and-white image files, as depicted in For the experiments, I constructed closed-set, partial, and open-set domains as follows: Closed-set domain adaptation: G can be shared in target contrastive learning for efficiency.The results of both experiments in I also analyzed the effect of the target domain contrastive learning loss function on domain adaptation. To do this, I added a target domain contrastive learning loss to ‘SO’ that applied unsupervised classification loss function only to the source domain. This is marked ‘SO+Target_Contrastive’. In Universal domain adaptation (UDA) is an important research topic for the efficient use of trained models in various image sensors. I found that the baseline method does not have a direct training loss for the target domain to improve the discriminative power. I hypothesized that if the source and target domains are too different, the feature extraction network does not obtain discriminative feature representations for the target domains. To overcome the limitations of the synthetic data, the information about the target domain data should be fully utilized to learn feature representations. To do this, I used contrastive learning  in the t


Drosophila being uniquely suited to causally link between these levels. The exact nanoscale distribution of voltage-gated Ca2+ channels relative to release sites (“coupling”) at pre-synaptic active zones fundamentally steers the release of the synaptic vesicle. Unc13A and B were found to be either tightly or loosely coupled across Drosophila synapses. In this review, we reported recent findings on diverse aspects of Drosophila Unc13A and B, importantly, their nano-topological distribution at active zones and their roles in release site generation, active zone assembly, and pre-synaptic homeostatic plasticity. We compared their stoichiometric composition at different synapse types, reviewing the correlation between nanoscale distribution of these two isoforms and release physiology and, finally, discuss how isoform-specific release components might drive the functional heterogeneity of synapses and encode discrete behavior.The so-called active zones at pre-synaptic terminals are the ultimate filtering devices, which couple between action potential frequency and shape, and the information transferred to the post-synaptic neurons, finally tuning behaviors. Within active zones, the release of the synaptic vesicle operates from specialized “release sites.” The (M)Unc13 class of proteins is meant to define release sites topologically and biochemically, and diversity between Unc13-type release factor isoforms is suspected to steer diversity at active zones. The two major Unc13-type isoforms, namely, Unc13A and Unc13B, have recently been described from the molecular to the behavioral level, exploiting  While short-term facilitation is typical for synapses with a high initial release probability, short-term depression is often observed at ones with a low initial release probability  are released from a specialized region of the plasma membrane called active zones (AZs). Although AZs largely share the same complement of proteins, the probability of SV release varies tremendously across different neurons and even between AZs formed by the same neuron  domain and a C2C domain  fly C domain . This arC domain . The cenC domain , is key C domain . HoweverC domain . In mammC domain  and is eC domain . A CAPS 2+ channels activate the Ca2+ sensor synaptotagmin on the SVs to trigger fusion. The efficacy of synaptic transmission depends largely on the distance between SVs and voltage-gated Ca2+ channels. The two major fly isoforms of Unc13, Unc13A, and Unc13B are found co-expressed within individual AZs but astonishingly with different nanoscopic patterns. The nano-topology was first mapped at the peripheral neuromuscular junctions (NMJs) of Drosophila larvae using dual-color super-resolution-stimulated emission depletion (STED) microscopy  in Drosophila  of Drosophila. Concretely, the A isoform was found at 74–83 nm from the center of AZ, while the B isoform was, with a distance of 102–115 nm, at different relay synapses within the Drosophila olfactory system, including olfactory receptor neuron (ORN), projection neuron (PN), and Kenyon cell (KC) output synapses  imaging on PN-derived AZs in the lateral horn (LH) showed that Unc13A localized at about 60 nm relative to the AZ center, and the B isoform spots were still found at further distances at inhibitory PN (iPN) output synapses. These were the same as the coupling distances at the terminals of cholinergic excitatory PN (ePN) in the LH , Liprin-α, and the glutamate (E), leucine (L), lysine (K), and serine (S) rich protein (ELKS)-family BRP. The Unc13A/B nano-topology design present across Drosophila brain are expressed in all neuropile areas, their relative abundance varies greatly among different types of synapses. Within the antennal lobe, the first processing center of the fly olfactory system, ORN-derived AZs display a high release probability  output synapses appear as Unc13B-dominated  component could be isolated when applying the nicotinic agonist imidacloprid or curare. The fast curare-sensitive component depressed more rapidly than the imidacloprid-sensitive component, presumably mediated by distinct types of acetylcholine receptors or distinct states of the same receptor   was gratefully acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 ) significantly increased the ratio of epothilone D (These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.The online version contains supplementary material available at 10.1186/s12934-021-01578-3. Nonribosomal peptides (NRPs) and polyketides (PKs) are two prominent families of natural products, some of which exhibit high bioactivity , 2. NonrSorangium cellulosum produces epothilone A and B, while epothilone C and D are produced by inactivation or deletion of epoK, which is an epoxidase gene in the epothilone biosynthesis gene cluster (MIBiG-ID BGC0000989) [in vivo to improve our understanding of the AT domain. The AT engineering mainly include three strategies: swapping domains, exchanging modules, and site-directed mutagenesis of PKS genes. EPOAT4 has been used as a donor AT in the engineering of 6-dEB synthase (DEBS) [in vivo. The genetic manipulation in the original epothilone producer is difficult. In previous studies, epothilone PKS was modified in heterologous host Myxococcus xanthus by the inactivation of KR, DH or ER domains in EpoD, resulted in production of novel epothilone derivatives but in lower yields for use in industrial production [Epothilones are members of the NRP/PK family synthesized by hybrid NRPS/PKS synthetases, which stabilize microtubules in parallel with taxol and have been used as anticancer agents . The nat0000989) . Epothil0000989) . The mar0000989) , 21. Mix0000989) . We wante (DEBS) , 23, butoduction , 24, 25.oduction –29.Moreover, module 9 of the epothilone PKS (EPOM9) is a sufficiently reducing module with a complete modifying region, and we observed that the DH and ER domains of module 9 (EPODH9 and EPOER9) are inactive Fig. . InactivSchlegelella brevitalea DSM 7029 and obtained mutant strains that produced high levels of epothilone C and D by the deletion of epoK [S. brevitalea. We significantly increased the proportion of epothilone D and obtained three strains that produced only epothilone C. We also successfully deleted the inactive DH and ER domains in EPOM9 and fused ΨKR with KR through an ~ 25-residue linker to generate a productive epothilone PKS and simplified the completely reducing region to a partly reducing region in EPOM9 without a decrease in the production level. Finally, our study further explored the importance and the sequence flexibility of ΨKR-KR-linker.In our previous work, we successfully expressed epothilone PKS in the heterologous host  of epoK . The higS. brevitalea 104-1 produced 27.7 mg/l epothilone D (4) and 19.9 mg/l epothilone C (3) (4:3 = 1.4:1) as a consequence of EPOAT4 incorporating either MMCoA or MCoA extender units during polyketide chain elongation [Engineering the AT specificity can alter the carbon skeleton of the polyketide by selecting different acyl-CoA substrates. To compare three strategies in altering AT specificity, we first exchanged the module in which EPOAT4 is located. The previously optimized high-yield epothilone producer ongation . We exchS. cellulosum produced minor congeners harbouring MCoA at C16, C8, C6, or C4 of the epothilone structure [In engineering the EPOAT4 domain, both replacement of the entire module and domain swapping are possible. Considering that all chimaeric epothilone PKSs obtained through module exchange have lower product yields, we performed domain-swap experiments. We engineered EPOAT4 using a fruitful AT-swap strategy reported in 2016 by Yuzawa et al. . We repltructure . Then, wSubsequently, we replaced EPOAT4 of the epothilone PKS with three MCoA-specific AT domains derived from the same PKS, including EPOAT3, EPOAT5, and EPOAT9  produced only epothilone C  and downstream KS (KSn) domains during chain translocation [n−1-KSn interaction, as it has been suggested that mutation of the residue in ACPn−1, which was predicted to affect the ACPn−1-KSn recognition, led to increased turnover [The activity levels of our module engineered systems were drastically diminished even though we retained appropriate covalent linkers to connect heterologous modules. We speculate that this result is attributable to the impaired specific interactions between upstream ACP  has also been described in previous studies , 37, 45.In conclusion, functional hybrid epothilone PKSs were generated, and product distributions were altered by swapping the EPOAT4 domain and by site-directed mutation of the conserved motif in AT. Furthermore, we simplified module 9 of epothilone PKS by the deletion of inactive domains without decreasing the production and proved the importance and the sequence flexibility of ΨKR-KR-linker. These results enhance our understanding of PKS domains and provide new insights into PKS engineering.S. brevitalea DSM 7029 strains were cultivated at 30 °C in CYMG broth or on CYMG agar [sacB gene was introduced for negative selection [Escherichia coli DH10B strains were grown at 37 °C in Luria-Bertani (LB) broth or on LB agar. Antibiotics were used where required at the following final concentrations: ampicillin, 100 µg/ml; spectinomycin, 50 µg/ml; apramycin, 50 µg/ml; kanamycin, 50 µg/ml; and hygromycin B, 150 µg/ml for E. coli and 100 µg/ml for S. brevitalea DSM 7029.All bacterial strains used in this study are listed in Table YMG agar . Sucroseelection . The EscepoAT2, epoAT6, epoAT7, epoAT8, epoAT3, epoAT5, and epoAT9 genes were cloned from the genome of S. brevitalea 104-1 by PCR. The rapAT1 and eryAT6 genes were amplified from the genomes of Streptomyces hygroscopicus and Saccharopolyspora erythraea, respectively. These fragments were ligated into pJM5 individually using In-Fusion cloning to form the recombinant plasmid pJM6-pJM14. After verification by restriction digestion analysis and sequencing, all recombinant plasmids were transformed into the parental strain S. brevitalea 104-1. After growing at 30 °C for 2.5 days, recombinant single-crossover colonies were selected on plates containing apramycin. We confirmed the integrated mutants by PCR and transferred the colonies into 2 ml of antibiotic-free CYMG broth at 30 °C. To force these mutants to undergo a second round of recombination, in the next three days, we transferred 10 µl of the culture into 2 ml of antibiotic-free fresh CYMG broth three times, and 1–2 % sucrose was added in each step. Then, we replicated the colonies grown on CYMG agar plates without antibiotics on CYMG agar plates with apramycin. Colonies that grew only on antibiotic-free CYMG agar plates were screened for being double-crossover mutants. The expected up-AT4swap and AT4swap-down fragments were obtained from a double crossover by PCR , MMR2024 ERYM6), MMR2027 (RAPM4), and MMR2026 RAPM10)  in the 2-l shake flask was inoculated with 25 ml of preculture. The fermentation medium and extraction methods were described in our previous work . We filtm/z = 478.4 and 290.2 for epothilone C, 492.4 and 304.2 for epothilone D. A dilution series of epothilone standards was created to generate a concentration standard curve for quantification , and the alignments were produced using ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ).The amino acid sequences were aligned using ClustalW (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to generate all tertiary structure models [Phyre2  of LC–MS analyses of epothilone production in EPOM4-swap mutants. Figure S4. Multiple sequence alignment of AT domains used in this study. Figure S5. Extracted ion chromatogram (EIC) of LC–MS analyses of epothilone production in some AT-swap mutants to further confirm the production of epothilones. Figure S6. Extracted ion chromatogram (EIC) of LC–MS analyses of epothilone production in AT-swap mutants MMR2012 and MMR2018. Figure S7. HPLC analysis of the extracts from the parental and site-directed mutant strains. Figure S8. Junctions for ER domain deletion constructs. Figure S9. Junctions for DH domain deletion constructs. Figure S10. PCR analysis for the confirmation of AT-swap mutants. Figure S11. PCR analysis for the confirmation of module-exchange mutants. Figure S12. DNA sequences of the PCR products of site-directed mutants. Figure S13. DNA sequences of the PCR products of ΨKR9-KR9-linker deletion mutant (MMR2053) and replacement mutant (MMR2054). Figure S14. Standard curves calculated based on the peak areas of epothilone standards of different concentrations.


Panthera pardus) and tigers (P. tigris) in and near Bardia National Park (BNP), Nepal. Drivers of carnivore depredations of livestock were influenced by carnivore species, animal husbandry practices, season, and deterrent technique. Leopards killed more livestock than tigers, and the likelihood of livestock depredations was not affected by the number of livestock owned or preventative measures used to reduce depredations.People in rural Nepal are experiencing increased livestock depredations from large carnivores; however, limited information is available on factors influencing livestock depredations. We quantified potential factors influencing livestock depredations by leopards (Panthera pardus) and tigers (P. tigris) near (<1 km) and far (>1 km) from this protected area. Overall, 1476 hoofed livestock were reportedly depredated by leopards, and 209 by tigers, during 2015–2019. The number of hoofed livestock killed by leopards each season was, at least, 86% higher than the number killed by tigers. More livestock were killed at BNP irrespective of carnivore deterrent techniques used. Due to severe effects created by livestock depredations near BNP, we recommend using more efficacious deterrent techniques when practical, in addition to improved livestock husbandry practices such as night penning.Wildlife attacks on livestock near human settlements are increasing due to the proximity of humans to protected areas. These attacks are often severe due to depredations of livestock adversely affecting the livelihoods of people. The nature of carnivore depredations on livestock can differ based on the carnivore species, animal husbandry practices, season, and deterrent technique. We surveyed people living near Bardia National Park (BNP), Nepal, to compare hoofed livestock depredations by leopards ( Panthera pardus) appear more prevalent than conflicts with species that are less adaptable . As a c tigris; ,15,16. D tigris; .People in rural Nepal often use traditional livestock practices for their livelihoods with few alternatives. Following government relocation programs before the 1980s, the number of people living near protected areas has increased. Improved habitat protection, bans on hunting, and public awareness programs have resulted in increased populations of large cats in some protected areas of Nepal ,18. Cons2, and the average elevation is about 415 m above sea level. The climate is seasonal and is typically defined as spring (March–May), summer (June–August), fall (September–November), and winter (December–February).We conducted this study in the Barahtal Rural Municipality (BRM) buffer zone of Bardia National Park, Karnali Province, Nepal . The buffer zone of BNP comprises 507 kmShorea robusta, saj Terminalia alata, khair Acacia catechu, simal Bombax ceiba, sissoo Dalbergia sissoo, and tooni Toona ciliata. The area also supports diverse wildlife including Asian elephants Elephas maximus, tigers, leopards, and swamp deer Rucervus duvaucelii [About 70% of the park is forested with dominant plant species including sal vaucelii . There avaucelii  which deWe collected data from 2 January to 12 February 2020 using a questionnaire survey with people living in the BNP buffer zone. We used the number of households in the BRM buffer zone of BNP to estimate the number of households to interview using a 95% confidence interval with a 5% margin of error . We estiWe interviewed only people >18 years old from a household. We did not discriminate based on education level, gender, ethnicity, or religion. We collected demographic data including age, gender, education , family size, and occupation. We asked people whether they experienced conflicts with carnivores and to characterize these conflicts . We asked them the number of hoofed livestock they owned and the number killed by leopards and tigers during 2015–2019. Respondents reported livestock depredations by leopards or tigers based on their experience, evidence  near the kill site, and evidence on the carcass  including differences between species for killing prey . The location of each kill site was recorded, and the distance from each site to the BNP boundary was measured using QGIS. Finally, we asked respondents about the number and type of deterrents used to mitigate the risk of conflicts with leopards and tigers. All aspects of this study were approved by the Department of National Parks and Wildlife Conservation, Nepal (permit DNPWC-67/77-105).We used chi-square and Kruskal–Wallis tests for binary and numeric responses, respectively, to examine differences between people living near (<1 km from the BNP boundary) and far (>1 km from the BNP boundary) from the protected area. We used the location of each livestock depredation and estimated the distance to the BNP boundary using GIS .We used generalized linear models with Poisson distribution to identify factors influencing livestock depredation by leopards and tigers using data for 2015–2019. Factors included distance to BNP, number of livestock (cattle and goats) owned, number of techniques used to mitigate conflicts, and season. We defined seasons as spring (March–May), summer (June–August), autumn (September–November), and winter (December–February). We ranked models using the Akaike Information Criterion adjusted for small samples AICc , and AkaWe interviewed 300 households (147 near BNP and 153 far from BNP); demographic characteristics of respondents near and far from BNP were not different . Most reχ2 = 3.681, p < 0.055; χ2 = 116.11, df = 3, p < 0.001; n = 626), followed by summer (n = 451), spring (n = 355), and fall (n = 342). More livestock were reportedly killed far from the park boundary during winter  was greater near BNP, due to the greater abundance of large carnivores [Leopard depredations of livestock were substantially greater than depredations caused by tigers, irrespective of the season or proximity to BNP. We suspect this marked difference in the frequency of depredations is a consequence of leopards being more common than tigers ,30. The rnivores ,34,35. Lrnivores .In our study area, more livestock depredations occurred during winter. However, in eastern and western areas of Bardia National Park, more livestock depredations from tigers and leopards were reported during summer and spring . LivestoMitigation techniques used in this study were ineffective in deterring livestock depredations by leopards and tigers. Using lights such as a fox light can be effective as a visual deterrent of leopard depredations of livestock ; howeverAnimal husbandry practices can also influence the frequency and occurrence of livestock depredations. Large carnivores, especially leopards, more frequently kill smaller-sized hoofed animals such as goats and calves ,43. GoatLivestock depredations by large carnivores can potentially limit carnivore populations through retaliatory killing . HoweverLivestock depredations by leopards and tigers in our study area and other remote areas can be frequent and adversely affect the livelihoods of people. We encourage the use of more effective carnivore deterrent techniques to mitigate this risk. Specifically, we recommend that people in our study area use lighting in and around corrals at night to reduce depredations, particularly in winter when livestock are most vulnerable. Further, we recommend consideration of alternate animal husbandry practices such as keeping mixed livestock  in the same corral at night to decrease the vulnerability of smaller-bodied species.


This study proposed a rotary back extrusion (RBE) process for an open punch, which is used to produce high-performance 7075 aluminum alloy cup-shaped piece. The RBE experiment was carried out on the Gleeble-3500 testing machine at 400 °C and compared with the conventional back extrusion (CBE). The microstructure was analyzed by optical microscope, scanning electron microscope and DEFORM-3D simulation software. The results shown that compared with CBE, RBE can significantly increase the equivalent strain value and deformation uniformity of 7075 aluminum alloy cup-shape pieces. RBE deformation increases the accumulated strain of the piece, and the rotation of the die causes the piece to produce shear strain, which increases the overall strain of the cup-shape piece. The proportion of dynamic recrystallization increases, and the grain refinement was obvious. The micro-hardness value of the RBE sample is higher than that of the CBE sample, which could be the result of grain refinement strengthening. What is more, RBE and CBE have different metal flow laws. Aluminum and aluminum alloys have the characteristics of high specific strength, low density, strong corrosion resistance, and good molding effect. These features prompt broad application for the alloys in the aviation, aerospace, transportation, weapon industries, etc. Therefore, the innovative research and development technology of aluminum alloys, especially the forged ones, is a basic technique and has been listed as a key development target of national defense science and technology ,2,3,4. 7However, 7075 aluminum alloy has poor plasticity at room temperature and is difficult to deform for the manufacturing of complex parts. Therefore, hot deformation is used to increase the formability and mechanical properties of 7075 aluminum alloy under different process parameters. The hot deformation process can significantly refine the alloy grain size, and increases the comprehensive mechanical properties of the aluminum alloy ,11,12. THowever, our previous research is mainly concentrated in the formation of the microstructure and texture of the magnesium alloy RBE process, and the evolution of 7075 aluminum alloy microstructure and performance is unclear, which will limit the further development and exploration of the RBE process. In addition, 7075 aluminum alloys have good plastic toughness and processing performance, is widely used in aerospace and military sector. Therefore, this paper will study the evolution of the 7075 aluminum alloy in the RBE process, and compare the CBE, revealing the deformation behavior of the 7075 aluminum alloy RBE process and the structure property evolution mechanism.The material used in this study was extruded 7075 aluminum alloy. The round bar was solution treated and heat treated at 480 °C for 24 h , aged at2O3 suspension, and then corroded with Keller reagent of 2.5 mL nitric acid, 1.5 mL hydrochloric acid, 1 mL hydrofluoric acid, and 95 mL distilled water [Using optical microscope (OM), scanning electron microscope (SEM), and electron backscatter diffraction (EBSD), the microstructure evolution of 7075 aluminum alloy cup-shaped piece during CBE and RBE were studied. The symmetrical longitudinal section of the sample was observed. OM and SEM were prepared on Zeiss Axio-A2m microscope and Hitachi-SU5000 SEM, respectively. Before the observation, the specimen was grounded with sandpaper, mechanically polished with Aled water . The EBSDEFORM 3D-V12.0 software was used to analyze the deformation behavior of 7075 aluminum alloy during CBE and RBE processes ,21. FirsA Vickers micro hardness tester was used to measure the hardness distribution of the alloy. The test conditions were carried out with 200 gf load and 15 s holding time. Based on different locations of the deformed specimen, the hardness sampling points were selected, and the average value was taken to improve the accuracy of the experimental results. The selected area was shown in For the preparation of 7075 aluminum alloy cup-shaped parts, a corresponding finite element model was established. Through the comparison of the two processes of CBE deformation and RBE deformation, the metal flow mode, strain distribution characteristics and the change law of forming load during the forming process of cup-shaped parts were analyzed.In order to further explore the equivalent strain distribution law of 7075 aluminum alloy cup-shaped parts under rotating back-extrusion deformation, according to the flow characteristics of the cup-shaped parts, three typical areas were selected: bottom zone, corner zone, and wall zone . The corAccording to the equivalent strain change law of the CBE and RBE cup-shaped parts, the RBE deformation significantly increased the cumulative strain of the piece and promoted the uniformity of the overall deformation of the component. Through the analysis of the metal flow law of the cup-shaped parts, the reasons for the difference in the equivalent strain value and distribution under the two deformation modes could be better explained. In order to quantitatively characterize the difference in metal flow between the two deformation methods, the point tracking function is used to analyze the metal flow law in different areas of the piece during the deformation process. Select the six characteristic points in the contact area of the punch and the piece within the diameter of the punch for point tracking analysis. P1, P2, P3 are the three points on the left side of the piece, P4, P5, P6 are the three points on the right side of the piece, P7, P8 are the two points in the middle of the piece.The distribution of the characteristic points before and after deformation is shown in Figure 7The results show that points P1–P6 flow to the wall area after conventional back-extrusion, while points P7 and P8 only move axially with the punch. In the rotating back extrusion, the 8 points are all on the wall, and P7 and P8 are close to the bottom. This also showed that the rotating back extrusion can eliminate the dead zone of deformation, promote the flow of the bottom metal to the wall, and make the overall structure more uniform. Moreover, it can be found that the wall area is approximately 30° inclined to the original wall. Just like rowing, after the water in front is opened by the oars, it will go back to the original place again. However, due to the increased shear stress, the rotating back extrusion does not have this phenomenon.In the bottom area of the cup-shaped piece, area A was the deformation dead zone, with an average grain size of about 92.2 μm, with coarse grains. This area belonged to the deformation dead zone observed in the simulation, because the metal in this area had poor fluidity and low deformation, dynamic recrystallization was not easy to occur, so the grains were relatively coarse. The microstructure in region B was equiaxed, with a grain size of 73.2 μm, which was about 31% smaller than the initial grains, indicating that the metal flow in this region was intense and the cumulative strain increased. Area C reflected the flow of the alloy, and as the deformation progresses, the metal in the area C would flow to the area D. The average grain size of area C was 87.1 μm, which is about 17.9% smaller than the initial grains. Area C was a mixed microstructure combining original deformed grains and dynamically recrystallized grains. The deformation in the middle of the wall of the CBE cup-shaped part is low, and the dislocation density inside the crystal grain is small, and the deformation and energy storage are small, which is not conducive to the generation of dynamic recrystallization. In addition, it could be observed that only a small amount of dynamic recrystallized grains were produced along the grain boundaries of deformed grains. This was due to the accumulation of dislocations caused by grain boundaries that could hinder the movement of dislocations. The plugging dislocation could not only provide nucleation sites for the generation of dynamic recrystallization, but also cause grain boundary migration and ultimately promote the generation of new crystal grains.For the elliptical region of the cup-shaped piece, the average grain boundary disorientation in the regional elliptical region was reduced from 17.8 μm in the conventional back extrusion to 14.9 μm in the rotary back extrusion, although the rotary back extrusion sample had a higher dynamic recrystallization ratio. However, the shear strain increased the deformation inhomogeneity inside the new dynamic recrystallization, and the increase in the degree of lattice distortion led to an increase in the dislocation density, and a large number of dislocations promoted the generation of LAGB, so the average grain boundary disorientation decreased.In order to further explore the deformation mechanism of 7075 aluminum alloy under rotating reverse extrusion deformation, the deformed grains in the EBSD map were selected for dynamic recrystallization mechanism analysis.Compared with conventional back extrusion, rotary back extrusion can significantly increase the equivalent strain value and deformation range of the cup-shaped piece. The rotating back-extrusion deformation can not only drive the bottom metal of the cup-shaped piece to rotate and increase the accumulated strain of the piece, but also causes the piece to produce shear strain, which increases the overall strain of the cup-shaped piece.Rotating back extrusion deformation can significantly refine the grains, increase the proportion of dynamic recrystallization, promote metal flow, and eliminate the “deformation dead zone” in conventional back extrusion deformation, which the grain size is reduced by 92.0% compared with the CBE, but more deformation heat will also be generated in the rotating back extrusion deformation, which will cause the grains in the forming zone to recover and grow up statically.Discontinuous dynamic recrystallization is the main dynamic recrystallization mechanism of rotary back-extrusion deformation. A large number of continuous dynamic recrystallization mechanisms occur in the shear zone of the cup-shaped piece, and less in other areas. The micro hardness values of different regions of the rotating back-extrusion cup-shaped parts are all higher than those of the conventional back-extrusion samples, which is mainly caused by the strengthening of fine grains.The metal flow in the wall area of the direct back-extrusion deviates from the original wall by about 30°, while the rotating back-extrusion does not have such a phenomenon due to the increased shear stress.The microstructure and texture evolution of 7075 aluminum alloy in rotating back extrusion and direct back extrusion experiments at 400 °C and an extrusion speed of 0.1 mm/s were systematically studied. The main conclusions are summarized as follows:


UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.Loss of the maternal   UBE3A allele  is a neurodevelopmental disorder characterized by severe developmental delay, motor dysfunction, absence of speech, and highly penetrant epilepsy , 2. WhilA allele . UBE3A ebstrates  and, genbstrates , 6, a nobstrates  and possbstrates , 9. Due pression , mature pression , 12. TheUBE3A expression in neurons. This can be achieved through small molecules (UBE3A-ATS). Processed from a long noncoding RNA that initiates from SNRPN promoters on the paternally inherited chromosome, UBE3A-ATS silences paternal UBE3A in cis in neurons  transfer of a functional gle dose , 22. AAVgle dose , 24. Thegle dose . In the gle dose , 26. Thegle dose , 27–29.UBE3A unsilencing. Our work provides a rationale for future AS clinical trials based on hUBE3A gene transfer and offers a positive outlook for both symptom relief and the tolerance of hUBE3A overexpression.Here, we report on the toxicity and efficacy of a one-time intracerebroventricular (ICV) administration to neonatal AS model mice of a recombinant AAV9-derived PHP.B vector  encodingmUbe3a) and human (hUBE3A) transcripts produces distinct UBE3A protein isoforms, which differ in their extreme amino N-termini  across brain regions during development. However, unlike mUbe3a short/long ratios, they remained constant throughout aging, showing no decrease in the adult brain. Of the 2 long hUBE3A species, isoform 3 was far more abundant; isoform 2 expression fractions were near zero in many instances (https://doi.org/10.1172/jci.insight.144712DS1). Collectively, these data support that short/long UBE3A isoform ratios are maintained with remarkable spatiotemporal consistency in the developing mouse and human brain.Alternative splicing of mouse  sequences contiguously within the same reading frame. Because each isoform is translated from an independent start codon, we differentially manipulated Kozak sequence strengths with the goal of favoring short hUBE3A (isoform 1) expression. The resulting expression cassette, termed hUBE3Aopt /mL of PHP.B/hUBE3Aopt vector to neonatal AS model mice, which come to lack neuronal expression of UBE3A (hUBE3Aopt transcripts in PHP.B/ hUBE3Aopt-injected WT (WT + AAV) and AS (AS + AAV) mice but not vehicle-treated controls. In situ hybridization of hUBE3Aopt probes in AS + AAV brain tissues revealed a neuron-specific punctate signal that was absent from WT brains  that exclusively express long UBE3A, we were able to reliably identify bands belonging to both long and short hUBE3A in AS + AAV samples, thereby establishing that PHP.B/hUBE3Aopt is in fact a dual isoform-expressing vector. Quantification evinced a short/long expression ratio of 2.7:1, which was comparable with both the 3.5:1 ratio we obtained for WT + vehicle controls , 42cis-aelopment  33). Co. Cocis-aelopment . AS miceelopment , enablinelopment , subcellelopment . Taken t14 vg/mL PHP.B/hUBE3Aopt. We administered a modified version of a behavioral test battery suitable for preclinical drug testing in AS model mice   . We therin (WFA) . Comparein (WFA) , WFA stain (WFA) . The PNNin (WFA) ; that wein (WFA) , is reflin (WFA) . CollectUBE3A and other genes (Dup15q syndrome is an autism spectrum disorder (ASD) caused by duplications and triplications of the 15q11.2–q13.1 chromosomal region that encompasses er genes . hUBE3A er genes . We founer genes . This dier genes .Evidence of hUBE3A overexpression was scarce in the thalamus , cerebelhUBE3A expression was evenly distributed across most subregions of AS + AAV hippocampi, with the exception of the inner half of the dentate gyrus granule cell layer, in which it was notably absent . The neuP = 0.64), albeit significantly rightward-shifted (versus AS + AAV elevation: P < 0.0001). Succinctly, equivalent UBE3A immunofluorescence values associated with a significantly lesser degree of dentate gyrus atrophy in WT + AAV versus AS + AAV mice  brain. In so doing, we revealed 3 remarkably well-conserved UBE3A expression features across species: (a) highly consistent ratios of short to long isoform expression during brain development; (b) dynamic subcellular localization in neurons, whereby UBE3A becomes increasingly nuclear as maturation progresses; and (c) ubiquitous neuronal expression with early developmental onset. These parameters guided our design of a potentially novel dual-isoform hUBE3Aopt vector, in which we manipulated Kozak sequence strengths to bias expression in favor of short UBE3A, successfully recapitulating ~3:1 short/long protein isoform representation. hUBE3A gene transfer now shares with paternal UBE3A unsilencing approaches –17 the cA growing literature supports that hUBE3A reinstatement should ideally be administered during the early postnatal period for maximal therapeutic effect in AS . Indeed,UBE3A is the only 15q11.2–q13.1 gene with exclusively maternal monoallelic expression in neurons, and Dup15q syndrome is most commonly associated with maternally inherited 15q11.2–q13.1 duplications and triplications  were subjected to a battery of behavioral tests beginning at 10–14 weeks of age, spanning a period of 4 months, administered in the following order: open field, marble burying, rotarod, fear conditioning, and nest building. Most treated mice (n = 69) also underwent an additional flurothyl kindling and rechallenge analysis. Upon the completion of behavioral experiments, precisely 1 hour following flurothyl rechallenge when applicable, mice were sacrificed for histological analyses of protein expression. Numbers of experimental mice were based on previous behavioral studies of the same AS mouse model in which rigorous statistical power analyses were performed . Neonatal (P0.5–P2) AS mice and WT littermate controls were randomly assigned to treatment with vehicle or PHP.B/hUBE3Aopt at a dose of 1.6 × 10erformed . No animUbe3a-KO allele were originally generated in the laboratory of A. Beaudet  by crossing congenic C57BL/6J WT males to paternal Ube3a-deficient females (Ube3am+/p−), which themselves are phenotypically normal  relative to long hUBE3A isoform 3 (TTTTATGG). The hUBE3Aopt cDNA was subcloned into plasmid pTRs-KS-hSYN-GFP-BGHpA to generate pTRs-KS-hSYN-hUBE3Aopt-BGHpA. pTRs-KS-hSYN-GFP-BGHpA was, itself, the product of subcloning a human synapsin promter (hSYN) into plasmid pTRs-KS-CBh-GFP-BGHpA .A codon-optimized human UBE3A cDNA, hUBE3Aopt, was designed using a commercially available algorithm tool (Atum). hUBE3Aopt encodes both hUBE3A isoform 1 and hUBE3A isoform 3, which are translated from independent start codons within a single reading frame. Kozak sequences were modified to enhance production of short hUBE3A isoform 1 , and the subsequent 2 rounds were done at 90,000 rpm . Purified vectors were exchanged into storage buffer containing 1× PBS, 5% sorbitol, and 350 mM NaCl.PHP.B/hSYN-EGFP vectors were produced by HHMI-Janelia Viral Tools using a polyethyleneimine triple-transfection protocol in 293 cells grown under serum-free conditions. Vectors were purified through 3 rounds of CsCl density gradient centrifugation, each carried out for 16 hours at 8°C. The initial round was performed at 65,000 rpm (14 vg/mL) to the ventricles, resulting in a total dose of 1.6 × 1011 vg injected per pup. The addition of Fast Green dye  to the virus solution facilitated the visualization of successful injections. Following injection, pups were warmed on an isothermal heating pad with home-cage nesting material before being returned en masse to their nursing dam.P0.5–P2 mouse pups were cryoanesthetized on wet ice for 3 minutes; they were then transferred to a chilled stage equipped with a fiber optic light source for transillumination of the lateral ventricles. A 10 μL syringe fitted with a 32-gauge, 0.4-in-long sterile syringe needle  was used to bilaterally deliver 0.5 μL of purified PHP.B/hUBE3Aopt vector , fear conditioning, and flurothyl kindling assays. In the figures, all values are expressed as means ± SEM unless otherwise specified. A P value less than 0.05 was considered significant. GraphPad Prism 8.3 software  was used for statistical analyses.One-way ANOVA followed by Tukey’s post hoc test was performed for open-field, WFA immunofluorescence analysis in naive mice, and GFAP immunofluorescence analysis was performed in kindled mice. Welch’s 1-way ANOVA followed by Dunnett’s post hoc test was carried out for marble burying, WFA immunofluorescence analysis was performed in kindled mice, GFAP immunofluorescence analysis was performed in naive mice, and quantification of UBE3A immunofluorescence was performed by brain region, as the Brown-Forsythe test indicated unequal variances in each case. To analyze ordinal nest building scores, the Kruskal-Wallis test was used, followed by Mann-Whitney Animal studies followed NIH guidelines and were performed in strict compliance with animal protocols approved by the IACUC of the UNC at Chapel Hill.E. coli–based ubiquitination assays. MTS performed and analyzed immunofluorescence staining experiments and analyses. NWM performed in situ hybridization experiments. KDR designed and generated PHP.B/hSYN-EGFP vectors. YE contributed to experimental design and advised on bioinformatic analyses. DGA contributed to experimental design. SJG oversaw AAV vector production and designed experiments. BDP helped design experiments and contributed to data analysis. All authors edited the manuscript.MCJ designed and performed experiments, analyzed data, and wrote the manuscript. CS designed and validated AAV vectors. JMS designed and performed bioinformatic analyses. CRD performed and analyzed behavioral studies. AMP designed and performed