The double mutant fin219-2phyA-211 showed a synergistic effect on seedling development under FR light. Further evidence revealed that FIN219 and phyA antagonized with each other in a mutually functional demand to modulate hypocotyl elongation and expression of light- and JA-responsive genes. Moreover, FIN219 interacted with phyA under prolonged FR light, and MeJA could enhance their interaction with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) in the dark and FR light. FIN219 and phyA interaction occurred mainly in the cytoplasm, and they regulated their mutual subcellular localization under FR light. Surprisingly, the fin219-2 mutant abolished the formation of phyA nuclear bodies under FR light. Overall, these data identified a vital mechanism of phyA–FIN219–COP1 association in response to FR light, and MeJA may allow the photoactivated phyA to trigger photomorphogenic responses.Integration of light and phytohormones is essential for plant growth and development. FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT 1 (JAR1) participates in phytochrome A (phyA)-mediated far-red (FR) light signaling in Arabidopsis and is a jasmonate (JA)-conjugating enzyme for the generation of an active JA-isoleucine. Accumulating evidence indicates that FR and JA signaling integrate with each other. However, the molecular mechanisms underlying their interaction remain largely unknown. Here, the  Interaction between plant growth and defense responses is critical for plant survival. However, the molecular mechanisms underlying their integration in response to environments remain elusive. Light provides plants with energy resources for photosynthesis and a signal for optimized growth and development. The phyA is the photoreceptor for perceiving far-red (FR) light in regulating different aspects of plant development. FIN219/JAR1 is a JA-conjugating enzyme that produces the physiologically active JA-isoleucine hormone, which triggers JA-regulated responses, including defense. Here, our findings reveal that phyA and FIN219 interact with each other, exogenous JA treatment enhances their interaction, and phyA-mediated FR light and FIN219-modulated JA signaling pathways are integrated in an antagonistic and mutually functional demand manner to regulate the photomorphogenic responses of seedling development, including inhibition of hypocotyl elongation, anthocyanin accumulation, chlorophyll content, and expression of light/JA-responsive genes. This regulatory relationship between both proteins may link plant growth and defense trade-offs upon exposure to various stresses. The integration of light and various hormone signals dramatically affects plant growth and development –3. The sPlants contain different photoreceptors to sense various light wavelengths, including red (R)/far-red (FR) light perceived by phytochromes and blue light by cryptochromes . These pArabidopsis has five phytochromes (phyA to phyE) to perceive the R and FR light . The phyAlternatively, phyA may interact directly with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a master repressor of photomorphogenesis, under FR light . This inVEGETATIVE STORAGE PROTEIN 1 (VSP1) as well as levels of 12-oxo-phytodienoic acid (OPDA)  and COP1 polyclonal antibodies [We extracted total proteins with grinding buffer , VSP1 (At5g24780), PDF1.2 (At5g44420), JAZ1 (At1g19180), CHS (At5g13930), RBCS-1A (At3g18780), PHYA (At1g09570) and ACTIN 2 (At3g18780) as an internal control to normalize the expression levels.Total RNA was isolated using Rezol C&T (Protech Technologies). RNA (2 μg) was treated with DNase to prevent possible genomic DNA contamination and used as a template for cDNA synthesis with the ABI cDNA transcription kit (#4368814). Each PCR reaction contained 0.5 μg cDNA, 0.25 μM gene-specific forward/reverse primers, and 1XiQ SYBR Green supermix (Bio-Rad). We detected fluorescence signals with CFX96 Touch deep well real-time PCR detection system (Bio-Rad). Three-step PCR program was used: (1) 95°C, 3min. (2) 95°C, 10s; 60°C, 30s; 72°C, 30s. (40 cycles). (3) Program for melting curves. We analyzed all the quantification data with CFX Manager software (Bio-Rad). Gene-specific primers  were usep35S: PHYA-GFP and p35S: FIN219-GFP for transfection. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) staining solution (10 μg/ml DAPI in PBS). We observed the protoplasts with light microscopy  and used an RS Photometrics CoolSNAP camera  to obtain images with the corresponding IM50 software.We analyzed transient expression in Arabidopsis protoplasts according to the Jen Sheen laboratory protocol . ProtoplNicotiana benthamiana was incubated in the cabinet for four weeks under long-day conditions. We cloned full-lengths of FIN219 or FIN219-N or FIN219-C regions and PHYA coding sequences into pEarleyGate201-YFPN and pEarleyGate202-YFPC, respectively [A. tumefaciens C58C1 and transiently expressed in N. benthamiana by Agrobacterium infiltration. After the infiltration, the plants were incubated at 22°C under white light for two days and transferred to FR light for one day. We detected the signal with a fluorescence microscope (Nikon H600L with DS-Ri2 camera).The ectively  and tranPHYA-GFP/Col-0 and PHYA-GFP/fin219-2 were grown on Murashige and Skoog (MS) medium containing ethanol or MeJA (50 μM) at 22°C under continuous dark or FR light (2 μmol m-2 s-1) conditions for three days. We observed nuclear speckles of hypocotyl cells with Nikon H600L microscopes.Seedlings of Harvested seedlings were extracted with 100 μl of 80% acetone for 16 h at 4°C in the dark with vigorous mixing. We added an amount of 50 μl extract to 450 μl absolute alcohol and used a 200 μl sample for spectrophotometric analysis. Then we measured the absorbance at 660, 647, and 720 nm using Tecan Infinite 200 PRO (Tecan) with 96-well plates. Total chlorophyll content was determined according to the extinction coefficient calculated as described .We weighed harvested seedling samples, ground them with liquid nitrogen, and extracted the total plant pigments. The anthocyanin content was quantified using absorbance (A) at 530 nm and 657 nm with the Tecan Infinite 200 PRO (Tecan) and normalized to fresh weight. We determined the total anthocyanin content as described equation .phyA mutants . We sent the eluted CBP-PHYA recombinant proteins to LTK BioLaboratories to produce monoclonal antibodies. The specificity of PHYA monoclonal antibodies was determined by using recombinant proteins of the full-length PHYA (CBP-PHYA-F), the N-terminal, and the C-terminal domains of PHYA (CBP-PHYA-N and CBP-PHYA-C), wild-type Col-0 and  mutants .We conducted statistical analyses with IBM SPSS Statistics version 22. Growth and bioassay data were analyzed using one-way ANOVA with posthoc Tukey’s test to determine the statistically significant means (P<0.05). Different alphabets represent a statistically significant difference.S1 FigA-B) qRT-PCR analysis of FIN219 (A) and PHYA (B) transcript levels in the indicated genotypes under dark. (C-D) qRT-PCR analyses of FIN219 (C) and PHYA (D) transcript levels in the indicated genotypes under FR light (2 μmol m-2 s-1). Seedlings were grown under dark (A-B) or FR light (C-D) without or with 50 μM MeJA for 3 days. Actin 2 was an internal control. Data are mean ± SE of three experiments.((TIF)Click here for additional data file.S2 FigPHYA overexpression transgenic lines in Col-0 and phyA-211 mutant backgrounds as shown in the figure were grown under FR light (2 μmol m-2 s-1) for 3 days, then used for total RNA extraction and subjected for qRT-PCR analysis. Actin 2 was an internal control. Data are mean ± SE of three experiments.The seedlings of wild-type Col-0 and various (TIF)Click here for additional data file.S3 FigA) Chlorophyll content in the FR-blocked greening of indicated seedlings grown on GM plates under FR light (2 μmol m-2 s-1) without or with 50 μM MeJA for 3 days followed by white light for 1 day. (B) Anthocyanin content in seedlings grown on GM plates under FR light (2 μmol m-2 s-1) without or with 50 μM MeJA for 3 days. (C) Effect of MeJA on chlorophyll content of seedlings grown on GM plates without (Control) or with 5 μM MeJA treatment under white light (70 μmol m-2 s-1) for 7 days. (D) Effect of MeJA on the germination rate (%) of seeds sown on GM plates without (Control) or with 50 μM MeJA treatment under cFR light (2 μmol m-2 s-1) for 3 days. Data are mean ± SE of three biological replicates. Different lowercase letters represent significant differences by ANOVA at P < 0.05.((TIF)Click here for additional data file.S4 FigA)Schematic diagrams of five different deletions of PHYA apoprotein as prey showed PHYA interaction with FIN219 as bait through its C-terminal domain. (B) Schematic diagrams of three different deletions of FIN219 as prey showed FIN219 interaction with PHYA as bait through its N-terminal domain. Yeast cells were transformed with PHYA-AD and FIN219-BD, grown on selective medium, and exhibited β-galactosidase activity. PAS: Per/Arndt/Sim domain; HKRD: histidine kinase-related domain; AD: active domain; NC: N-terminal coiled-coil domain; CC: C-terminal coiled-coil domain; BD: binding domain.((TIF)Click here for additional data file.S5 FigA) and then transferred to FR light (2 μmol m-2 s-1) for different times (B-C). Total proteins 2 mg extracted from the seedlings were immunoprecipitated with phyA monoclonal antibodies, then probed with PHYA and FIN219 monoclonal antibodies. I: input proteins; S: supernatants after immunoprecipitation; P: pellets after immunoprecipitation.Col-0 seedlings were grown in the dark with 50 μM MeJA for 3 d ((TIF)Click here for additional data file.S6 FigphyA mutant were differentially regulated by FR light fluences. The seedlings of Col-0, phyA-211 and fin219-2 were grown under different fluences of FR light for 3 days. The extracted proteins were subjected for Protein gel blot analysis. The primary antibodies were PHYA and FIN219 monoclonal antibodies. Tubulin was a loading control. The number below each blot represents the level of the indicated protein. The level of wild-type Col-0 was arbitrarily set to 1.Protein gel blot analysis indicated that FIN219 levels in (TIF)Click here for additional data file.S7 FigA) Photograph of purified recombinant CBP-PHYA proteins. Purified recombinant proteins of the full-length and N-terminal and C-terminal domains of PHYA fused with a calmodulin binding peptide  were separated by 10% SDS-PAGE and stained with Coomassie blue dye (left panel). Protein gel blot analysis of recombinant CBP-PHYA proteins with different phyA monoclonal antibodies. PHYA monoclonal antibody #2 specifically recognized the full-length and N-terminus of PHYA  or PHYA monoclonal antibody #5 for the C-terminus of PHYA . Dilution factor: 10,000X. (B) Protein gel blot analysis of PHYA protein detection specificity in seedlings of the indicated genotypes grown in the dark for 3 d. All phyA mutants were in a Columbia background. phyA: phyA-1; phyA-T: SALK_014575 mutants. Total protein (60 μg) was loaded in each lane and blots were probed with monoclonal antibody #2 against PHYA at dilution ratio 5000X.((TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


Taniborbactam and xeruborbactam are dual serine-/metallo-beta-lactamase inhibitors (BLIs) based on a cyclic boronic acid pharmacophore that undergo clinical development. Recent report demonstrated that New Delhi metallo-beta-lactamase (NDM)-9  deprivation) is resistant to inhibition by taniborbactam constituting pre-existing taniborbactam resistance mechanism. Using microbiological and biochemical experiments, we show that xeruborbactam is capable of inhibiting NDM-9 and propose the structural basis for differences between two BLIs. Enterobacterales worldwide (NCT03840148). With their study, the authors raised awareness of differences in sensitivity of MBLs to new inhibitors, including pre-existing resistance to cefepime-taniborbactam. This finding is of concern as it demonstrates that NDM-9 has an advantage either in the presence or in the absence of selective pressure from cefepime-taniborbactam.Recent studies suggest that NDM [metallo-beta-lactamase (MBL) of the New Delhi group] variants are evolving to overcome Zn (II) deprivation, a condition that can be elicited by the immune system in response to infection . One of orldwide . Recent orldwide  demonstrorldwide  that has administered by the IV or oral route (as a prodrug form). In these studies, xeruborbactam was found to be safe and well tolerated at exposures that exceeded non-clinical PK-PD targets Fig. 1).  targets . The spe targets . Compare targets , 12. TheKlebsiella pneumoniae and one isolate of Escherichia coli producing NDM-9  guidelines  and an increased cefepime-xeruborbactam (8 µg/mL) MIC values. These strains carried four amino acids, YRIN, insertion in PBP3 associated with a decreased susceptibility of cephalosporins and monobactams but not of meropenem  as a part of various worldwide surveillance studies. KP1280 (0106), (KP1297 0143), EC1100 (0128), and EC1104 0151) were obtained from the idelines . The MICng NDM-9 . Meropen3, EC11001 were obng NDM-9 . No sucheropenem , 17. Of te M07 20 guidelineropenem . Based o50 of xeruborbactam (and taniborbactam) inhibition of imipenem hydrolysis in cell lysates prepared from either NDM-1 (KP1297) or NDM-9 (KP1671) producing strains. Bacterial lysates were prepared as previously described ” equation using Prizm software.Inhibition of NDM-9 by xeruborbactam was also demonstrated in biochemical experiments. We determined ICescribed ; imipene50s of xeruborbactam inhibition of imipenem hydrolysis by NDM-1 (0.77 ± 0.12 µM) or NDM-9 (1.2 ± 0.1 µM) were essentially the same. Taniborbactam inhibited NDM-1-meditaed hydrolysis (IC50 = 0.24 ± 0.052 µM) but not NDM-9-mediated hydrolysis (IC50 >60 µM).The ICAvailable crystallography data , 12 prov., NDM-4 and NDM-6) that also evolved to withstand Zn (II) deprivation (In conclusion, we have shown that xeruborbactam retains high potency against NDM-9 as well as against other NDM variants (e.grivation . This fi


More importantly, pAD1016 vaccination in mice, rabbits and pigs generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These preliminary data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad-spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy of further translational study as a COVID-19 vaccine candidate.Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants currently pose a global challenge to the control of the COVID-19 pandemic. We previously reported a pVAX1-based DNA vaccine candidate, pAD1002, that encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 wildtype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1 RBD-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ Effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools for controlling the ongoing pandemic coronavirus disease 2019 (COVID-19), which has caused more than 630 million infections with more than 6.5 million deaths worldwide since late 2019 ,5. In th+-CD4 and CD8 T cells against WT SARS-CoV-2 as well as Delta variant in volunteers aged between 18 and 60 years [SARS) and SARS-CoV-2 Omicron BA.1 (RBDBA1). When delivered by either microneedle array patch (MAP) or electroporation, the pAD1002 plasmid elicited high titer NAbs against heterologous sarbecoviruses including SARS-CoV-1, SARS-CoV-2 WT and its variants Beta, Delta and Omicron BA.1, BA.2, and, relatively poorly, BA.4/5 [BA1-encoding sequence in pAD1002 with that of BA.4/BA.5. Mouse antibodies (Abs) elicited by plasmid pAD1016, in either a two-dose DNA vaccination procedure, or a prime-boost scheme with the DNA vaccine as a booster dose, effectively cross-neutralized multiple Omicron subvariants, including BA.4/5, BF.7 and BQ.1. pAD1016-induced rabbit Abs effectively neutralized BA.2, BA.4/5, BF.7 and XBB pseudoviruses. Moreover, after immunization with pAD1016 twice, pigs produced serum Abs capable of neutralizing BQ.1.1 pseudovirus. Our RBDSARS chimera approach paves the way for expedited vaccine development to catch up with the continually mutating SARS-CoV-2 virus.The spike (S) protein of SARS-CoV-2 is the main target for COVID-19 vaccines. It contains the receptor-binding domain (RBD) responsible for human ACE2 (hACE2) receptor binding and mediating virus entry ,13. Neut60 years . In 2021, BA.4/5 . However, BA.4/5 . In the chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif (ARE), repeat sequences  and restriction sites that may interfere with cloning. The resulting synthesized and optimized cDNA was cloned into pAD1002, replacing the BA.1 RBD-encoding DNA fragment. Restriction enzyme analysis and DNA sequencing were performed to confirm the accuracy of construction. Plasmids were transformed into E. Coli strain HB101. Single colonies underwent expansion in one-liter flasks for culturing in LB broth. Plasmids were extracted, purified by MaxPure Plasmid EF Giga Kit , and dissolved in distilled water at 1 mg/mL final concentration. Purity of the plasmids was measured by an agarose gel electrophoresis and a UV detector at a range of 1.8–2.0 OD260 nm/280 nm. Endotoxin contamination in plasmid samples was below 30 EU/mg by the LAL test.Preparation of pVAX1-based plasmid pAD1002 was as previously described . The cloDissolvable microneedle array patches (MAP) each laden with 20 μg pAD1016 DNA was fabricated as previously described . For theInactivated WT SARS-CoV-2 vaccine was a gift from Sinovac Biotech Co. Ltd, Beijing, China.WT primary Ab  diluted 1:1000 in 5% milk-0.05% PBS-Tween 20 and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary Ab . Chemiluminescence detection was performed with the ECL Prime Western Blotting System and acquired by the ChemiDoc Imaging System .HEK293 cells pre-plated in a 6-well plate were transiently transfected with 2.5 μg DNA vaccine plasmids with Hieff TransTM Liposomal Transfection Reagent . Two days later, the cells were pelleted and lysed in immunoprecipitation assay buffer. Cell lysates and supernatants were separated by SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed by using rabbit anti-RBDSARS-CoV-2 RBD-specific quantitative reverse transcription-PCR (qRT-PCR) assays were performed by using a FastKing One Step Probe RT-qPCR kit  on a CFX96 Touch real-time PCR detection system . Sequences of the 3′ and 5′ end probes are: AAGCTGAACGACCTGTGCTTCA and GGCAGCTTGTAGTTGTAG.Female BALB/c and C57BL/6 mice (6–8 weeks of age) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.  and maintained under specific pathogen-free (SPF) conditions at the animal facilities of Advaccine Biologics (Suzhou) Co. New Zealand white rabbits, purchased from Shanghai Somglian Experimental Animal Company , were housed in the Grade I animal facilities of Advaccine Biologics Co. . All animal experiments were performed in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology Ethics Committee and approved (document No. 2021070102) by the Ethics Committees of the company.®2000 and electrode  with two sets of pulses with 0.2 amp constant current. All intramuscular (IM)+EP-delivered vaccines were primed on day 0 and boosted on day 14 unless otherwise indicated. Blood samples were collected on days 0, 14, 21 and/or 28.EP-assisted DNA immunization was performed in mouse and rabbit quadriceps injected with 20 μg (for mice) or 0.5 mg (for rabbits) DNA (1 mg/mL in 30 μL SSC), followed by EP using Inovio CELLECTRA2SO4, and the absorbance measured at 450 nm and reference 620 nm using a microplate reader .Antibody titration was performed on sera obtained by retro-orbital bleeding from mice or venous bleeding from rabbit’s ears. The ELISA plates were functionalized by coating with the recombinant RBD of wildtype SARS-CoV-2 virus  at 1 μg/mL, incubated 18 h at 4 °C and subsequently blocked with 3% BSA-0.05% Tween 20-PBS (PBST) for 1 h at room temperature. Serial diluted serum samples were then added in triplicate wells, and the plates were incubated for 1 h at room temperature. After a double wash with PBST, horseradish peroxidase (HRP)-conjugated Ab against murine , or rabbit  IgG was added and then developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate . The reaction was stopped with 2 M of H2 for 72 h. The cells were then lysed using Bright-Glo™ Luciferase Assay , and relative luminance unit (RLU) was measured using an automated luminometer. Fifty percent pseudovirus neutralization titer (pVNT50) was determined by fitting nonlinear regression curves using GraphPad Prism and calculating the reciprocal of the serum dilution required for 50% neutralization of infection. These assays were performed in a BSL-2 facility of Advaccine. Pseudovirus neutralization results were verified using Vero cells by Gobond Testing Technology.The pseudovirus microneutralization assay was performed to measure neutralizing antibody levels against prototype SARS-CoV-2 and variants. Pseudovirus stocks of SARS-CoV-2 prototype, variants Delta, BA.2, BA.4/5, BF.7, BQ.1 and XBB were purchased from Gobond Testing Technology, and were aliquoted for storage at −80 °C. hACE2 stable expressing HEK293T cells (prepared in our lab) were used as target cells plated at 10,000 cells/well. SARS-CoV-2 pseudoviruses (50 μL) were incubated with heat-inactivated (56 °C for 30 min) and 1/3 serial diluted mouse sera (50 μL) for 90 min at room temperature; then, the sera-pseudovirus mixtures were added to hACE2-HEK293T cells and allowed to incubate in a standard incubator 37% humidity, 5% COAlphaFold2 was used for structure prediction with the required homology modeling databases running on ColabFold. The pLDDT plots generated and the obtained structures were further visualized by PyMol 2.4.5 freshly prepared mouse splenocytes, or LN cells, were plated into each well and stimulated for 20 h with pooled overlapping 15-mer peptides (10 μg/mL) covering respective SARS-CoV-1 or SARS-CoV-2 Omicron BA4/5 RBDs at 37 °C in a 5% CO2 incubator. PMA(phorbol-12-myristate-13-acetate)/Ionomycin was used for positive controls. The plates were processed in turn with a biotinylated detection antibody. Spots were scanned and quantified using AID ImmunoSpot reader . IFN-γ spot-forming units were calculated and expressed as SFUs per million cells.Spleens and draining lymph nodes (LN) from immunized mice were collected and used to prepare single cell suspension in RPMI-1640 medium supplemented with 10% FBS and penicillin/streptomycin. ELISpot was performed using mouse IFN-γ ELISpot PLUS kits  according to the manufacturer’s protocol. Briefly, 5 × 10t-tests and two-way ANOVA tests to analyze experiments with multiple groups and two independent variables. Significance is indicated as follows: * p < 0.05; ** p < 0.01. Comparisons are not statistically significant unless indicated.Statistical analyses were performed with GraphPad Prism software version 9 . Error bars indicate the standard error of the mean (SEM). We used Mann–Whitney BA1-encoding DNA sequence in pAD1002 with that of BA.4/5 (SARS/BA5 polypeptide was readily detectable by ELISAs in culture supernatant of the transfectant cells (WT-cross-binding) IgG responses in BALB/c and C57BL/6 mice , known to play pivotal roles in protection against viral infections in vivo [+ T cells bearing T cell receptors (TCRs) specific for a H-2Db-restricted predominant CTL epitope (the “S” epitope) in the RBD of SARS-CoV-2 [+ cells responsive to SARS-CoV-1 and SARS-CoV-2 BA.1/2 RBD peptides  followed by a booster dose of either pAD1016  or the inactivated virus vaccine. SARS/BA1 chimera, here we demonstrate the modification of pAD1002 by replacing the RBDBA1-encoding sequence with that of RBDBA5. The resulting plasmid, pAD1016, generated Abs capable of neutralizing multiple Omicron subvariants including BA.4/5, BF.7, BQ.1 and XBB in mice and rabbits, thereby overcoming the inability of pAD1002 in covering the recent emerging Omicron subvariants. In addition, pAD1016 was able to induce NAbs against Omicron subvariants BF.7 and BQ.1.1 in pigs  replacement approach in DNA vaccine development merits further translational studies.


Klebsiella pneumoniae carbapenemase 2–producing K. pneumoniae. Plasmid recombination and chromosomal integration formed a novel virulence plasmid and provided an additional promoter for blaSHV-12, leading to blaSHV-12 overexpression and ceftazidime/avibactam resistance. Genetic rearrangement contributed to convergence of hypervirulence and ceftazidime/avibactam resistance.We identified a novel ceftazidime/avibactam resistance mechanism in sequence type 11  Klebsiella pneumoniae pose therapeutic challenges to public health  provides an antimicrobial strategy (http://www.chinets.com/Data/AntibioticdrugFast), 9.9% of K. pneumoniae carbapenemase (KPC) 2–producing K. pneumoniae (KPC-KP) displayed CAZ/AVI resistance  and capsular polysaccharide regulator RmpA2. pVir-KP0714 was 99.97% identical to reference plasmid pOXA1_020030 (GenBank accession no. CP028791) from K. pneumoniae strain WCHKP020030 at 74% coverage. Both ends of pVir-KP0714 were absent from pOXA1_020030 but were highly homologous to another plasmid, pLAP2_020030 (GenBank accession no. CP028792), from WCHKP020030 -Ia, which exhibited 100% identity and 100% query coverage with the reference plasmid pLAP2_020030 (KP0714 possessed a novel IncFIB(K)-type virulence plasmid pVir-KP0714, encoding siderophore aerobactin -Ia provided an addition promoter P2 for blaSHV-12 (blaSHV-12 by using a pConj working vector-based genetic engineering approach (blaSHV-12 in KP0714 was ≈20-fold higher than in ΔP1 and ΔP2 mutants (blaSHV-12 resulting from an additional promoter, and the original promoter P1 was also necessary for the biological function of P2.However, we observed substantial structural changes in this chromosomal insertion fragment compared with pLAP2_020030  Figure 2laSHV-12  Figure 2approach  Table 1. mutants  Figure 2K. pneumoniae strain CR-HvKP4 (Because a novel virulence plasmid pVir-KP0714 was formed through plasmid recombination, we determined the virulence potential of KP0714 by using a mouse infection model and human neutrophil phagocytosis assay (CR-HvKP4  Figure 2In conclusion, KP0714 high-level resistance to carbapenems and CAZ/AVI, compensating for decreased carbapenem hydrolyzation activity of KPC variants (Klebsiella pneumoniae.Additional results for study of genetic rearrangement and ceftazidime/avibactam resistance in hypervirulent


Herein, we report the complete genome for an avian infectious bronchitis virus isolated from cecal tonsils of California layers in 2021. This whole-genome sequence belongings to genotype GVIII, previously classified as a unique variant. Gammacoronavirus and family Coronaviridae, causes a contagious, endemic disease  in chickens associated with respiratory and urogenital consequences and severe economic losses (Avian infectious bronchitis virus (IBV), of genus c losses –3. Itsc losses . Since, c losses , 6. Hereg, 45 min) and reconstituted in 200 µL VTM. Total RNA was extracted from the allantoic fluid using the MagMax-96 Viral Isolation Kit . ds-cDNA was generated using the total RNA, random hexamers, and the Maxima H Minus Double-Stranded cDNA Synthesis kit (ThermoFisher) and then purified using AMPure XP beads (1:1) . A library was generated using the ONT Ligation Sequencing Kit . The cDNA was end-repaired using the NEBNext Ultra II DNA End Prep and Repair kit , bead purified (1:1), and eluted in nuclease-free H2O. NEBNext Quick T4 DNA ligase (New England Biolabs) ligated AMX-sequencing adapters (ONT) to the cDNA. Adapter-ligated DNA was bead purified (1:2.5), washed twice with short fragment buffer (ONT), and eluted in elution buffer (ONT). Each library was loaded individually onto Flongle flow cells R9.4.1 on a GridION sequencer (ONT) and run for 24 h with super-accuracy basecalling and Q10+ read quality. IBV/Ck/USA/CA/21-1883 returned 35,117 reads with >500X IBV genome coverage and IBV/Ck/USA/CA/21-1926 returned 80,179 reads with >1,000X coverage.In late 2021, two cecal tonsils collected from 33- and 34-week-old  white Leghorn layers at a California farm experiencing increased mortality and decreased egg production were submitted to the California Animal Health and Food Safety Laboratory. The tissues were IBV positive by RT-qPCR , and theBioinformatics were performed in the Geneious Prime suite 2022.1.1 (Biomatter Ltd.). Reads were mapped using MiniMap2  to strai


Pancreatic cancer is the eighth leading cause of cancer-related deaths worldwide. Chemotherapy including gemcitabine, 5-fluorouracil, adriamycin and cisplatin, immunotherapy with immune checkpoint inhibitors and targeted therapy have been demonstrated to significantly improve prognosis of pancreatic cancer patients with advanced diseases. However, most patients developed drug resistance to these therapeutic agents, which leading to shortened patient survival. The detailed molecular mechanisms contributing to pancreatic cancer drug resistance remain largely unclear. The growing evidences have shown that noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are involved in pancreatic cancer pathogenesis and development of drug resistance. In the present review, we systematically summarized the new insight on of various miRNAs, lncRNAs and circRNAs on drug resistance of pancreatic cancer. These results demonstrated that targeting the tumor-specific ncRNA may provide novel options for pancreatic cancer treatments. Pancreatic cancer (PC), a highly lethal human malignancy, ranks the fourth leading cause of cancer-related death in the United States and the eighth worldwide. PC patients had only 5% overall survival rate at 5-years after diagnosis . A totalNoncoding RNAs (ncRNAs) transcripts, a series of RNA molecules lacking of proteins coding potentials, constitute about 70% of human genome and modulate most signaling pathways, physiological and pathological processes . MultiplConsidering the critical and complicated roles of ncRNAs in PC drug resistance development, herein, we systematically summarized the underlying molecular mechanisms how miRNAs, lncRNAs and circRNAs confer drug resistance in PC.miRNAs, a subclass of sncRNAs, negatively regulate target gene expression through binding to the 3′-untranslated region (3′-UTR) of the target mRNA and are involved in multiple cellular process, such as cell difference, proliferation, apoptosis, cell cycle progression, angiogenesis, EMT and CSC formation. Several dysregulated miRNAs, functioning as oncogenes or tumor suppressors, have been identified to be involved in resistance development to therapeutic agents in PC . CompareGEM, a kind of cytosine nucleoside derivative, is currently the first-line standard chemotherapeutic agent to treat PC patients. However, only a few PC patients can maintain sensitivity to GEM chemotherapy and subsequently, inevitable drug resistance often leads to low response rate and poor treatment efficacy. A variety of miRNAs have been found to be associated with GEM resistance in PC. Multiple mechanisms, such as diminished apoptosis, increased DNA repair, disordered cell cycle, decreased intracellular drug accumulation due to increased expression of drug efflux transporters, EMT, as well as CSCs, play crucial roles in miRNAs-mediated development of GEM resistance in PC. Oncogenic and tumor suppressor miRNAs involved in GEM resistance are summarized in details ; Table 2Bim, could potentiate GEM sensitivities, activate caspase-3 and promote apoptosis in human PANC-1 and BxPC3 PC cell lines (Dikkopf-1 (Dkk1), Kremen2, secreted frizzled related protein 2 (sFRP2) and activating the Wnt/β-catenin signaling in human MIAPaCa-2 and PSN-1 PC cells (phosphatase and tensin homologue deleted on chromosome ten (PTEN)/RECK, programmed cell death 4 (PDCD4), Bcl-2, FasL, matrix metalloproteinase-2 (MMP-2), MMP-9 and VEGF (platelet-derived growth factor (PDGF), MMP-3, MMP-9 and chemokine (C-C motif) ligand 7 (CCL7) (BAX and adaptor protein Src Homology 2 Domain Containing 1 (SHC1), miR-365 has been shown to potentiate the resistance against GEM in PC cells  and cylindromatosis (CYLD) as well as activation of the Hippo signaling  . ThroughPC cells . MoreovePC cells . In addiignaling . Interesignaling . In addiignaling . RecentlP1 in PC . By cont pathway .cadherin 1 (CDH1) expression (PTEN expression (TAp63 in PC (Bcl2-related ovarian killer (BOK) and EMT marker vimentin and N-cadherin, exogenous expression of miR-296-5p has been found to weaken the apoptosis induced by GEM, indicating that targeting miR-296-5p may have therapeutic potential to overcome GEM resistance of PC patients  and Krüppel-like factor 6 (KLF6), respectively . Plasma ignaling . miR-317ignaling .survivin, cyclin D1 (CCND1) and multi-drug resistance 1 (MDR-1) transcription, as well as reducing protein expression of N-cadherin, slug and vimentin  (High mobility group box 1 (HMGB1) and ubiquitin specific peptidase-22 (USP22) and, thus, reducing autophagy, respectively  . miR-494s (PUMA) . In addis (PUMA) . It has ectively . Similar S phase .E-cadherin, Z O -1, Zinc finger E-box binding homeobox transcription factor 1 (ZEB1) and Snail in PC , RRM1 and ZEB1  and Integrin β1 (ITGB1), respectively (RRM2 (ZNF32 (hypoxia-inducible factor 1ɑ (HIF-1ɑ)  .As a thymidylate synthase inhibitor, 5-FU is commonly used for PC treatments in clinic. 5-FU leads to apoptosis and cell cycle arrest through interfering DNA replication, RNA function and protein synthesis. It has been demonstrated that several oncogenic or tumor suppressor miRNAs contribute to the resistance to 5-FU in PC .PTEN and PDCD4  binding cassette subfamily B member 1 (P-gp), ATP binding cassette subfamily C member 1 (MRP1), and ATP binding cassette subfamily G member 2 (BCRP)  .pleiotropic growth factor (PTN) expression  in PC cells (forkhead transcription factor O subfamily 3a (FoxO3a) in PC cells   and clusterin (CLU) (glucose transporter protein type 1 (GLUT1) expression and GLUT1-mediated glycolysis in ASPC-1 and PANC-1 PC cells , targeted therapy drugs and immunotherapy agents are also used during PC clinical treatments. It has been found that several tumor suppressor miRNAs, including miR-137, miR-142 and miR-212, could weaken ADM resistance of PC. For example, through targeting PC cells . InteresPC cells . OncogenPC cells . In humapression . miR-100 (FGFR3) . In addiin (CLU) . Tumor sPC cells . Laura apecimens . Interespecimens .mitogen-inducible gene 6 (MIG6) in the process of transforming growth factor β (TGFβ)-mediated EMT. Moreover, the ratio of MIG6 mRNA to miR-200 (MIG6 mRNA/miR-200) was negatively correlated with erlotinib response not only in cancer cell lines with diverse tissue origins in vitro, but also in xenografts derived from PC patients carrying wild-type EGFR in vivo (fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) (erythropoietin-producing hepatocellular receptor 2 (EphA2), tumor suppressor miR-124 has also been found to improve erlotinib sensitivity in Capan-1 PC cells with K-RAS mutations  . Throughutations . Table 4LncRNAs could be divided into four types according to their location in the genome: intronic lncRNAs, intergenic lncRNAs, divergent lncRNAs and antisense lncRNAs . AccumulSimilar with miRNAs, several lncRNAs have been shown to participate in GEM resistance in PC, including oncogenic lncRNAs and tumor suppressive lncRNAs molecules .HOXA transcript at the distal tip (HOTTIP), glutathione S-transferase mu 3, transcript variant 2 (GSTM3TV2), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), plasmacytoma variant translocation 1 (PVT1) and DiGeorge syndrome critical region gene 5 (DGCR5).Multiple oncogenic lncRNAs have been shown to contribute to GEM resistance in PC, such as HOXA13 in PDAC cells. In contrast, silencing HOTTIP could lead to enhanced sensitivity of PC cells to GEM (L-type amino acid transporter 2 (LAT2) and oxidized low-density lipoprotein receptor 1 (OLR1). Moreover, the increased levels of GSTM3TV2 in PC tissues have been significantly associated with worse prognosis, indicating that GSTM3TV2 may be a novel prognostic marker and therapeutic target in PC  could enhance GEM resistance via functioning as a ceRNA through sponging miR-3163 to inhibit the Wnt/β-catenin signaling and modulate expression of DNA topoisomerase 2-alpha (TOP2A) (HIF1A-AS1 (antisense RNA1 of HIF-1α) has been found to prevent GEM sensitivity of PC cells through activating the AKT/YB1/HIF-1α signaling and promoting glycolysis , which was delivered by hypoxic pancreatic stellate cells (PSCs)-derived exosomes (HPSC-EXO), has also been shown to enhance GEM resistance in PC  has been found to confer GEM resistance via diminishing SMAD family member (Smad4) expression in PC  . LncRNA ycolysis . Interesce in PC . Throughon in PC . Via spoPC cells .Accumulating evidences demonstrated that CAFs are critically involved in chemoresistance . LINC004ZEB1 and CSC markers including Lin28, Nanog, Sox2 and Oct4  and stimulating intracellular reactive oxygen species (ROS) (SBF2-AS1 could promote EMT and GEM resistance in PC via sponging miR-142-3p and up-regulating expression of twinfilin 1 (TWF1)  . In addi1 (TWF1) .HLA complex P5 (HCP5) and SNHG14 could confer GEM resistance of PC through sponging miR-214-3p and miR-101 and activating autophagy , a crucial cell type in tumor micro-environment, may contribute to drug resistance in multiple neoplasms via producing protective cytokines or influencing gene expression . It has growth arrest-specific 5 (GAS5) has been demonstrated to suppress GEM resistance of PC cells through inhibiting miR-221 expression and increasing SOCS3 expression  expression (HOX transcript antisense gene RNA (HOTAIR) has been demonstrated to confer resistance to TNF-related apoptosis-inducing ligand (TRAIL) via interacting with the epigenetic regulator EZH2 and inhibiting TRAIL receptor death receptor 5 (DR5) expression. Silencing HOTAIR could enhance TRAIL-induced apoptosis in TRAIL-resistant PC cells. On the contrary, HOTAIR over-expression could inhibit apoptosis induced by TRAIL in TRAIL-sensitive cells, indicating that HOTAIR may act as a potential therapeutic target to conquer TRAIL-resistance in PC  and three downregulated circRNAs  were successfully verified by the qRT-PCR assay. Among which, chr14:101402109-101464448 + and chr4:52729603-52780244+ have been found to be highly expressed in plasma of PC patients, who showed no response to GEM treatment. Silencing these two circRNAs could restore GEM sensitivity of PANC-1-GR cells . SimilarRASSF1 expression  axis .To date, no report has shown that oncogenic circRNAs could involve in the resistance to other drugs. In the aspect of tumor suppressor circRNAs, only circ_0013587 has been found to reverse erlotinib resistance through modulating the miR-1227/E-cadherin signaling in PC cells .http://clinicaltrials.gov, no clinical trials based on ncRNAs therapeutics in PC were found currently. The combined therapeutic modalities based on the manipulation of ncRNAs’ levels and traditional treatments, i.e. chemotherapy, molecular targeted therapy or immunotherapy, may be a promising approach to overcome drug resistance and help to improve prognosis of advanced PC patients. Nevertheless, a serious issue is how to select the appropriate target molecules from a huge number of ncRNA candidates. More importantly, clinical studies and translational studies are also needed for conquering drug resistance in PC.Many ncRNAs have been shown to be involved in the pathogenesis and progression of PC, indicating the potential roles of ncRNAs as biomarkers for early diagnosis, as promising prognostic and predictive markers for the identification of candidates amenable to adjuvant treatment, enabling a personalized clinical approach. Interestingly, a number of studies attempted to investigate the diagnostic value of circulating miRNAs in PC . Recentl


C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on  Once autophagosomes form, they fuse with lysosomes, creating autolysosomes to degrade their contents.Organelle integrity and cellular proteostasis depend heavily upon a variety of degradative mechanisms for macromolecular turnover. Among these, one key pathway is macroautophagy (hereafter referred to as autophagy), a mechanism that can remove whole defective organelles and cytoplasmic protein aggregates as they build up, ameliorating the cellular dysfunction that can occur if such components accumulate. Autophagy begins with the phagophore, a 2, a clathrin coat, the severing enzyme dynamin, and kinesin motor activity), an allele we generated by CRISPR-based genome engineering to mimic the human late onset Parkinson’s associated allele N855S[rme-8(pw22[N861S]) bears an equivalent point mutation to N855S, changing asparagine 861 to serine (N861S).(b1023ts). Tempera(b1023ts). For mosele N855S. rme-8(prme-8(ts) mutant on late endosomes and lysosomes using an mScarlet::RAB-7 marker expressed in the mechanosensory neurons. Importantly, in young adult (adult Day 1) rme-8(b1023ts) mutants we noted the appearance of RAB-7 vesicles bearing highly elongated tubules, a morphology not observed in control animals (rme-8(N861S). Interestingly, we observed a similar accumulation of elongated RAB-7-positive tubules in aged animals in late adulthood (adult Day 9), but not in young adults  mutants, measuring a highly significant increase in LMP-1 tubule length (rme-8(+) backgrounds , a transmembrane marker of lysosomes . We notee length . We notee length , and in e length . These pkgrounds . We extekgrounds . These drme-8(b1023ts) phenotype in animals expressing a touch neuron-specific rme-8(+) minigene. Indeed, we observed that expressing wild-type RME-8 in just the six touch neurons rescued touch neuron lysosomal tubule accumulation . Neurme-8(b1023ts) mutants in LMP-1::mSc; mNG::LGG-1 double labeled strains  mutants, but a reduced percentage of LMP-1-positive area that colocalized with LGG-1 signal in rme-8(b1023ts) mutants (rme-8(b1023ts) mutants  in  strains  and in s strains . We note mutants . We also mutants . Taken trme-8 mutants suggests a failure to recycle LMP-1 out of the autolysosome, further supporting the hypothesis that the elongated tubules accumulating in rme-8 mutant neurons represent an accumulating ALR intermediate P2), phosphatidylinositol 3-phosphate (PI(3)P), as well as the coat protein clathrin and the membrane severing GTPase dynamin, have been suggested to function in ALR, in addition to their more well-known roles in other trafficking steps ) for 24–30 h prior to imaging. Live animals were picked into 2 uL of 10 mM levamisole on the center of a cover slip. After two minutes, 5% agarose pads were placed on top. Fluorescence images were obtained using a spinning-disk confocal imaging system: Zeiss Axiovert Z1 microscope equipped with X-Light V2 Spinning Disk Confocal Unit (CrestOptics), 7-line LDI Laser Launch (89 North), Prime 95B Scientific CMOS camera (Photometrics) and oil-immersion objective (100X). Fluorescence images were captured using Metamorph 7.10 software. Z series of 25–30 optical sections were acquired using a 0.2μm step size in order to image through the entire soma. Exposure times ranged from 50ms to 300 ms based on the fluorescent protein being imaged such that images acquired were at minimum in the 12-bit and did not become saturated in the 16-bit range in any genetic background.Prior to imaging, L4 hermaphodites maintained at 15°C were picked to fresh OP50 plates and plates were temperature shifted to 25°C  were manually thresholded and the average, maximum, and integrated intensity measurements recorded using Metamorph’s “Region Measurements” tool. Each worm was treated as one data point.Maximum projections were also used for length measurements. Lengths were measured and recorded using Metamorph’s “region measurements” and using the line tool to trace the length of every puncta/ tubule in the axon. The length of every visible puncta/ tubule within each axon was measured and counted as a data point. Analysis of tubule elongation was restricted to axons due to the poor spatial resolution of puncta within the soma. For each experiment and genetic background, 15–25 animals were analyzed.Colocalization analysis was performed using MetaMorph 7.7 colocalization plugin, whereby intensities in each channel were thresholded and analyzed.DNAJC13 shRNA plasmid(sc-77968-SH) and Control shRNA Plasmid-A(sc-108060) were from Santa Cruz Biotechnology. GFP-LC3B, GFP-LAMP1, and mRFP-LC3 constructs were prepared as previously described–76). E642 on polyornithine- and fibronectin-coated coverslips. Neurons were grown overnight in a plating medium  supplemented with 100× L-glutamine in Neurobasal medium (Invitrogen). Starting at DIV2, cultures were maintained in a conditioned medium with half-feed changes of neuronal feed  every 3 days. Neurons were co-transfected with Control shRNA (Cat. Sc-108060) or DnaJC13 (RME-8) shRNA  with GFP-LC3, GFP-LAMP1, or GFP-LAMP1 and mRFP-LC3 at DIV5 using Lipofectamine 2000 (Invitrogen) before live-cell imaging 7–12 days after transfection before quantification analysis. For some experiments, 24-hour incubation with lysosomal inhibitors E64D (40 μM) and Pepstatin A (40 μM) was applied to suppress lysosomal degradation.Cortices were dissected from E18–19 mouse embryos as described–79). Cor2, 1.2 mM MgCl2, 3 mM KCl, and 145 mM NaCl, pH 7.4. The temperature was maintained at 37°C with an air stream incubator. Confocal images were obtained using an Olympus FV3000 oil immersion 60x oil immersion lens  with a sequential-acquisition setting, using 488 nm excitation for GFP-LC3 or GFP-LAMP1 and 543 nm for mRFP-LC3. Images were acquired using the same settings below saturation at a resolution of 1,024 × 1,024 pixels (8-bit). Eight to ten sections were taken from the top-to-bottom of the specimen and brightest point projections were made. Time-lapse sequences of 1,024 × 1,024 pixels (8 bit) were collected at 2 s intervals with 1% intensity of the laser to minimize laser-induced bleaching and cell damage while maximizing pinhole opening. Time-lapse images were captured at a total of 100 frames. All recordings started 6 min after the coverslip was placed in the chamber. Images were imported to Adobe Photoshop and morphometric measurements were performed using NIH ImageJ. The thresholds in all images were set to similar levels. Measured data were imported into Excel software for analysis. Data were obtained from at least three independent experiments.For live-cell imaging, cells were transferred to Tyrode’s solution containing 10 mM HEPES, 10 mM glucose, 1.2 mM CaClAll results were graphed using GraphPad Prism 9 software, generating scatter plots depicting individual data points. Bars represent mean with SEM. Significance was measured by Student’s unpaired t-test when comparing 2 strains/conditions or Ordinary one-way ANOVA when comparing >2 strains, unless otherwise noted. Data were considered statistically different at P < 0.05. P < 0.05 is indicated with single asterisks, P < 0.01 with double asterisks, P < 0.001 with triple asterisks, and P < 0.0001 with quadruple asterisks.Supplement 1rme-8(ts) C. elegans ALM soma shows persistent lysosomal tubulation.Movie S1. Dynamics of LMP-1::mNG labeled lysosomes in  Time lapse image sequences of ALM neurons expressing LMP-1 are shown. An elongated tubule emanating from a lysosome vesicle can be seen. The tubules expands and contracts, but does not sever. Images were collected at 1s increments for 45s.Supplement 2C. elegans ALM soma.Movie S2. Dynamics of LMP-1::mNG labeled lysosomes in wild-type Time lapse image sequence of ALM neurons expressing LMP-1 in an rme-8(+) background is shown. A lysosome in the lower portion of the soma can be observed producing tiny transient tubular processes. Images were collected at 1s increments for 90s.Supplement 3Movie S3. Dynamics of lysosomal organelles marked by GFP-LAMP1 in the proximal processes of the cortical neuron expressing DNAJC13(RME-8) shRNA.Cortical neurons were transfected with GFP-LAMP1 and RME-8/DNAJC13 shRNA at DIV5, followed by time-lapse imaging at DIV16. Note that GFP-LAMP1-indicated lysosomal organelles appear as elongated tubules and undergo oscillatory movement in proximal processes to the neuronal soma (right). Time-lapse sequences were collected at 2-sec intervals during a 200-s observation.Supplement 4Movie S4. Dynamics of GFP-LAMP-1-labeled lysosomes in the proximal processes of control cortical neuron expressing Control shRNA.Cortical neurons were transfected with GFP-LAMP1 and Control shRNA at DIV5, followed by time-lapse imaging at DIV16. Note that lysosomal organelles appear as small puncta in the neuronal process and undergo dynamic movement in a predominant retrograde direction toward the soma (left). Time-lapse sequences were collected at 2-sec intervals during a 200-s observation.Supplement 5


Tac1-expressing glutamatergic neurons in ventrolateral division of ventromedial hypothalamus (VMHvl) drive intermale aggression. Aggression is increased due to increases of Ube3a gene dosage in the VMHvl neurons when modeling autism due to maternal 15q11-13 triplication. Targeted deletion of increased Ube3a copies in VMHvl reverses the elevated aggression adult mice. VMHvl neurons form excitatory synapses onto hypothalamic arcuate nucleus AgRP/NPY neurons through a NRXN1-CBLN1-GluD1 transsynaptic complex and UBE3A impairs this synapse by decreasing Cbln1 gene expression. Exciting AgRP/NPY arcuate neurons leads to feedback inhibition of VMHvl neurons and inhibits aggression. Asymptomatic increases of UBE3A synergize with a heterozygous deficiency of presynaptic Nrxn1 or postsynaptic Grid1 (both ASD genes) to increase aggression. Targeted deletions of Grid1 in arcuate AgRP neurons impairs the VMHvl to AgRP/NPY neuron excitatory synapses while increasing aggression. Chemogenetic/optogenetic activation of arcuate AgRP/NPY neurons inhibits VMHvl neurons and represses aggression. These data reveal that multiple autism genes converge to regulate the VMHvl-arcuate AgRP/NPY glutamatergic synapse. The hypothalamic circuitry implicated by these data suggest impaired excitation of AgRP/NPY feedback inhibitory neurons may explain the increased aggression behavior found in genetic forms of autism.The circuit origins of aggression in autism spectrum disorder remain undefined. Here we report  A feedback circuit in the hypothalamus that inhibits aggression is impaired by converging autism genetic defects. Autism spectrum disorders (ASDs) are early onset behavioral disorders defined by impaired social communication, increased repetitive behaviors and restrictive interests. The condition is associated with variable comorbidities such as heightened irritability including aggressive behaviors. Heightened irritability can include excessive tantrums and self-injurious aggressive behaviors that often require medical treatment. One effective medication is risperidone, an agent that acts as an atypical antipsychotic providing one of the few FDA approved treatments for irritability behavior in ASD (Esr1), progesterone receptor (Pgr), and neuropeptide substance P (Tac1). VMHvl Esr1 and Pgr neurons were shown to promote aggressive behavior while a role for VMHvl Tac1 neurons in regulating aggression is so far only suggested by a recent single cell-seq study showing increased expression of immediate early genes in response to aggressive behavior  technique to measure in vivo calcium dynamics in individual VMHvl Tac1 neurons in freely moving mice . As a positive control, Pgr-Cre expressing neurons in VMHvl were also tested. Administering CNO (1 mg/kg i.p.) dramatically increased total attack time and attack numbers in both Tac1-Cre and Pgr-Cre male mice when compared to animals receiving saline  triple the dosage of UBE3A gene expressed in mature neurons where the paternal allele is silenced. We studied Ube3a transgenic mice with additional copies of mouse full-length Ube3a gene designed to model this strongly penetrant genetic form of ASD (Ube3a-1x mice (heterozygous for the genomic Ube3a transgene insert) modeling maternal 15q11-13 interstitial duplication and Ube3a-2x mice (homozygous for the genomic Ube3a transgene insert) modeling maternal extranumerary isodicentric chromosome 15 (idic15)  impairs social interactions and vocalizations while Ube3a-1x mice display more limited deficits  , A and B protein –22. We ptermates . In the termates .Ube3a-3xFLAG mouse line (LoxTB-Ube3a mouse) where the extra copies of full-length Ube3a are expressed only in the presence of Cre recombinase. By inserting a LoxP-flanked translational blocker cassette (LoxTB) into intron 1 of the mouse Ube3a gene with a C-terminal FLAG tag (Ube3a-3xFLAG), the LoxTB cassette disrupts expression of all Ube3a splice variants. When Cre recombinase is expressed in specific cell types (through cell-type specific promoters), it deletes the LoxTB cassette, permitting cell-type specific expression of the FLAG-tagged UBE3A  mice since Sf1-Cre is expressed predominantly in VMH  mice  mice . The resTac1 neuron in VMHvl might underlie this UBE3A-augmented aggression, AAV-hSyn-DIO-Ube3a (untagged) was stereotactically injected into VMHvl of Tac1-Cre male mice  in exon 12 of the mouse Ube3a transgene  displayed heightened aggression . Targeting UBE3A to the nucleus selectively in VMHvl Tac1+ neurons, we observed that Tac1-Cre mice injected in VMHvl with AAV-DIO-Ube3a-NLS:AAV-DIO-GFP exhibited heightened aggression compared to littermate Tac1-Cre mice injected only with AAV-DIO-GFP alone , no increase of aggression relative to littermate mice injected with AAV-DIO-GFP was observed  reduces synaptic transmission from VTA glutamatergic neurons to medium spiny neurons of nucleus accumbens while impairing sociability . We found that Cbln1 mRNA is repressed in VMH of Ube3a-NLS7-1x transgenic mice compared to wild type littermates  when compared to mock antibody  from wild type mouse cortex using an anti-UBE3A antibody, and quantified the levels of UBE3A-bound Cbln1 in VMH is sufficient to increase aggression behavior, we combined homozygous floxed Cbln1 (Cbln1flx/flx) mouse with Sf1-Cre to delete Cbln1 in VMH neurons  increased attack behavior  and postsynaptic GluD1  and GRID1 in 89 cases (UBE3A gene copy numbers are likely underestimated as autism cases diagnosed with extranumerary isodicentric chromosome (idic15) by cytogenetics are often excluded.We previously used a bioinformatics strategy to examine the physical interactions between the proteins encoded by UBE3A-regulated and other ASD genes and discovered that CBLN1 physically interacts with two other gene products frequently deleted in ASD, presynaptic NRXN1 (encoded by 89 cases ). UBE3A Nrxn1 or Grid1 heightens aggression in mice as previously reported  , ventralNST , ven-2x mice , B and ECbln1 mRNA and Nrxn1 mRNA are highly expressed in VMHvl neurons, while, in hypothalamus, Grid1 mRNA is enriched in arcuate nucleus, in addition to its expression in multiple other brain regions  increases aggression in fly  or GABAergic (Vgat-Cre:Grid1flx/flx) neurons failed to increase aggression (Grid1 in POMC neurons (Pomc-Cre:Grid1flx/flx), a major neuron subtype in arcuate nucleus, failed to increase aggression (Grid1 in AgRP neurons (Agrp-Cre:Grid1flx/flx) increased aggression  but not in arcuate POMC neurons (Pomc-Cre) increased aggression when injecting AAV-hSyn-TATAlox-Grid1-shRNA (vs. AAV-hSyn-TATAlox-scrambled-shRNA)  , L to N.Cbln1-expressing glutamatergic cerebellar granule cell neurons form excitatory synapses onto local inhibitory neurons in a Grid1-dependent manner (Grid1 in arcuate NPY/AgRP neurons impairs glutamate synaptic transmission onto NPY/AgRP neurons. Whole–cell patch clamp recordings of arcuate NPY/AgRP neurons were guided using the Npy-hrGFP transgene (A receptor antagonist bicuculline. Loss of Grid1 reduced mEPSC frequency in arcuate NPY/AgRP neurons in homozygous Grid1 knockout mice with Npy-hrGFP (Grid1 in AgRP neurons (Agrp-Cre:Grid1flx/flx) or knockdown of Grid1 in arcuate AgRP neurons (AAV-hSyn-TATAlox-Grid1-shRNA vs. scrambled) also reduced mEPSC frequency  was injected into the arcuate nucleus of Agrp-Cre male mice  to inhibit these neurons increased attack behavior in these mice   . Injectie, i.p.) , G and HAgrp-Cre crossed to Ai32 mice   . We foun2 (ChR2) , D to G.2 (ChR2) . This saale mice . These rGrid1 impaired glutamatergic synaptic transmission onto arcuate NPY/AgRP neurons. We examine whether a loss of other components of the NRXN1-CBLN1-GRID1 transsynaptic complex or an increase of nuclear UBE3A also impairs such synapses. mEPSC frequency of arcuate NPY neurons was reduced in mice with homozygous Nrxn1 deletion, Cbln1 deletion in VMH (Sf1-Cre:Cbln1flx/flx), or increased nuclear UBE3A (Ube3a-NLS7 mice) with no change in mEPSC amplitude  recorded in the presence of antagonists of GABAA (bicuculline) and NMDA  receptors were blocked by AMPA ionotropic glutamate receptor antagonist CNQX , or deleting CBLN in VMHvl (via co-injection of AAV-VGluT2-Cre and AAV-hSyn-DIO-ChR2 in VMHvl of Cbln1flx/flx mice) also markedly reduced light-evoked AMPA EPSC amplitude (via co-injection of AAV-hSyn-DIO-Cbln1 with AAV-Ube3a-NLS) recovered the evoked EPSC amplitude repressed by the increased nuclear UBE3A . Other more complex pathways should also be considered. A study reported arcuate NPY/AgRP neurons may repress aggression through their projections to medial amygdala neurons with secondary projections to the bed nucleus of stria terminalis , providing a biological proof-of-concept example for how genetic interactions could give rise to behavioral deficits in neuropsychiatric diseases by acting on multiple members of a molecular pathways such as a transsynaptic complex. Importantly, the elevated aggression caused by increases of UBE3A can be rescued by viral vector-based Cbln1 expression in VMHvl neurons. Also removing the elevated UBE3A expression in VMHvl during adulthood rescues the heightened aggression. Behavioral reversibility in adulthood highlights the plasticity of this circuits and implicates its potential as a future therapeutic target to treat irritability and aggressive behaviors in ASD. Our findings, that multiple genetic forms of ASD disrupt VMHvl-arcuate AgRP/NPY neuron glutamate synapses and that augmenting the aggression-limiting feedback inhibition from arcuate to VMHvl reduces aggression pave the way for precision medicine approaches specifically directed at this neuronal circuitry.We establish that an array of converging genetic defects including increases of UBE3A or a loss of NRXN1-CBLN1-GRID1 transsynaptic complex members that are impacted by ASD-associated copy number variations impair excitatory glutamatergic synapses from VMHvl to arcuate NPY/AgRP neurons while increasing aggressive behaviors. Heterozygous Ube3aNLS mice and LoxTB-Ube3a mice were generated using bacterial artificial chromosome (BAC) recombineering techniques as described previously . KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) was used in 6 μ l triplicate reactions containing 2.5 ng genomic DNA and 200 nM primers. Absolute Ube3a copy numbers were calculated using the 2^−(ΔΔCT) method, multiplying by a factor of two . The following lines were bred into an FVB/NJ genetic background for 6 or more generations. Grid1KO mice were generously provided by Dr. Jian Zuo . Cbln1flx/flx mice were a generous gift from Dr. Masayoshi Mishina (VGluT2-Cre (Slc17a6tm2(cre)Lowl), VGaT-Cre (Slc32a1tm2(cre)Lowl), Agrp-Cre (Agrptm1(cre)Lowl) mice and Npy-hrGFP (B6.FVB-Tg(Npy-hrGFP)1Lowl/J) mice were provided as a generous gift from Dr. Bradford Lowell (BIDMC). The Sf1-Cre (Tg(Nr5a1-cre)7Lowl), Pomc-Cre (Tg(Pomc1-cre)16Lowl), Tac1-Cre (Tac1tm1.1(cre)Hze), Esr1-Cre (Esr1tm1.1(cre)And), Pgr-Cre (Pgrtm1.1(cre)Shah), and ePet-Cre (Tg(Fev-cre)1Esd) lines of mice, and Nrxn1α KO, Ai9 (RCL-tdT), Ai32/ChR2-EYFP . Chemically-modified sgRNAs were obtained from Synthego; gRNA target 1: 5’-TCCAGCCCCAGGATATAAGG-3’, gRNA target 2: 5’-CGGTTCCTTCACAGACCACG-3.’ The single-stranded DNA homology-directed repair template containing the loxP-flanked Grid1 exon 4 and homology arms was synthesized by Integrated DNA Technologies. CRISPR/Cas9-edited founder mice were generated at the BIDMC Transgenic Core Facility by microinjection of FVB/NJ zygotes with a mix of 200 ng/μl SpCas9-NLS, 50 ng/μl sgRNA1, 50 ng/μl sgRNA2 and 15 ng/μl ssDNA. Candidate founder mice were crossed to FVB/NJ to obtain germline-transmitted F1 mice. F1 mice with correct editing at the target locus were identified by sequence validation and used to establish the floxed Grid1 mouse line.The Harvard Medical Area Standing Committee on Animals and the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center approved all mouse protocols. Mice were housed at the Center for Life Sciences barrier animal facility in sex-matched groups of 3–5 with ad libitum food and water access. Unless otherwise specified, littermate controls were used and cohorts were male only. Ube3a transgenic mice, eviously . Male an Mishina  . pAAV-hSyn-DIO-Ube3a-NLS was then sent to the University of North Carolina Vector Core for AAV particle production with serotype 9. To generate the pAAV-mMeCP2-DIO-EGFP-W3SL vector, first, the W3SL enhancer sequence was amplified from Addgene Plasmid #61463 with a 5’ XhoI site addition and subcloned into EcoRI and KpnI sites of Addgene Plasmid #61591 to replace bGHpA. Next, the mMeCP2 promoter sequence was amplified from Addgene Plasmid #60957 with a 3’ SalI site addition and subcloned into XbaI and SpeI sites. Then, the DIO-EGFP cassette was amplified from Addgene Plasmid #50457 and subcloned into SalI and EcoRI sites: pAAV-mMeCP2-DIO-EGFP-W3SL. To generate Cre-conditional shRNA AAV constructs, the U6-TATAlox-CMV-EGFP-TATAlox cassette was amplified from pSico (Addgene Plasmid #11578) and subcloned into XbaI and XhoI sites of pAAV-mMeCP2-DIO-EGFP-W3SL to replace mMeCP2-DIO-EGFP. An HpaI site within W3SL was destroyed using the Q5® Site-Directed Mutagenesis Kit (NEB): pAAV-U6-TATAlox-CMV-EGFP-TATAlox-W3SL. For Grid1 knockdown, three Grid1 and one scrambled control shRNA hairpin sequences using TCTGCTT loop sequence were subcloned individually into HpaI and XhoI sites of pAAV-U6-TATAlox-CMV-EGFP-TATAlox-W3SL. shRNA target sequences were selected from the Broad Institute RNAi Consortium database:AAV2-CMV-CreGFP, AAV2-CMV-GFP, AAV2-hSyn-DIO-GFP, AAV2-hSyn-DIO-hM3D(Gq)-mCherry, AAV2-hSyn-DIO-hM4D(Gi)-mCherry and AAV2-EF1α-DIO-ChR2(E123T/T159C)-EYFP were purchased from the University of North Carolina Viral Vector Core. AAV2-hSyn-DIO-Cbln1, AAV2/9-hSyn-DIO-Ube3a, AAV2/9-hSyn-mVglut2-mCherry and AAV2/9-hSyn-mVglut2-mCherry-2A-Cre were generated as previously described . AAV2/9-Grid1-A (GCTGAGAATATCCTTGGACAA)Grid1-B (GTGCTCATATTCGTGTTGAAT)Grid1-C (CGTTACAAAGGGTTCTCCATA) andScrambled (CCTAAGGTTAAGTCGCCCTCG).Grid1 shRNA target plasmids were combined in equimolar ratio and packaged together in a single preparation. The AAV-hSyn-DIO-GCaMP7f-WPRE (AAV9 virus) are from Addgene.AAV-mMeCP2-DIO-EGFP-W3SL and AAV-U6-TATAlox-CMV-EGFP-TATAlox- Scrambled - shRNA W3SL vectors were packaged using AAV9 serotype at Boston Children’s Hospital Viral Core. For the AAV2/9-U6-TATAlox-CMV-EGFP-TATAlox-Grid1-shRNA-W3SL virus, the three different Anesthesia was induced in a chamber with isofluorane/oxygen following which mice were placed into a stereotaxic frame fitted with a continuous isofluorane delivery system. A single midline vertical scalp incision revealed skull landmarks. Stereotactic measurements were used to make 0.7mm wide burr holes over the entry point for bilateral viral injections directed at the VMHvl , VMHdm  and arcuate nucleus , all relative to bregma. 1 μl (or 0.5 μl for AAV2-EF1α-DIO-ChR2(E123T/T159C)-EYFP) of virus was infused at 0.2 μl/min through a 33 gauge Hamilton needle connected to an automated infusion pump. Following each infusion, the needle remained in place for 5 minutes. The incision was sutured closed and a single injection of 10 mg/kg meloxicam dissolved in saline was administered intraperitoneally for perioperative analgesia. All behavioral measurements were conducted approximately 28 days following surgery, and accurate targeting to the VMHvl and arcuate was confirmed by immunohistochemistry. Electrophysiological recordings were conducted at least 30 days after the surgery. The accuracy of injection sites at VMHvl and arcuate were also confirmed under microscopy when patch clamp recordings were performed.2+ imaging experiments in Tac1-Cre male mouse, a craniotomy was performed as described in stereotactic surgery above, and saline was repeatedly applied to the exposed tissue to prevent drying. The dura was then carefully removed with a 30-gauge beveled syringe needle. AAV-hSyn-DIO-GCaMP7f or AAV-hSyn-DIO-GCaMP7f + AAV-hSyn-DIO-Ube3a-NLS (300 nL) virus were injected unilaterally into VMHvl of Tac1-Cre male mice as described in stereotactic surgery procedures at the coordinates as . After finishing the virus injection, a ProView™ Lens Probe  was implanted above the virus injection site with coordinates . The bottom of the lens was situated approximately 200 to 300 μm directly above the VMHvl. The portion of the lens extending ~2 to 3 mm above the skull surface was fixed with dental cement . A silicone elastomer  was applied to the top of the lens to protect the imaging surface from external damage. Four to six week later after virus injection and lens implantation, baseplate  was installed to support the miniaturized microscope. For this procedure, mice were anesthetized with isoflurane, and the silicone mold around the lens was carefully detached. Debris was removed from the exposed lens with compressed air canisters, and lens paper with ddH2O was used to clean the top of the lens. Next, the miniature microscope  with the baseplate attached was positioned above the implanted lens with an adjustable gripper . The microscope was lowered toward the top of the lens by micromanipulator until the field of view was in focus. To ensure that the objective lens was completely parallel and aligned with the implanted lens, the angle of the microscope’s position was adjusted by manually tilting the scope within the adjustable gripper. Once the field of view was in focus and the scope was parallel with the lens, the magnetic baseplate was cemented around the implanted lens. Finally, a baseplate cover  was secured into the baseplate with a set screw to protect the lens until imaging. One week later after installation of baseplate, a female mouse was added to the cage of the Tac1-Cre male mouse for one week for preparing resident intruder (R-I) test. Then the in vivo Ca2+ imaging was performed in freely moving animal when R-I tests were conducted. For each imaging session, the nVista3 microscope was connected to the baseplate on the cranium and fixed in place by the baseplate set screw. Animals acclimate in their home cage for 20 min prior to the starting imaging sessions. Calcium images were acquired using Inscopix nVista3 Data Acquisition Software v1.3.1 with collecting frame rate 10 Hz with an average exposure time of 65 ms. The analog gain on the image sensor was set to 6, while the LED power was set as 1 mW. The acquired data by the Inscopix microscope were analyzed using Inscopix Data Processing Software (IDPS version 1.6) to visualize calcium spike movies, extract calcium traces, threshold events and calculate calcium spike frequencies. The ΔF/F0 = [F(t) − F0]/F0, where the baseline image (F0) was calculated using mean frame of the input movie, and calcium imaging data were normalized as relative changes in fluorescence. The cell images and traces were identified by running PCA/ICA algorithm or manual ROI. The calcium spikes were calculated after deconvolution of cell traces. The amplitude value of the calcium spikes was export as excel file from the IDPS. The calcium spike score  was used to comparing calcium events in resident Tac1-Cre male mice expressing AAV-DIO-GCaMP7f + AAV-DIO-Ube3a-NLS to mice only injected AAV-DIO-GCaMP7f.To prepare animals for in vivo CaResident intruder paradigm : mature in vivo Ca2+ imaging with inscopix during resident-intruder test, aggression behavior was recorded with a Basler color camera, which was controlled by the Ethovision XT 15 software (Noldus). Inscopix Ca2+ imaging was synchronized with the behavior recordings through a USB I/O box which also controlled by Ethovision XT 15. Inscopix Data Processing Software was used to analyze the Ca2+ imaging data. Synchronous video recordings were used to manually score aggression behaviors.For Mice were anesthetized with an intraperitoneal injection of tribromoethanol and then perfused with ice-cold PBS followed by cold 4% paraformaldehyde. Brains were frozen in optimal cutting temperature (OCT) compound  after cryoprotected in 15% sucrose followed by 30% sucrose (each for 24h). 16μm sections were cut on a cryostat and slide-mounted. When an anti-mouse secondary antibody was used, sections were blocked with MOM reagent (Vector) and then sections were permeabilized with 0.1% Triton X100 and blocked with 10% normal goat serum with 1% BSA in PBS. The FLAG tag, GFP and mCherry were probed respectively with FITC-conjugated anti-FLAG , Green Fluorescent Protein (GFP) antibody , Red Fluorescent Protein Antibody . The brain sections were incubated with antibodies in blocking solution at 4°C temperature overnight. Sections were washed and when required, incubated with Alexa-conjugated secondary antibodies  for 3 hours at room temperature, and then mounted in Vectashield with DAPI (Vector). Fluorescent images were taken using a LSM510 confocal microscope (Zeiss), FV1000 Confocal microscope (Olympus) or the VS120-SL 5 Slide Scanner (Olympus). Images were processed using Fiji image J software.For c-fos quantifications , Fig S4,Ube3aNLS7-1x mice (n=6 each) were placed in a brain matrix (ASI Instruments) and razor blades were placed at 1mm intervals to obtain brain slices containing VMH. Punches were obtained with a 0.5mm punch core needle (Harris Unicore) and immediately frozen to −20C and stored at −80C. RNA was isolated using Trizol Reagent (ThermoFisher) and column-purified using the RNAeasy Protect Mini Kit (Qiagen). First strand cDNA synthesis was carried out with M-MLV Reverse Transcriptase (ThermoFisher) and Oligo (dT) 20. Expression of Cbln1 was quantified using RT-qPCR with Power SYBR Green Master Mix (ThermoFisher) and a Bio-Rad CFX 384 Real-Time System. Primer pairs were selected through Primer3 with specificity confirmed with Primer-BLAST. Primer sequences are listed in Supplementary Table2. Cbln1 expression between genotypes was determined using the ΔΔCT method with Syn1 as a reference gene. Melt-curve analysis and agarose gel electrophoresis were used to confirm a single PCR product of the appropriate amplicon size.Brains were rapidly removed and submerged in ice-cold phosphate buffered saline (PBS) for 20–30s. For the isolation of cortex (ChIP studies), two “slabs” of total cortex were dissected using curved forceps. For VMH punches, the PBS-cooled brains from wild type and Cbln1 promoter luciferase construct was purchased from GeneCopoeia, Inc. (Cat# MPRM22597-PG02), which encodes Gaussia luciferase driven by a 1331 bp genomic fragment upstream of the coding sequence of the murine Cbln1 gene (Accession: NC_000074.6). The Ube3a expression construct was generated by amplifying the coding sequence of human Ube3a isoform III from Plasmid #37605 (Addgene) with primers 5’-TCTTCCACTAGTGCCACCATGGCCACAGCTTGTAAAAGATC-3’ and 5’-TCTTCCGGATCCTTACAGCATGCCAAATCCTTTGG-3’ and subcloning into the SpeI and BamHI sites of the pLVX-IRES-mCherry vector (Clontech Cat#631237). HEK293T cells were transfected in 96-well using Lipofectamine 3000 Reagent (ThermoFisher) with 50 ng Cbln1 promoter-Gluc plasmid and 50 ng of either pLVX-IRES-mCherry or pLVX-Ube3a-IRES-mCherry per well (n = 6 each condition). 48 hours after transfection, Gaussia luciferase activity was developed using the Secrete-Pair Gaussia Luciferase Assay Kit  and measured with the BioTek Synergy 2 luminescence plate reader.The n = 3) and chromatin was prepared using the SimpleChIP plus Enzymatic Chromatin IP Kit . UBE3A-bound chromatin was immunoprecipitated using rabbit anti-UBE3A antibody (Bethyl Laboratories). Rabbit IgG was used as a mock control. The ratio of UBE3A-bound Cbln1 promoter DNA relative to input chromatin was quantified using qRT-PCR with primers 5’-CTCGCCGCTCCTAATAACAA-3’, 5’- CCACCCTCCAGCCAATC-3’ and FAM/ZEN-labeled probe 5’-ACAGGGCAACCATTGGCTCG-3’ with Taqman Gene Expression Master Mix (ThermoFisher).25 mg cortical tissues were dissected from wild type mice  and filled with (mM): 130 K gluconate, 10 HEPES, 5 NaCl, 1 MgCl2, 0.02 EGTA, 2 MgATP, 0.5 NaGTP and 10 mM sodium phosphocreatine at a pH of 7.3 and osmolality of 275 to 290 mOsm. Filled patch pipettes had resistances of 3–5 MΩ. Synaptic currents were recorded in voltage clamp mode by an Axon MultiClamp 700B amplifier (Molecular Devices). Data were filtered at 1 kHz, digitized at 10 kHz with DigiData 1440A interface (Molecular Devices) and acquired by Clampex 10.5. Series resistance was monitored with a 5 mV hyperpolarizing step (5 ms). Resting membrane potential and action potentials (APs) were recorded in current clamp mode with APs induced by injecting 200 ms square wave pulse of positive currents ranging from 40 to 400 pA. An optical fiber  was used to deliver optical stimulation. The fiber was placed 200 μm from the site of recording and coupled to a diode-pumped 473 nm laser (CrystaLaser Company) to control stimulus intensity light pulse duration (5ms) was controlled by a Master-8 stimulator and light pulse frequency (0.1 Hz) was determined and triggered by the Clampex 10.5. The evoked EPSCs in neurons-expressing channelrhodpsin-2 (ChR2) were induced by the laser light (30 mW)(A antagonist bicuculline (10 μM) and NMDA receptor antagonist D-APV (30 μM) were present to isolated the AMPA receptor currents. 30 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used in some studies to block and confirm AMPA receptor currents. Miniature EPSCs (mEPSCs) were recorded at a holding potential of −70 mV in the presence of GABAA antagonist bicuculline (10 μM) and tetrodotoxin . mEPSCs were detected and analyzed using Mini Analysis Program . Amplitude and area thresholds were set to 5 pA and 20 fC, respectively. The peak amplitude and inter-event interval of mEPSCs from 60 second episodes were used to generate cumulative probability plots, and the statistical significance was determined by Kolmogorov-Smirnov test.Mice were deeply anesthetized with isofluorane, following which mouse brains were rapidly removed and submerged in an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 126 choline chloride, 2.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 8 MgSO4, 0.2 CaCl2, 20 glucose, and 46 NaHCO3, equilibrated with 95% O2/5% CO2. Coronal brain slices containing the VMHvl or arcuate nucleus (300 μm thick) were made using a Leica VT1200S microtome (Leica Microsystems). The slices were transferred into a holding chamber containing ACSF comprised of (in mM): 124 NaCl, 3 KCl, 1 MgSO4, 1.25 NaH2PO4, 2 CaCl2, 25 Glucose, and 26 NaHCO3, equilibrated with 95% O2/5% CO2. The slices were maintained at 33°C temperature for 30 minutes and then kept at room temperature until recorded. To increase viability of neurons, 5 μM glutathione, 500 μM pyruvate, and 250 μM kynurenic acid were added to the choline chloride replacement ACSF and ACSF in the holding chamber. During recordings, slices were perfused (2 ml/min) with ACSF without glutathione, pyruvate and kynurenic acid. Whole cell recordings were performed from neurons located in the VMHvl or arcuate nucleus under visual control on an upright microscope with differential interference contrast and infrared illumination. Arcuate NPY neurons were identified by GFP fluorescence labeled by Cbln1 and other target genes was quantified using RT-qPCR with Power SYBR Green Master Mix (ThermoFisher) and a Bio-Rad CFX 384 Real-Time System. Primer sequences are listed in Supplementary Table 2. Fold change in Cbln1 expression between genotypes was determined using the ΔΔCT method with Syn1 as a reference gene.The brain slices were prepared as described in electrophysiological recordings. Then single tdTomato fluorescent labeled neuron (Ai9 mice) were aspirated from the VMHvl and collected into 5 μl of 2x CellsDirect Reaction Mix . cDNA was generated and preamplifed 20 cycles for target sequences and treated with ExoSAP-IT  as previously described . ExpressP < 0.05 was considered statistically significant with ns indicating non-significant, *P <0.05, **P < 0.01, ***P <0.001 and ****P <0.0001.Sample sizes are shown in figure legends in parentheses and were chosen based on a power analysis designed to exceed a statistical power (1-beta) of 80% and to meet or exceed sample sizes typically employed in similar mouse behavioral experiments. Behavioral studies performed in a blinded fashion and the code broken after the testing when data was analyzed. Where possible, animals were placed into groups through a randomization process. No animal are excluded from the data analysis. For resident–intruder aggression tests, an unpaired two-tailed Student’s t-test was used to determine statistical significance when comparing two groups. Comparisons across two groups before and after CNO treatment were analyzed by paired two-tailed Student’s t-test. Multiple groups were analyzed using 1-way ANOVA followed by Bonferroni’s Multiple Comparison Correction. 2-way Repeated-Measures ANOVA followed by Multiple Comparison was used to analyze c-fos quantification data  and F. OMice created for this manuscript have been deposited at JAXS.Supplement 1