LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.Hair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the DOI:http://dx.doi.org/10.7554/eLife.24318.001 Hair cells in the mammalian cochlea convert sound-induced vibrations into mechanical signals to enable our sense of hearing. Each hair cell contains at the apical surface a bundle of stereocilia that are organized in rows of decreasing height . Tip linWe know little about the transport and targeting mechanisms that regulate the precise configuration of proteins within the tip-link complex. Stereocilia contain bundles of parallel actin filaments with their barbed ends facing toward the tips of stereocilia. No vesicles have been observed within stereocilia. Membrane proteins and cytoplasmic components are thus thought to be transported into stereocilia at least in part by actin-based molecular motors of the myosin family . AccordiLRTOMT gene are associated with profound non-syndromic hearing loss at the DFNB63 locus  has catechol-O-methyltransferase activity and a point mutation in the murine gene that reduces this activity causes deafness  in the Tomt gene (referred to as addTomt)  of the Tomt gene  in-frame deletion near the N-terminus  . Our pretability . To moreomt gene  and injeterminus . The secof mTOMT . Analysiadd/addTomt, D12/D12Tomt and del/delTomt mice at 3–5 weeks of age. The ABR thresholds to broadband click stimuli of control wild-type animals were at 26 ± 3 dB , consistent with a recessive mode of inheritance of the auditory phenotype.Next, we carried out measurements of the auditory brain stem response (ABR) of homozygous offspring of 6 ± 3 dB . In cont6 ± 3 dB . Recordispectrum . Hearingf1 and f2) are presented. Outer hair cells (OHCs) amplify the distortions and they are propagated back through the middle ear and ear canal and can be measured in sound pressure waveforms , which are mechanical distortions generated in the inner ear when two primary tones 0-P1 and determined protein localization at P8 by immunostaining with antibodies to HA or GFP . mTOMT-HTomt-deficient mice. Whole mount staining of the dissected sensory epithelium of add/addTomt, D12/D12Tomt and del/delTomt mice at P5 revealed that the sensory epithelia in mutant mice were patterned into three rows of OHCs and one row of IHCs with no obvious structural abnormalities   . We detey (HPLC) . Serotony (HPLC) .Tomt-GFP cDNA and introduced a point mutation in a tyrosine residue that is conserved within the methyltransferase domain of mTOMT and mCOMT and critical for enzymatic activity (Tomt-Y108A-GFP)  were at 541 ± 41 pA. Partial rescue of transduction could be a consequence of differences in expression levels of transgenes expressed from heterologous promoters compared to endogenous protein levels in wild-type hair cells or modified function due to epitope tags, but further studies are necessary to address this point (see discussion). Nevertheless, the fact that both mTOMT-GFP and mTOMT-Y108A-GFP rescued transduction to a similar extent, while the methyltransferase mCOMT-GFP or the putative TOMT methyltransferase domain (mTOMT-MT-GFP) could not, suggests that methyltransferase activity is not essential for TOMT function in hair cells.Next, we wanted to test more directly the extent to which methyltransferase activity is important for mTOMT function in hair cells. We took advantage of our 08A-GFP)  (Zhang a08A-GFP) . We also08A-GFP) . The GFP08A-GFP) . We then culture . OHCs fr culture . Mechano culture  but stilTo further define the mechanism by which TOMT affects transduction, we tested the hypothesis that mTOMT might interact with putative components of the mechanotransduction channel of hair cells  or with tip-link proteins . We therefore co-expressed TOMT carrying either a C-terminal HA-tag (TOMT-HA) or a C-terminal FLAG tag (TOMT-FLAG) together with TMC1, TMC2, TMIE and TMHS/LHFPL5 in HEK293 cells. TMC1 and TMC2 contained N-terminal Myc tags, TMIE carried a C-terminal HA tag, and LHFPL5/TMHS was tagged at the N-terminus with HA. We also co-expressed TOMT-FLAG with the tip-link cadherins CDH23 and PCDH15. Significantly, epitope-tagged versions of TOMT (TOMT-FLAG or TOMT-HA) could be co-immunoprecipitated with Myc-TMC1, Myc-TMC2, TMIE-HA, HA-TMHS/LHFPL5 and PCDH15, but not with CDH23  that might be involved in the transport of PCDH15, TMHS/LHFPL5, TMIE, and TMC1/2 still need to be defined, although MYO7A can bind to PCDH15 and might participate in PCDH15 transport . HowevermTOMT differs significantly from other proteins implicated in shuttling components of the tip-link complex into stereocilia since mTOMT, unlike TMHS/LHFPL5 and MYO7A, cannot be detected within stereocilia. Based on the localization of mTOMT in the cytoplasm, it seems more likely that mTOMT acts in a transient transport compartment prior to the insertion of proteins into the membrane of stereocilia. Expression and localization studies in heterologous cells are consistent with this model.We and others have shown that TMC1/2 proteins are retained in an intracellular compartment of heterologous cells that were transfected to express TMC1/2  were generated using ENU mutagenesis as described . The sgRNA-target genomic DNA sequence (TACGCACCAGCAGTCGGAAA) was chosen based on proximity to the start codon and a low number of potential off-target sites. Two complementary oligonucleotides encoding the sgRNA-target sequence and its reverse complement were synthesized, annealed and ligated into the BbsI restriction enzyme site of pX330 (Addgene #42230). Plasmid DNA was purified from bacterial clones containing sequence-verified px330-Tomt sgRNA target sequence. DNA template for in vitro transcription containing T7 promoter, sgRNA target sequence, and tracrRNA was produced by PCR and purified using Phenol-Chloroform precipitation. Chimeric guide RNA was synthesized by in vitro transcription using Ambion MEGAshortscript T7 kit, and RNA was purified using Ambion MEGAclear kit. One-cell embryos were microinjected into pronuclei with 5 ng/µL Cas9 mRNA (TriLink) and 5 ng/µL Tomt chimeric guide RNA diluted in nuclease-free TE buffer. After injections, embryos were cultured for 1 day and then implanted into pseudopregnant mice. Pseudopregnant mice were allowed to give birth, offspring resulting from embryo injections were tail-clipped at P21, and genomic DNA was collected. Genomic DNA was screened using PCR and sequencing to determine presence of insertions or deletions. Founder mice containing 12 bp deletions (D12Tomt) and 77 bp deletions (delTomt) in exon 2 of Tomt were bred with C57BL/6 (RRID:IMSR_JAX:000664) mice, and offspring were screened to verify germ-line transmission of mutations. All animal experiments were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine (#M016M271).escribed . CRISPR/Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements were performed as described in 2, 5 mM MgCl2, and 0.9% NaCl. After perfusion, temporal bones were removed from the skull and postfixed in 4% PFA in SEM Buffer for 30 min at room temperature with gentle rocking. The temporal bone was placed in SEM buffer and the bony cochlear shell, tectorial membrane and Reissner’s membrane were removed, leaving the Organ of Corti on the modiolus. The samples were then placed in SEM buffer containing 4% PFA and 2.5% glutaraldehyde and incubated overnight at RT. The samples were washed in SEM buffer with no fixative, and then dehydrated in a series of EtOH treatments. Samples were treated to critical point drying using an Autosamdri-815A (Tousimis) or Baltec 030, mounted on stubs with carbon-coated tape, and coated with Iridium or Chromium using a sputter coater . Imaging was performed using a Hitachi S-4800-ll Field Emission Scanning Electron Microscope or a Leo/Zeiss Field Emission Scanning Electron Microscope. Inner and Outer Hair cells were selected from mid-apical regions of the cochlear spiral. Tip links were quantified as the numbers of tip links per stereocilial column from Hair Cells.Scannin electron microscopy (SEM) methods were performed as described . P7-P8 m2 and 50 µg/mL Ampicillin, sectioned into three equal pieces representing apex, middle and base, and plated on the bottom of 35 mm tissue-culture dishes containing culture media composed of 1x DMEM/F12, 10% Fetal Bovine Serum (FBS) and 50 µg/mL Ampicillin. Cochlear explants were incubated at 37°C with 5% CO2. After 4 hr, the media was replaced with fresh culture media with or without 1 mM Gentamicin. The dishes containing the explants were returned to the incubator for 24 hr, fixed by incubating for 30 min in 1x HBSS containing 1.5 mM CaCl2 and 4% PFA. After fixation, explants were immunostained for MYO7A and phalloidin and imaged to analyze hair cell survival. For catecholamine treatments with Gentamicin, explants were pretreated for 24 hr with catecholamines or inhibitors prior to gentamicin treatment. Media was then replaced with fresh media with or without 1 mM Gentamicin and catecholamines or inhibitors. Explants were treated for an additional 24 hr, followed by fixation, immunostaining and imaging as above.Cochleas from postnatal day 5 (P5) wild-type and mutant animals were dissected in 1x HBSS containing 0.1 mM CaCl2PO4, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 glucose, and 10 K-HEPES, pH 7.4. The patch pipette was filled with intracellular solution . The membrane potential was measured under current-clamp model by patch-clamping P6 Outer hair cells at 0 pA. The whole-cell outward currents were elicited from P6 Outer hair cells by a series of command potentials from −90 mV to 60 mV for 150 ms in 10 mV steps  from a holding potential of −70 mV. For nonlinear capacitance, Organs of Corti were dissected from the cochlea of postnatal mice (P16). The apical region was mounted onto a recording dish containing in (mM):100 NaCl, 5.8 KCl, 1.5 MgCl2, 20 TEA, 20 CsCl, 2 CoCl2, 0.001 tetrodotoxin, pH 7.4. The patch pipette was filled with (in mM): 140 CsCl, 5 TEA, 2 MgCl2, 10 EGTA, pH 7.2. Voltage-dependent capacitance was determined by Lock-in function of Patchmaster (HEKA). After compensation of membrane capacitance, Command sinusoid  was summed to voltage ramps ranging from −150 to 100 mV during capacitance measurement, 16 cycles were integrated to generate each data point. The data were fitted with a Boltzmann function: NLC=Clin+Qmax/{[α*exp((V-V1/2)/α)]/[1+exp((V-V1/2)/α)]^2}, where Clin is the residual linear capacitance, V is the membrane potential, Vh is voltage at half-maximal non-linear charge transfer, Qmax is the maximal voltage sensor charge moved through the membrane electrical field, and α is the slope factor of the voltage dependence. Reverse polarity currents were elicited from OHCs using a fluid jet from a pipette (tip diameter of 10–15 µm). Sinusoidal force stimulus was applied to a 27-mm-diameter piezoelectric disc to produce fluid jet. The position of the pipette delivering the fluid jet was positioned at the modiolar side of the hair bundles and adjusted to elicit maximal mechanical stimuli-induced currents. For BAPTA treatments, Organs of Corti were perfused with 5 mM BAPTA for 10 min prior to recording. The sinusoids (40 Hz) was generated with Patchmaster 2.35 software (HEKA) and filtered at 1.0 kHz with 900CT eight-pole Bessel filter .Mechanotransduction currents were recorded following our published procedures . In whol2, and openings were made in the bony cochlear shell at the apex and through the oval and round windows. The temporal bones were incubated in 1x HBSS containing 4% PFA and 1.5 mM CaCl2 for 30 min at RT. The temporal bones were washed in 1x PBS, the bony cochlear shell was removed, and the Organ of Corti was separated from the modiolus and collected for immunostaining. Reissner’s membrane and the Tectorial membrane were removed, and the Organ of Corti was permeabilized in PBS containing 5% BSA and 0.5% Triton for 30 min at RT. After permeabilization, the tissue was blocked in PBS containing 5% BSA for 1 hr. The tissue was incubated in PBS containing 5% BSA and primary antibodies (see below) overnight at 4°C. The tissue was then washed three times in PBS, and incubated for 1–2 hr at RT in PBS containing 5% BSA, secondary antibodies , and phalloidin conjugated to various Alexa Fluor dyes . After secondary antibody incubation, the tissue was washed three times and mounted on slides using ProLong Diamond (Invitrogen). Whole mounts were imaged using 60x and 100x lenses on a widefield fluorescence deconvolution microscope . For immunocytochemistry, Heterologous cells (HEK293 (RRID:CVCL_0045) or COS-7 cells (RRID:CVCL_0224)) were plated to 70–90% confluence in six-well plates. HEK293 cells  and COS-7 cells  were obtained from ATCC. Cells were authenticated using STR profiling and verified to be free of Mycoplasma contamination. Cells were transfected with plasmids using Lipofectamine 2000, incubated for 24 hr, re-plated on glass coverslips in 24-well plates, and incubated for an additional 24 hr. Cells were fixed in 4% paraformaldehyde in 1X PBS for 10 min, permeabilized in 0.25% Triton for 10 min, and then processed for immunostaining and imaged as above.Whole mount cochleas were dissected, fixed, and immunostained as described . BrieflyAB_10015251), Rabbit anti-Cdh23 , Rabbit anti-Myc , mouse anti-Espin , mouse anti-HA , mouse anti-Myc , Chicken anti-GFP . The Rabbit anti-Whirlin antibody (1:500) was generated by Covance  against a peptide specific for the mouse Whirlin protein. The antibody was affinity-purified against the peptide used as an immunogen.Primary antibodies were as follows: Rabbit anti-Myosin VIIa , Rabbit , Rabbit , RabbitAB_2534084), Goat anti-Rabbit F(ab’)2 488 , Goat anti-Rabbit F(ab’)2 Alexa Fluor 555 , Goat anti-Rabbit Alexa Fluor 555 Superclonal , Goat anti-Mouse F(ab’)2 Alexa Fluor 647 .Secondary antibodies were as follows: Goat anti-Mouse F(ab’)2 Alexa Fluor 488  to the sensory epithelium of explants. Four square wave pulses were applied with an electroporator  at 1 s intervals with a magnitude of 60 V and duration of 15 ms. After injectoporation, the media in the dishes was changed to fresh culture media containing 1x DMEM/F12, 10% Fetal Bovine Serum (FBS) and 50 µg/mL Ampicillin. Dishes were returned to the 37°C incubator and incubated for 24 hr, followed by fixation, immunostaining, and imaging as above.Exogenous DNA plasmids were expressed in cochlear hair cells via injectoporation as published . CochleaTomt-HA or Tomt-GFP was cloned into pAAV-CAG-GFP (Addgene #37825) between the XbaI and EcoRI sites. 250 µg pHelper (generous gift of Anton Maximov), 125 µg pAAV-RC1 (Cell Biolabs), and 125 µg pAAV-CAG-Tomt-HA or pAAV-CAG-Tomt-GFP in OptiMEM were used with 700 µL of 1 mg/mL PEI  in OptiMEM to transfect ten 150-mm petri dishes containing HEK293 cells at 70% confluency. After 3 days, cells were lysed, collected, and AAV viral particles were purified using AAVpro kit (Clontech). Purified AAV vectors were stored at −80°C. For in vivo injections, anesthetized P0-P2 mouse pups were injected with a Hamilton syringe with a beveled tip containing 1–2 µL of AAV. The needle was inserted through the skin into the temporal bone, containing the inner ear. After injection, the pups were allowed to recover from anesthesia, returned to their cage and allowed to survive until P7-P8, after which they were sacrificed and cochleas were fixed, immunostained, and imaged as above.AAV serotype 2/1 viral vectors were produced by PEI transfection in HEK293 cells. add/addTomt mice were dissected and snap frozen on dry ice in eppendorf tubes. Each tube contained two temporal bones pooled together and represented one animal. For tissue extraction, the frozen temporal bones were homogenized using a tissue dismembrator, in 0.1M TCA, containing 0.1 M sodium acetate, 0.1 mM EDTA, 5 ng/ml isoproterenol  and 10.5% methanol (pH 3.8). Ten microliters of homogenate was set aside for assay of protein concentration using BCA protein assay kit (Thermo Scientific). The samples were then centrifuged and the supernatant was collected for analysis via HPLC.Temporal bones from P5 wild-type and Catecholamines and other biogenic amine levels were determined by an HPLC assay utilizing an Antec Decade II (oxidation: 0.65) electrochemical detector operated at 33°C. Twenty microliter samples of the supernatant were injected using a Water 2707 autosampler onto a Phenomenex Kintex  C18 HPLC column (100 × 4.60 mm). Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1M TCA, 0.1 M sodium acetate, 0.1 mM EDTA and 10.5% methanol (pH 3.8). Solvent was delivered at 0.6 ml/min using a Waters 515 HPLC pump. Using this HPLC solvent the biogenic amines elute in the following order: Noradrenaline, Adrenaline, DOPAC, Dopamine, 5-HIAA, HVA, 5-HT, and 3-MT. HPLC control and data acquisition were managed by Empower software. Absolute catecholamine levels were normalized to protein concentration values and reported as ng catecholamine/mg of protein.The tissue extraction, protein assay and HPLC measurements were performed by the Vanderbilt Neurochemistry Core Facility.2, 10% glycerol, 1% Triton X-100, 1 mM EDTA and a Roche Complete Protease Inhibitor Tablet. After lysis, lysates were centrifuged at 20,000 rcf for 15 min at 4°C. Supernatant was collected and precleared with EZ View Red Protein A Agarose (Sigma). At this point, 10% of the lysate was set aside for use as an input control. The rest of the lysate was immunoprecipitated for 1 hr at 4°C using EZ View Red HA Affinity Gel or EZ View Red Flag M2 Affinity Gel. After immunoprecipitation, the affinity gel was washed three times with CoIP buffer and boiled in Lamelli Buffer containing beta-mercaptoethanol to elute protein complexes. Eluted immunoprecipitated protein was run in parallel with input lysate on 4–20% SDS-PAGE gels (Biorad) and transferred to PVDF membranes using iBlot2 (Thermo Scientific) or using traditional wet transfer. Membranes were blocked for 1 hr with 1% Bovine Serum Albumin (BSA) in 1X TBST . Membranes were incubated with primary antibodies (see below) in 1% BSA in 1X TBST at 4°C overnight. After primary antibody incubation, membranes were washed three times with 1X TBST, followed by incubation for 1 hr in solution containing secondary antibodies (see below) in 1% BSA in 1X TBST. Membranes were washed three times in 1X TBST and then imaged with Clarity or Clarity Max Substrate (Biorad) on a G-Box ECL imager (Syngene).Co-immunoprecipitations were performed as described . BrieflyAB_331789), mouse anti-Flag M2 , rabbit anti-MYC , rabbit anti-HA , rabbit anti-PCDH15-CD2 , and ra. SecondExpression vectors for MYC-TMC1, MYC-TMC2, CDH23, PCDH15-CD2, HA-LHFPL5/TMHS and TMIE-HA were described previously . pCMV-LRTomt coding sequence (based on NCBI Consensus Coding Sequence # CCDS40044.1) using PCR primers containing HA tag, EcoRI, and NotI. HA-Tomt was ligated into pCAGEN (Addgene #11160) digested with EcoRI and NotI. PCR primers were: (i) 5’-AAAAAAAAAAGAATTCGCCACCATGTACCCATACGATGTTCCAGATTACGCTTCCCCTGCCATTGCACT-3’ containing EcoRI sequence, Kozak sequence and HA tag; (ii) 5’-AAAAAAAAAAGCGGCCGCTCAGCCGGGTCCAGTATAGG-3’ containing NotI sequence.Mouse cochlear cDNA was used as a PCR template to amplify Tomt, without the final stop codon (TomtΔstop). pPBCAG-GFP (generous gift of Joseph LoTurco) was used as a PCR template to amplify GFP (GFPΔstart), without the start codon. Assembly PCR was used with TomtΔstop and GFPΔstart as templates to create an in-frame fusion, and the product of this reaction was ligated into pCAGEN digested with EcoRI and NotI. PCR Primers were: (i) To amplify TomtΔstop: 5’-AAAAAAAAAAGA ATTCGCCACCATGTCCCCTGCCATTGCAC-3’ containing EcoRI sequence, and 5’-AACAGCTCCTCGCCCTTGCTCACGCCGGGTCCAGTATAGGTGA’−3; (ii) To amplify GFPΔstart: 5’-GTGAGCAAGGGCGAGGAG-3, and 5’-AAAAAAAAAAGCGGCCGCTTA CTTGTACAGCTCGTCCATGCC-3’, containing NotI sequence; (iii) Assembly primers to amplify Tomt-GFP 5’-AAAAAAAAAAGAATTCGCCACCATGTCCCCTGCCATTGCAC-3’ containing EcoRI sequence 5’-AAAAAAAAAAGCGGCCGCTTACTTGTACAGCTCGTCC ATGCC-3’, containing NotI sequence.Mouse cochlear cDNA was used as a PCR template to amplify Tomt-GFP, using primers containing XbaI and EcoRI. Tomt-GFP was ligated into pAAV-CAG-GFP (Addgene #37825) digested with XbaI and EcoRI and gel purified to remove GFP. PCR Primers were: 5’-AAAAAAAAAATCTAGAGCCACCATGTCCCCTGCCATTG CAC-3’ containing an XbaI sequence. 5’-AAAAAAAAAAGAATTCTTACTTGTACAGCTC GTCCATGCC-3’ containing an EcoRI sequence.pCAGEN-TOMT-GFP was used as a PCR template to amplify Tomt, using PCR primers containing HA tag, XbaI and EcoRI. Tomt-HA was ligated into pAAV-CAG-GFP (Addgene #37825) digested with XbaI and EcoRI and gel purified to remove GFP. PCR primers were: (i) 5’-AAAAAAAAAATCTAGAGCCACCATGTCCCCTG CCATTGCAC-3’ containing XbaI sequence and Kozak sequence; and (ii) 5’-AAAAAAAAAAGAATTCTCAAGCGTAATCTGGAACATCGTATGGGTAGCCGGGTCCAGTATAGGT-3’ containing EcoRI sequence and HA-tag.Mouse cochlear cDNA was used as a PCR template to amplify Comt, without the final stop codon (ComtΔstop). pPBCAG-GFP (generous gift of Joseph LoTurco) was used as a PCR template to amplify GFP (GFPΔstart), without the start codon. Assembly PCR was used with ComtΔstop and GFPΔstart as templates to create an in-frame fusion, and the product of this reaction was ligated into pAAV-CAG-GFP (Addgene #37825) digested with XbaI and EcoRI and gel purified to remove GFP.Mouse cochlear cDNA was used as a PCR template to amplify ComtΔstop: 5’-AAAAAAAAAATCTAGAGCCACCAT GCTGTTGGCTGCTGTCTCAT-3’ containing XbaI sequence, and 5’-CTCCTCGCCCTT GCTCACGGACTTCACGGGGCTGCT-’3; (ii) To amplify GFPΔstart: 5’-AGCAGCCCC GTGAAGTCCGTGAGCAAGGGCGAGGAG-3’, and 5’-AAAAAAAAAAGAATTCTTACTT GTACAGCTCGTCCATGCC-3’, containing EcoRI sequence; (iii) Assembly primers to amplify Comt-GFP: 5’-AAAAAAAAAATCTAGAGCCACCATGCTGTTGGCTGCTGTCT CAT-3’ containing XbaI sequence, and 5’-AAAAAAAAAAGAATTCTTACTTGTACAGCT CGTCCATGCC-3’, containing EcoRI sequence.PCR Primers were: (i) To amplify Tomt-GFP DNA sequence were t322g and a323c. PCR primers to make t322g and a323c mutations:Site-directed mutagenesis to modify pCAGEN-Tomt-GFP to pCAGEN-Tomt-Y108A-GFP was performed according to instructions using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). Mutations in (i)5’-GTGTAGAGTATCCACAGGCTGTGCCCAGCTCCAGCA-3’ and (ii) 5’-TGCTGGAGCTG GGCACAGCCTGTGGATACTCTACAC-3’.GFPΔstart) between EcoRI and NotI sites. pCAGEN-TOMT-GFP was used as a template to amplify the putative Tomt methyltransferase (MT) domain using PCR primers containing EcoRI and KpnI. Tomt MT domain was ligated into modified pCAGEN-MCS-GFPΔstart was digested with EcoRI and KpnI. PCR primers were: 5’- AAAAAAAAGAATTCGCCACCATGGTGCTGGAGCTGGGCA-3’, containing an EcoRI sequence, and 5’-AAAAAAAAGGTACCCAATACAGTGGCGCCATGT- 3’, containing a KpnI sequence.pCAGEN was modified to contain a multiple cloning site containing NheI, SacI, XmaI, and KpnI sites followed by GFP (from pPBCAG-GFP) with a deleted start codon (0)/F0, where F0 is the averaged fluorescence baseline at the beginning. Transduction current-displacement curves (I(X)) were fitted with a three-state Boltzmann model  and Igor pro 6 . Calcium signal (△F/F) was calculated with the equation: ; Gregory I Frolenkov (Reviewer #2).Thank you for submitting your article "The Catecholamine Methyltransferase mTOMT is Critical for Mechanotransduction by Cochlear Hair Cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Tomt mutants.Cunningham et al. perform comprehensive analysis of roles of the catecholamine methytransferase family gene mTOMT in mechanosensory hair cells. Using a combination of in vivo and in vitro analyses, histology and physiology, they demonstrate a requirement for mTOMT in hair cell mechanotransduction. The authors show that mTOMT mouse mutants have profound hearing loss through ABR recordings and that their outer hair cells fail to function shown through a loss of DPOAEs. These defects appear to be due to a loss of mechanotransduction in Tomt mutant mice. It has previously been hypothesized that TOMT's role in hair cells was due to impaired catecholamine degradation. The authors provide data that this is likely not the case due to the fact that application of exogenous catecholamines and pharmacological inhibition of catecholamine degradation have no effect on hair cell activity. Also, a TOMT construct with a point mutation eliminating TOMTs methyltransferase activity is able to rescue TOMT mutants to a similar extent as wildtype TOMT. They additionally show that TOMT is able to interact with a number of proteins important for mechanotransduction activity including PCDH15, LHPL5, TMIE and TMC2 and that TMC1 and TMC2 fail to properly localize to the stereocilia in Even though the mechanism of this regulation is unclear, this is the first identified molecule that may regulate trafficking of the MET channel components in the hair cells. Therefore, the study is significant despite all drawbacks described below.Essential revisions:Tomt mutants it is not sufficient for the emergence of reverse polarity currents that are normally seen in Tmc mutants. This major take-home message is based on three panels . The "low hanging fruits" are the resting membrane potential, voltage-gated ion currents, and electromotility (non-linear capacitance) of the OHCs. Without knowing the physiological condition of OHCs, it is hard to determine whether the lack of proper trafficking of TMC1 and TMC2 is a primary or secondary effect of TOMT deficiency.The MET function was investigated in OHCs  while ti Essential revisions:While this mislocalization is presumably responsible for mechanotransduction defects seen in Tomt mutants it is not sufficient for the emergence of reverse polarity currents that are normally seen in Tmc mutants. This major take-home message is based on three panels , out of del/+ mice but not in Tomtdel/del mice. We would like to emphasize that consistent with findings by other laboratories, it is much more difficult to express TMC1 in hair cells compared to TMC2 . Thus, the overall levels of TMC1 in the hair bundles are lower compared to TMC2, but TMC1 can clearly be localized to the bundle. We have revised the text to indicate that expression levels for TMC1 are low compared to TMC2.We provide now in Where is mTOMT localized within the cell? Does it promote overall stability or production of TMC1/2? That is, are overall levels of TMC1/2 higher in cells with mTOMT activity or is there differential localization?We have carried out additional experiments and have revised the text and figures accordingly: “We have now carried out a more detailed analysis of mTOMT localization in hair cells as well as in heterologous cells. […] On rare occasions (<1/10 cells) we observed expression of mHA-TOMT in stereocilia (data not shown). However, the significance of this finding is currently unclear.”mTomt locus thus providing a greater chance that the transgene is expressed at normal levels. Consistent with the data shown in the manuscript, we observed mTOMT expression throughout the cell body of hair cells. We could show the data in the current manuscript but would prefer not to do so because we generated the mice as the foundation for a new study to further define the mechanisms of TOMT function in hair cells. Nevertheless, we provide here for inspection a figure to the reviewers that supports the conclusions put forward in the current manuscript.We also would like mention that we have used CRISPR technology to generate a mouse line where the endogenous mTOMT protein was tagged with HA. The advantage of the mouse line is over injectoporation is that gene expression is driven by the genomic mTomt mutant mice since we used overexpression of epitope tagged proteins to support our conclusion . The "low hanging fruits" are the resting membrane potential, voltage-gated ion currents, and electromotility (non-linear capacitance) of the OHCs. Without knowing the physiological condition of OHCs, it is hard to determine whether the lack of proper trafficking of TMC1 and TMC2 is a primary or secondary effect of TOMT deficiency.add/addTomt mutants; (ii) in We agree with the assessment of the reviewer and have included additional experiments to address these points: (i) In Naturally, we cannot exclude that other proteins not studied here might be affected by mTOMT-deficiency, but there is an incredible specificity for effects on TMC1/2 without affecting other components of the mechanotransduction machinery of hair cells  and key proteins of hair bundles .The MET function was investigated in OHCs  while tiTomt mutants (Tomt mutants. (In the revised version we confirm quantitatively that tip-link numbers are not affected in OHCs of  mutants . We alsomutants. .Revised The electrophysiological data are now provided in


Bacillus subtilis AS2 that was isolated from heavy crude oil-contaminated soil samples from sludge pits of an Omani heavy-oil field. B. subtilis AS2 was able to biodegrade heavy crude oil and produce biosurfactant. In order to provide a better understanding of the biodegradation mechanism and biosynthesis of metabolites, the B. subtilis AS2 genome was sequenced and compared to those of other B. subtilis strains.Here, we report the draft genome sequence of  Bacillus bacteria are used in many processes due to their physiologic characteristics and ability to produce various metabolites (Bacillus were isolated from heavy crude oil (HCO)-contaminated soil samples   represents the use of bacteria to extract remaining oil volumes from depleting petroleum reservoirs by means of utilizing microbial products. The general bacterial mechanisms for accessing petroleum hydrophobic substrates are interfacial accession by direct contact of the cell, with the hydrocarbon and biosurfactant-mediated accession by cell contact with emulsified hydrocarbons . Bacilluabolites . Fifteen samples . BacilluC 1A700) , B. subtDe novo assembly of high-quality paired-end (PE) reads was accomplished using Velvet v1.2.10  (The DNA was extracted by using the UltraClean soil DNA isolation kit (Mo Bio Laboratories). A paired-end sequencing library was prepared using the NEBNext Ultra DNA library preparation kit. Ligated products were purified using Ampure XP beads. The product was PCR amplified as described in the kit protocol. The amplified library was analyzed in a Bioanalyzer 2100 (Agilent Technologies) using a high-sensitivity (HS) DNA chip per the manufacturer’s instructions. The library was loaded onto an Illumina platform (2 × 150-bp read length) for cluster generation and sequencing.  v1.2.10 , which p v1.2.10 . CDS werpcaC (benzoate) and atoD (aminobenzoate), which were not found in strain 168. The pcaC gene (benzoate) was not found in TU-B-10. There were no differences between AS2 and BSP1 or between AS2 and 6051-HGW. RO-NN-1 and TU-B-10 had nitrotoluene degradation gene K10678 nfsA, whereas AS2 and BSP1 did not.With respect to crude oil and petroleum hydrocarbon biodegradation, AS2 exhibited two degradation genes, With respect to biosurfactant biosynthesis, AS2 had surfactin family lipopeptide synthetase A, B, and C and iturin family lipopeptide synthetase A, B, and C. BSP1 and TU-B-10 had surfactin family lipopeptide synthetase B and C and iturin family lipopeptide synthetase A, B, and C. RO-NN-1 had surfactin family lipopeptide synthetase B and C and fengycin family lipopeptide synthetase A, B, C, D, and E.MUXL00000000. The version described in this paper is version MUXL01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux). Riemerella anatipestifer is a Gram-negative bacterium that belongs to the Flavobacteriaceae family  was isolated from the brains of diseased ducks from Chengdu, Sichuan, China, identified by our laboratory  broth or LB agar  at 37°C. The antimicrobial agents that were added to the media used for strain construction and selection were purchased from Sigma, and they were added to reach the following final concentrations: ampicillin (Amp), 100 μg/ml; cefoxitin (Cfx), 1 μg/ml; chloramphenicol (Cm), 25 μg/ml; kanamycin (Kan), 50 μg/ml; and spectinomycin (Spc), 80 μg/ml.All the plasmids and strains used in this study are listed in boratory  and cultThe DNA fragments were obtained by polymerase chain reaction (PCR). PCR was carried out in a Hybaid PCR thermocycler (Bio-Rad) using the DNA Polymerase (Takara) according to manufacturer’s instructions. All primers used in this study were synthesized by Invitrogen.R. anatipestifer CH-1 strains in the logarithmic phase using RNAiso Plus (Takara) according to the manufacturer’s instructions. The RNA was then reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Takara) with gDNA Eraser (Takara). PCR with SYBR® Premix Ex TaqTM II (Takara) was performed using the gene-specific primers P1-16S rRNA-F/P2-16S rRNA-R P3-raeBRT-F/P4-raeBRT-R and P5-B739_0874-F/P6-B739_0874-R. In the qRT-PCR analysis, 16S rRNA gene was used as an internal control. The relative gene expression was calculated using the 2-ΔΔCt method with Bio-Rad CFX Manager software -PCR was used. Total RNA was extracted from software . ExperimB739_0873USD and pRE112::B739_0872-B739_0873USD were used to delete the B739_0873 and both the B739_0872 and B739_0873 genes, respectively, by allelic exchange, according to previously described methods  cassettes from the plasmid pYES1. Each set of three PCR fragments was integrated using overlap PCR , which was ligated into pLMF03 at NcoI and XhoI sites to generate pLMF03::B739_0873. For the complementation analysis, the pLMF03::B739_0873 plasmid was introduced into the two R. anatipestifer CH-1 mutant strains by conjugation, as described previously  was defined as the ratio between the number of mutant and wild-type CFUs. All experiments were performed in triplicate.To evaluate the growth rates under non-competitive conditions, we monitored the growth curves for the wild-type strain, the RA-CH-1 Δd method . In vitr3 mutant . BrieflyR. anatipestifer CH-1 strains according to the Clinical and Laboratory Standards Institute guidelines (R. anatipestifer. The E. coli American Type Culture Collection (ATCC) 25922 strain was used for quality control. Antimicrobial agents were serially diluted twofold in TSB broth with concentrations ranging between 1 and 512 μg/ml. The turbidity of the inoculum was adjusted to 107 CFU/ml and 100 μl was added into every well. The 96-well microtiter plates were incubated at 37°C for 24 h. The lowest concentration that inhibited bacterial growth was considered to be the MIC. All tests were performed in triplicate.A twofold serial dilution assay was used to measure the minimal inhibitory concentration (MIC) of antimicrobial agents for idelines . The genm-chlorophenylhydrazone  was used to attempt to inhibit B739_0873 activity. First, a series of CCCP concentrations  were prepared to determine the optimal concentration for the full survival of the wild-type strain, the RA-CH-1 ΔB739_0873 mutant and the complemented strain RA-CH-1 ΔB739_0873 pLMF03::B739_0873. Subsequently, the MICs of aminoglycosides and detergents for these R. anatipestifer CH-1 strains were determined with or without CCCP.To determine whether the B739_0873 protein was driven by PMF, the PMF inhibitor carbonyl cyanide R. anatipestifer CH-1, RA-CH-1 ΔB739_0873 and RA-CH-1 ΔB739_0873 pLMF03::B739_0873 were grown in TSB broth and harvested at the exponential phase of growth, and then washed twice at room temperature with 50 mM phosphate buffer (pH 7.0), containing 1 mM MgSO4 and 0.4% (wt/vol) glucose. The washed cells were resuspended in the same buffer at a density of 1 mg (dry weight) per ml, including 32 μg/ml gentamicin. To determine the dry weight, cells harvested at the exponential phase of growth by centrifugation for 10 min at 8,000 rpm, removed the supernatant and dried to a constant weight at 60°C. At the 24-min time point, 5 μM CCCP was added to the cells suspensions to assess energy-dependent efflux. The samples were collected every 8 min, centrifuging at 12,000 g and 4°C for 1 min and the samples were immediately diluted into ice-cold phosphate buffer, followed by two washes and sonication on ice. Gentamicin uptake was measured using a Gentamicin ELISA Test Kit following the manufacturer’s instructions (Reagen).To further confirm that the B739_0873 protein was the inhibited efflux pump, a gentamicin accumulation assay was performed. Cells of R. anatipestifer CH-1 B739_0873 protein are vital for it to function. The point mutations were introduced into plasmid pLMF03::B739_0873 using the overlap PCR method  following the manufacturer’s instructions and then cloned into the pLMF03 plasmid to generate mutant recombinant plasmids, pLMF03::B739_0873D400A, pLMF03::B739_0873D401A, pLMF03::B739_0873K929E, pLMF03::B739_0873R959A, and pLMF03::B739_0873T966E. They were introduced into RA-CH-1 ΔB739_0873 by conjugation, as described previously   in the RR method . The muteviously . The traB739_0873 was used to express B739_0873 protein. Briefly, the primers P22-Truncated raeB-F/P23-Truncated raeB-R were used to amplify the truncated B739_0873 ORF, which was ligated into pET32a (+) at BamHI and XhoI sites to generate pET32a (+)::B739_0873.The recombinant plasmid pET32a (+)::E. coli BL21 (DE3) pET32a (+)::B739_0873 was grown overnight at 37°C in LB broth containing 100 μg/ml Amp. Subsequently, the LB broth containing 100 μg/ml Amp was inoculated with the overnight culture to an optical density at 600 nm (OD600) of 0.05 and grown at 37°C. Expression was induced by adding 0.6 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6 for 6 h at 37°C. Cells harvested by centrifugation for 10 min at 8,000 rpm and 4°C were suspended in lysis buffer  and then sonicated on ice. After centrifugation at 10,000 rpm and 4°C for 10 min, the supernatant of the lysate was loaded onto an equilibrated  nickel-nitrilotriacetic acid (NTA) column . By washing with lysis buffer  containing 20 and 50 mM imidazole, non-specific contaminants were removed. Subsequently, the B739_0873 protein was eluted with lysis buffer  containing 200 mM imidazole. Then the purified protein was placed in the dialysis membrane (Solarbio), soaked in phosphate-buffered saline (PBS) buffer (pH 7.4) and changed the buffer every other 1, 2, 4 h, the final dialyzed overnight, to eliminate any residual imidazole.Strain The purified B739_0873 His-tagged protein was used to generate a polyclonal antibody. Approximately 2 mg of B739_0873 protein emulsified in complete Freund’s adjuvant (Sigma) was used to immunize four rabbits  via intradermal injections. Subsequently, booster doses of 3 and 4 mg of B739_0873 protein were prepared in incomplete Freund’s adjuvant (Sigma), and the immunization was given after 2 and 3 weeks, respectively, using subcutaneous injections. A week after the last immunization, the antibody was collected from the ear vein of the rabbits and frozen at -80°C.B739_0873 pLMF03, RA-CH-1 ΔB739_0873 pLMF03::B739_0873, RA-CH-1 ΔB739_0873 pD400A, RA-CH-1 ΔB739_0873 pD401A, RA-CH-1 ΔB739_0873 pK929E, RA-CH-1 ΔB739_0873 pR959A, and RA-CH-1 ΔB739_0873 pT966A were grown at 37°C in TSB containing 1 μg/ml Cfx. Bacteria were harvested by centrifugation until they grew to exponential phase, suspended in the PBS buffer and then sonicated on ice. The sonicated cells were suspended in loading buffer and heated for 5 min at 100°C. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using TGX Stain-FreeTM FastCastTM Acrylamide Kit (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Non-specific binding sites were blocked with 5% skim milk (Solarbio) in TBS-Tween 20 (0.05%). The PVDF membranes were probed with B739_0873 rabbit polyclonal antibody (1:200), followed by a 1:3,000 dilution of a goat anti-rabbit IgG alkaline phosphatase-conjugated secondary antibody (Bio-Rad). The binding of antibodies to protein was revealed using a substrate for horseradish peroxidase (HRP)-based chemiluminescence Western blot detection following the manufacturer’s instructions (Takara).Strains RA-CH-1 ΔAll animals were handled in strict accordance with good animal practices, as defined by the local animal welfare bodies. The protocol for the animal work to be performed at Sichuan Agriculture University was reviewed and approved by the Sichuan Agriculture University ethics committee in September 2014.t-test. A value of P < 0.05 was considered significant.Statistical analysis was performed using GraphPad Prism version 7 software for Windows. The significance of the between-group differences was ascertained using Student’s B739_0873 gene of R. anatipestifer CH-1 consists of an ORF with 3,156 bp, which encodes a 1,051-amino acid protein with a putative molecular mass of 115 kDa. The B739_0873 protein is annotated as a cation/multidrug efflux pump in the National Center for Biotechnology Information (NCBI) database. A protein–protein Basic Local Alignment Search Tool (BLASTP) analysis of the B739_0873 amino acid sequence of different R. anatipestifer strains indicated 99–100% identity. Homologs of B739_0873 are also present in Riemerella columbina, Riemerella columbipharyngis, Flavobacteriaceae bacterium 3519-10, Salinimicrobium terrae, Chryseobacterium treverense, Chryseobacterium solincola, Salegentibacter salegens, and Salegentibacter agarivorans, and identities range from 77 to 83%. However, the function of all these homologs has not previously been characterized.The E. coli AcrB and 27% identity with Pseudomonas aeruginosa MexB. Furthermore, the essential residues in E. coli AcrB    and two extremely large periplasmic loops between helix 1 and 2 and between helix 7 and 8. These results are consistent with the general features of RND efflux pumps . The CluThr 978)  and P. aThr 976)  that areB739_0873 gene showed that the B739_0872 gene encodes a protein belonging to the MFP family (B739_0871 gene encodes a protein belonging to the outer membrane factor (OMF) family (B739_0870 gene encodes a tetracycline resistance repressor protein (TetR) family transcriptional regulator . Sequence alignments showed that B739_0870 shares 31% identity with E. coli AcrR, B739_0872 shares 24% identity with E. coli AcrA and 25% identity with P. aeruginosa MexA, and B739_0871 shares 20% identity with E. coli TolC and 23% identity with P. aeruginosa OprM.Analysis of the upstream regions of the P family , the B73) family  and the egulator . These tB739_0870-0871-0872-0873 genes as raeR-raeC-raeA-raeB and the B739_0870-0871-0872-0873 proteins as RaeR-RaeC-RaeA-RaeB described below.Overall, the bioinformatic analysis suggests that B739_0873 is a putative RND efflux pump. Thus, we designated the raeB, a mutated raeB was constructed in R. anatipestifer CH-1. To determine whether the inactivation of raeB affected the growth state of R. anatipestifer CH-1, both the growth curves and the CI values were evaluated. The results showed that RA-CH-1 ΔraeB had the same growth rate and CI value as the wild-type strain . To determine whether the inactivation of raeB affected the virulence of R. anatipestifer CH-1, the median lethal dose (LD50) was measured as described previously by 50 changes between the wild-type strain and RA-CH-1 ΔraeB (data not shown).To investigate the role of raeB mutant and the wild-type strain to a variety of antimicrobial agents with dissimilar structures , streptomycin, sulfamethoxazole, tetracycline, trimethoprim, Triton X-100, and vancomycin) was compared. It was shown that deletion of raeB increased susceptibility to all the aminoglycosides tested  and all the detergents tested [Triton X-100 (fourfold) and SDS (16-fold)] (Table 3). There were no MIC changes between the wild-type strain and the mutant strain for any of the tested cephalosporins, chloramphenicols, cationic dyes, quinolones, glycopeptides, lincosamides, macrolides, penicillins, rifampicin, sulfonamides, and tetracyclines . This result revealed that the changes were caused solely by raeB. Complementation of the RA-CH-1 ΔraeB mutant with plasmid pLMF03::raeB restored resistance to aminoglycosides and detergents (Table 3). This result indicated that raeB is involved in aminoglycoside and detergent resistance.In a second set of experiments, the susceptibility of the RA-CH-1 ΔraeB is regulated by aminoglycosides and detergents, sub-inhibitory concentrations of aminoglycosides and detergents were added to an R. anatipestifer CH-1 growth culture and the transcription of raeB was measured using qRT-PCR. As shown in Figure 2, the level of raeB expression was up-regulated by two- to sevenfold after treatment with Triton X-100, SDS, streptomycin, Kan, or gentamicin.To address whether B739_0873 mutant and the complemented strain RA-CH-1 ΔB739_0873 pLMF03::B739_0873. The results showed that the addition of CCCP (5 μM) decreased the MICs of streptomycin (eightfold), gentamicin (16-fold), and Kan (eightfold) for the wild-type strain and RA-CH-1 ΔraeB pLMF03::raeB. Similarly, the MICs of SDS and Triton X-100 for the wild-type strain and RA-CH-1 ΔraeB pLMF03::raeB decreased by 4- and 16-fold, respectively, in the presence of CCCP. In contrast, CCCP addition did not modify the MICs of streptomycin, gentamicin, Kan, SDS, and Triton X-100 for RA-CH-1 ΔraeB. The MICs of streptomycin, gentamicin, Kan, SDS, and Triton X-100 for the wild-type strain and RA-CH-1 ΔraeB pLMF03::raeB in the presence of CCCP were consistent with those evidenced for RA-CH-1 ΔraeB in the absence or presence of CCCP. These data indicated that an aminoglycoside and detergent efflux pump exists in R. anatipestifer CH-1.The final concentration of CCCP in TSB broth was 5 μM, which did not affect the growth of the wild-type strain, the RA-CH-1 ΔFigure 3, at the 24-min incubation time point, the accumulation level of gentamicin achieved a steady-state for all strains. The gentamicin accumulation level in the mutant strain RA-CH-1 ΔraeB was about six times higher than that in the wild-type strain and RA-CH-1 ΔraeB pLMF03::raeB. After CCCP was added to the cells containing gentamicin at 24-min, the accumulation of gentamicin increased in the wild-type strain and RA-CH-1 ΔraeB pLMF03::raeB; these accumulation levels were lower than that in RA-CH-1 raeB. In contrast, under our conditions, CCCP had no significant effect on the level of gentamicin accumulation in the mutant strain RA-CH-1 ΔraeB. In addition, the wild-type strain, RA-CH-1 ΔraeB, and RA-CH-1 ΔraeB pLMF03::raeB with no CCCP added at 24-min served as controls. These data indicated that RaeB pumped out gentamicin in an energy-dependent process, presumably coupled to the PMF.As shown in R. anatipestifer CH-1, five mutant recombinant plasmids, pLMF03::raeBD400A, pLMF03::raeBD401A, pLMF03::raeBK929E, pLMF03::raeBR959A, and pLMF03::raeBT966E, were constructed. These plasmids were introduced into RA-CH-1 ΔB739_0873, and the MICs for RA-CH-1 ΔraeB pD400A, RA-CH-1 ΔraeB pD401A, RA-CH-1 ΔraeB pK929E, RA-CH-1 ΔraeB pR959A, and RA-CH-1 ΔraeB pT966A were examined. It was shown that none of these mutant strains exhibited restored resistance to aminoglycosides and detergents (Table 3). To exclude the possibility that these results occurred because these mutant raeB genes were not expressed, we detected the expression of these mutant proteins by immunoblotting with antibody against the RaeB protein. The immunoblot analysis showed that all these RaeB mutant proteins expressed in RA-CH-1 ΔraeB  were detectable and indistinguishable from the RaeB protein expressed in RA-CH-1 ΔraeB .We investigated whether amino acid residues Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 are vital for the function of the RaeB protein of raeB is co-transcribed with raeA  as previously reported by raeB, the RA-CH-1 ΔraeA ΔraeB mutant was constructed. Antibiotic susceptibility testing showed that this mutant displayed MICs that were decreased by eightfold for streptomycin, 16-fold for gentamicin, eightfold for Kan, 16-fold for SDS, and fourfold for Triton X-100 (Table 3). However, complementation of the RA-CH-1 ΔraeA ΔraeB mutant with plasmid pLMF03::raeB did not restore resistance to any of these compounds (Table 3). These results indicate that both raeA and raeB are required for aminoglycoside and detergent resistance.We confirmed that E. coli  played an essential role in aminoglycoside and detergent transport. However, no structural information about the RaeB protein is currently available, so further studies determining whether these five amino acid residues are relevant to the proton transport process in R. anatipestifer CH-1 are needed.It has been demonstrated that that AcrB of function . In thisransport . We alsoEnterobacter cloacae, growth curves and competition experiments in vitro demonstrate that lack of the AcrB efflux pump poses a fitness cost to E. cloacae (raeB gene did not alter the fitness of R. anatipestifer CH-1. This may reflect a possible adaptation, in that other efflux pump systems may exist in R. anatipestifer CH-1 and were overproduced to compensate for the deletion. Additionally, efflux pumps AcrB in E. cloacae (Klebsiella pneumoniae (Vibrio cholerae (Mycobacterium tuberculosis (Salmonella typhimurium (50 was measured between the wild-type strain and the mutant strain RA-CH-1 ΔraeB, the result showed that deletion of the raeB gene had no significant impact on the LD50 of R. anatipestifer CH-1. The possibility that raeB is not the main virulence factor in R. anatipestifer CH-1 may account for this result. All these data suggest that RND efflux pumps have multiple different physiological functions in different species of bacteria.In some Gram-negative bacteria, RND efflux pumps have a role not only in antibiotic resistance, but also in bacterial fitness and virulence . As repo cloacae . However cloacae , AcrB ineumoniae , VexM ancholerae , MmpL11 rculosis , and Acrhimurium  contriburaeB and raeA-raeB R. anatipestifer CH-1 deletion mutants and assessed their antimicrobial resistance. D-KZ and XZ assessed the mRNA levels of the raeB and B739_0874 genes using RT-PCR. XZ, M-SW, and M-FL constructed the mutant recombinant plasmids, pLMF03:: raeBD400A, pLMF03::raeBD401A, pLMF03::raeBK929E, pLMF03::raeBR959A, and pLMF03::raeBT966E. XZ and M-FL constructed the RA-CH-1 ΔraeB complemented strain and the RA-CH-1 ΔraeA ΔraeB complemented strain. XZ, QY, and YW performed the CCCP inhibition assay and gentamicin accumulation assay. M-SW, K-FS, and X-YC constructed the bacterial growth curves and performed the in vitro competition experiments. X-XZ, R-YJ, and SC performed the LD50 determination for R. anatipestifer CH-1 and RA-CH-1 ΔraeB. XZ, A-CC, and FB drafted and revised the manuscript. All the authors read and approved the final version of the manuscript.A-CC and XZ conceived of and designed the project. XZ and M-FL constructed the 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.


HLA-G single-nucleotide polymorphisms as predictive biomarkers, but few analyzed HLA-G haplotypes. Here, we aimed to characterize HLA-G haplotypes and describe their association with asthmatic clinical features and sHLA-G peripheral expression and to describe variations in transcription factor (TF) binding sites and alternative splicing sites. HLA-G haplotypes were differentially distributed in 330 healthy and 580 asthmatic individuals. Furthermore, HLA-G haplotypes were associated with asthmatic clinical features showed. However, we did not confirm an association between sHLA-G and genetic, biological, or clinical parameters. HLA-G haplotypes were phylogenetically split into distinct groups, with each group displaying particular variations in TF binding or RNA splicing sites that could reflect differential HLA-G qualitative or quantitative expression, with tissue-dependent specificities. Our results, based on a multicenter cohort, thus support the pertinence of HLA-G haplotypes as predictive genetic markers for asthma.Human leukocyte antigen (HLA)-G, a HLA class Ib molecule, interacts with receptors on lymphocytes such as T cells, B cells, and natural killer cells to influence immune responses. Unlike classical HLA molecules, HLA-G expression is not found on all somatic cells, but restricted to tissue sites, including human bronchial epithelium cells (HBEC). Individual variation in HLA-G expression is linked to its genetic polymorphism and has been associated with many pathological situations such as asthma, which is characterized by epithelium abnormalities and inflammatory cell activation. Studies reported both higher and equivalent soluble HLA-G (sHLA-G) expression in different cohorts of asthmatic patients. In particular, we recently described impaired local expression of HLA-G and abnormal profiles for alternatively spliced isoforms in HBEC from asthmatic patients. sHLA-G dosage is challenging because of its many levels of polymorphism , thus many clinical studies focused on  Human leukocyte antigen-G (HLA-G), a member of the HLA class Ib  family, modulates natural killer (NK) cells and cytotoxic T-lymphocyte-mediated activity as well as B-lymphocyte proliferation. HLA-G is also involved in epithelial cell differentiation –3.Human leukocyte antigen-G is expressed by a restricted pattern of cells including human bronchial epithelium cells (HBEC) and displays interindividual variation expression in physiological conditions. The mechanisms responsible for tissue-specific expression and interindividual HLA-G expression variation are of utmost importance since HLA-G expression is associated with many diseases involving immune system surveillance and inflammatory disease, such as asthma , 4.Asthma is characterized by structural changes in the airways with abnormalities of the epithelium and concomitant recruitment, survival, and activation of inflammatory cells. In asthma, soluble HLA-G (sHLA-G) expression was reported to be higher in bronchoalveolar lavage (BAL) from asthmatic individuals and in both plasma and serum from atopic asthmatic children, whereas others reported no difference in non-allergic asthmatics –8. In BAHLA-G*01:04 has been associated with high HLA-G secretion and G*01:03 and G*01:05N with low HLA-G secretion  and that 3′UTR haplotypes display equivalent means of microRNA binding  binding sites and alternative splicing sites between HLA-G haplotypes that could account for their differential association with asthmatic features.We aimed to investigate the association of Cohorte Obstruction Bronchique et Asthme, INSERM Hôpital Bichat, CNIL 28/01/2008) were included in the study. DNA samples with known genotype were used as control. Biological samples included 590 samples of DNA, 582 of serum, and a further 528 of serum collected 1 year after the first sample. Patient data were categorized into biological and clinical data and asthma severity according to the Guidelines for the Diagnosis and Management of Asthma, NHLBI 2007. Healthy individuals (HI) and asthmatic patient characteristics are detailed in Table This study was carried out in accordance with the French Public Health Code art L1221-1). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Three hundred and thirty volunteer bone marrow donors from France were recruited, blood donations were collected, and DNA was extracted from a 200 µl total blood sample using the QIAmp Blood DNA Mini kit  in accordance with BSL-2 practices. A medical interview was carried out prior to blood donation to exclude donors with medical contraindications such as severe asthma, respiratory failure, or allergies. Five hundred and ninety asthmatic patients enrolled in a French asthmatic multicenter cohort (221-1. AlHLA-G gene was sequenced from position −1983 to +3447 by next-generation sequencing (NGS). A 5,430 pb long amplicon was generated by PCR; primer sequences were 5′AGGAGCTGACACAGGAGGAA3′ and 5′CAGCTGAGCAGTGACCACAT3′; and amplification was performed using the Long Range PCR Kit . PCR fragments were sequenced using an NGS platform .The HLA-G NGS data were analyzed through PolyPheMe, specific software developed by the Xegen Company (France). HLA-G allelic assignment at eight digits was based on the HLA sequences listed in the official IMGT/HLA database  according to the manufacturer’s protocol.Soluble HLA-G (sHLA-G), i.e., peripheral HLA-G expression, was quantified in sera from 582 asthmatic patients and 528 sera collected 1 year after the first sample. All serum samples were stored at −80°C prior to analysis. Measurement of soluble isoforms sHLA-G1 and sHLA-G5 was performed in duplicate on serum samples using the ELISA test , history of near-fatal asthma, asthma control, asthma control test score, asthmatic exacerbations within the last 12 months, emergency consultation for asthma within the last 12 months, hospitalization within the last 12 months because of asthma, intensive care unit stays, according to biological parameters , HLA-G predictors , and sHLA-G serology and differences between sHLA-G dosage results in sera collected at a 1-year interval. Univariate analyses were also performed on sHLA-G dosage and differences between sHLA-G dosage results in sera collected at a 1-year interval according to clinical and biological parameters such as gender, height, weight, BMI, occupational exposure, tobacco use, allergies, associated pathologies, maternal asthma, paternal asthma, treatment for asthma or for allergy , month of serum collection, and genetic parameters . The False Discovery Rate correction for multiplicity was applied; however, no power analysis was performed. Variables with p values below 0.05 and corrected p values below 0.20 in univariate analysis were further investigated in multivariate analysis. Association results with quantitative variables are expressed as estimates and standard errors (Std error), whereas association results with qualitative variables are expressed as odd ratios (OR) and 95% confidence intervals.Associations between patients’ asthmatic features, sHLA-G expression, and genetic polymorphism were tested with logistic regressions when the predicted variable was categorical and general linear regression when the predicted variable was quantitative. Univariate analyses were performed on each of these asthmatic features: eosinophil count  software version 7.0 (HLA-G full-length sequences were inferred using the neighbor-joining (NJ) method with 1,000 replicates. Evolutionary distances were computed using the p-distance method, and units were the number of base differences per site. Analysis involved gorilla HLA-G genomic sequence as an outgroup (accession number CU104658.1).sion 7.0 . EvolutiHLA-G haplotypes were checked for their tissue-specific expression (blood and lungs) in NCBI and Tissue-specific Gene Expression and Regulation (TiGER) databases  HI and 582 (98%) asthmatic patients. Thirty-seven SNPs, 1 base deletion, 1 base insertion, and a 14bp insertion were validated through Xegen pipeline analysis in the regulatory regions 5′URR and 3′UTR from position −1983 to +3447; all were previously described , 35, 36.HLA-G alleles were typed at maximum resolution (eight digits) and regulatory regions (UTR1–8) were typed at low resolution according to eight SNPs in 5′URR and at high resolution based on all 28 polymorphisms in the 5′URR and 12 polymorphisms in the 3′UTR. Fifteen haplotypes with frequency above 0.3% displayed 94% of the cumulated frequencies. Their description and frequencies are detailed in Table G*01:01:01:01 and G*01:03:01:02, which were associated with several haplotypes, and G*01:01:03:03 and UTR7/H16 that were exclusively associated together.Whereas high-resolution typing of regulatory region haplotypes did not discriminate between low-resolution typed haplotypes , some UTRs were refined . Several eight-digit alleles were associated with one haplotype, except for G*01:04:01 and H23-UTR3-G*01:04:04 were, respectively, higher and lower in HI than in asthmatic patients (p = 0.044 and p = 0.012); H04-UTR4-G*01:01:01:05 and H02-UTR4-G*01:01:01:05 were both higher in HI than in asthmatic patients (p = 0.015 and p = 0.009), finally H47-UTR6-G*01:01:01:04 was not observed in HI (p = 0.002) (Table H23-UTR3-N = 369) was associated with asthmatic exacerbation within the last 12 months  and eosinophil count (/mm3) . UTR7 (N = 76) was associated with acute asthma recurrence .Correlations between HLA-G alleles, UTR, or haplotypes and asthmatic patient data investigated in multivariate analysis are shown in Tables Serum sHLA-G measurements from asthmatic patients were 17.1 (0–493) UI/ml and 17.5 (0–404) UI/ml 1 year later (mean and range). Multivariate analyses revealed no association between peripheral sHLA-G expression and patients’ asthmatic features, biological and clinical data, or HLA-G genetics (data not shown).HLA-G alleles and haplotypes with a frequency above 0.3% were investigated using the NJ method , the second clade consists of the H23-UTR3 sequences (G*01:04:01 and G*01:04:04), the third clade includes UTR5-G*01:03:01:02 sequences , and the fourth clade assembles the UTR6, UTR4, and UTR1 sequences. This fourth clade had the most divergent sequences  as compared to a reference gorilla HLA-G sequence : the transition G>A at position −1155 impaired progesterone receptor (PR) and SOX5 binding sites. Besides this unique feature, this haplotype displayed similarities with H16 (UTR7-G*01:01:03:03) and H10 (UTR2-G*01:06/G*01:05N/G*01:01:02:XX). Comparing the most common sequences, some SNPs created new binding sites: one to two sites for SRF, one site for CUX1, two to three additional sites for POU2F1, one site for E2F, one site for glucocorticoid receptor, additional sites for SP1 (up to 10), one site for XFD-1, RXR, NR2C1, VDR, CDX1, and an additional site for HNF4. Conversely, some SNPs in H23, H16, and H10 disrupted binding sites for RUNX1, RFX1, and MYB. H16 and H10 differed from H23 and other haplotypes as their SNPs created a fixation site for Lmo complex, FOXI1, and GATA1 and disrupted a binding site for NF-E and TATA binding proteins.Interestingly, haplotype grouping based on their TF binding site profiles mirrored that observed on the phylogenetic tree Table . Hence, G*01:03:01:02) distinguished themselves by an additional fixation site for ELK and IRF1, and the disruption of a fixation site for STAT; H19 and H20 displayed a disruption of a IKZF1 fixation site. H21 displayed an increase in REL binding sites. Genetic polymorphism in H21, H19, and H20 created a site for NF-1 and SREBF1 which were also present in haplotypes H23, H16, and H10. H21, H19, H20, H16, and H10 had a genetic polymorphism that created a site for TEF. Haplotypes H21, H19, H20, H23, H16, H10, H04, and H02 displayed a fixation site for C/EBP.Haplotypes H21, H19, and H20 , H47 and H03 distinguished themselves by an additional site for REL and disruption of a fixation site for MEF-2. H46 and H49, H01, H05, and H54 and H04 and H02 displayed quite similar patterns, with the exception of H54 for which a fixation site for IKZF1 was disrupted, as for H19 and H20.Concerning haplotypes associated with UTR6 . HLA-G*01:01:01:01, HLA-G*01:01:01:04, and HLA-G*01:01:01:06 had a disrupted intronic cryptic acceptor site as compared to HLA-G*01:01:01:05 due to 99G>A at the beginning of intron 2 and 494A>C at the beginning of intron 3  in sera from 582 asthmatic patients and in 528 serum samples collected 1 year after the first sample. Associations between patients’ asthmatic features, sHLA-G expression, and genetic polymorphism were tested with logistic regressions.We fully characterized HLA-G haplotypes displayed statistically significant differential distribution between HI and asthmatic patients. Furthermore, multivariate analyses showed significant association of HLA-G haplotypes with asthma inflammation markers (eosinophil count) and asthma severity . We did not find any association between peripheral sHLA-G and genetic data in our asthmatic patient cohort; such an association described by us and others in physiological conditions may be masked in an inflammatory context as asthma. The lack of association between peripheral sHLA-G expression and asthmatic features supports the hypothesis that sHLA-G is not overexpressed as a systemic immune response to control local inflammation. These results reinforce the involvement of HLA-G in asthma and suggest that the modulation of local sHLA-G associated with asthma phenotypes and asthma inflammation markers described in Ref.  was associated with acute asthma recurrence. These sequences, the closest within the phylogenetic tree and according to TF binding site profiles, could lead to similar expression regulation. These results support the hypothesis of an impaired immunomodulation of the UTR2-HLA-G molecule, previously associated with a worse evolution of cystic fibrosis ; however, multiple correction testing did not reach statistical significance (pc = 0.22). One may speculate, however, that a larger cohort may lead to statistical significance; thus this haplotype might be a good candidate marker for inflammation, as we previously associated UTR3-HLA-G*01:04 with impaired long-term survival following lung transplantation (p = 0.044 and p = 0.012). These sequences differ at position 1827G>A in the last positions of exon 4 which, according to in silico predictive analysis, creates an exonic cryptic acceptor site. We previously reported alternatively spliced isoform expression of HLA-G in HBEC from asthmatic patients as compared to HI forms . No specific TF binding site was predicted by in silico analysis that could account for a putative protective effect; however, it could be due to a specific splicing process: when compared to its closest sequences . All subjects gave written informed consent in accordance with the Declaration of Helsinki. Asthmatic patients were enrolled in a French asthmatic multicenter cohort [Cohorte Obstruction Bronchique et Asthme (COBRA)].CaR, FC, CeR, FJ, PG, GM, NM, JP, CP, JC, LA-R, PC, DG, JDC: substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work. CaR, FC, CeR, FJ, PG, GM, NM, JP, CP, JC, LA-R, PC, DG, JDC: drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Mecp2308/y mice exhibited reduced heroin seeking behavior in CPP test. These data reveal a functional role of miR-218 and its target, MeCP2, in the regulation of heroin-induced behavioral plasticity.MicroRNAs (miRNAs) are a class of evolutionarily conserved, 18–25 nucleotide non-coding sequences that post-transcriptionally regulate gene expression. Recent studies implicated their roles in the regulation of neuronal functions, such as learning, cognition and memory formation. Here we report that miR-218 inhibits heroin-induced behavioral plasticity. First, network propagation-based method was used to predict candidate miRNAs that played potential key roles in regulating drug addiction-related genes. Microarray screening was also carried out to identify miRNAs responding to chronic heroin administration in the nucleus accumbens (NAc). Among the collapsed miRNAs, top-ranked miR-218 was decreased after chronic exposure to heroin. Lentiviral overexpression of miR-218 in NAc could inhibit heroin-induced reinforcement in both conditioned place preference (CPP) test and heroin self-administration experiments. Luciferase activity assay indicated that miR-218 could regulate 3′ untranslated regions (3′ UTR) of multiple neuroplasticity-related genes and directly target methyl CpG binding protein 2 (Mecp2). Consistently,  Drug addiction is a psychiatric disorder characterized by loss of control over drug consumption and compulsive drug taking despite serious negative consequences456Recent studies indicate that epigenetic regulation of gene expression plays an important role in neurogenesis, synaptic plasticity and neurological disorders891112151617In this study, we utilized bioinformatic approaches to predict potential key regulators in drug addiction. Among the top-ranked miRNAs, we found that miR-218 is down-regulated in response to chronic heroin administration. Lentiviral-mediated miR-218 overexpression significantly attenuated heroin-induced reinforcement in both conditioned place preference (CPP) and self-administration (SA) model. These effects were proposed to be mediated by suppression of target genes such as Mecp2, that participates in epigenetic control of gene transcription. Our observation provides a possible miRNA-mediated epigenetic regulatory mechanism in heroin addiction.etc. were top-ranked and previously reported172021First, we tried to screen for miRNAs involved in drug addiction-related regulatory networks. A miRNA could regulate gene expression either by directly targeting drug addiction-related genes, or by targeting regulatory elements (e.g. transcription factors) whose impact may propagate across the whole regulatory network. Thus we utilized a network propagation-based model (NP-method)Microarray screening was carried out to identify miRNAs responding to chronic heroin administration. As shown in Among these 16 candidate miRNAs, miR-181a, a miRNA previously implicated in cocaine-induced plasticity = 11.80, p = 0.003). Moreover, Bonferroni’s post hoc analysis revealed that rats with miR-218 overexpression in the NAc exhibited reduced preference to the heroin-paired chamber, compared with LV-control injected rats  or scramble control sequence (LV-control) into the NAc region . The expted rats .Next, we carried out heroin self-administration tests to further validate the role of miR-218 in heroin seeking behavior. Rats were trained to self-administer heroin for 8 consecutive days, then subjected to LV-miR-218 or LV-control injection. Consistent with CPP results, miR-218 overexpression significantly reduced heroin acquisition compared with the control group .Renilla luciferase and co-transfected the constructs with miR-218 mimic or scramble control in HEK293T cells. Reduced luciferase activity indicated that these genes were the directly regulated by miR-218 In the present study, we found that miR-218 was downregulated by chronic heroin use in NAc. Consistently, bioinformatic analysis indicated the predicted targets of miR-218 was enriched in addiction-related genes and involved in neuroplasticity. MiR-218 is encoded by an intron of the Slit gene and inhibits the expression of Robo1 which play important roles in axonal growth3638Mecp2308/y mice. However, Deng et al. found that Mecp2308/y mutant mice showed enhanced locomotor response to acute amphetamine administration and impaired CPP, but lentivirus mediated knockdown or overexpression of MeCP2 in NAc enhanced or inhibited amphetamine-induced locomotion and CPP respectivelyMecp2 Ser421Ala knock-in mice display both a reduced threshold for amphetamine sensitization and enhanced behavioral sensitivity to the reinforcing properties in cocaine self-administration, indicating that MeCP2 in NAc limited behavioral plasticity upon repeated psychostimulant exposure3050MeCP2 is a transcription factor binding to methylated cytosine residues in DNA and recruits histone deacetylases and other transcriptional repressors to silence target genes4546Taken together, our results reveal that miR-218 is involved in heroin induced behavioral plasticity possibly by regulating Mecp2. Overexpression of miR-218 in NAc decreased heroin conditioned preference and heroin self-administration. MiR-218 is likely to contribute to heroin related modifications, both transcriptionally and epigenetically. Our results provide a promising and novel approach to the treatment of heroin addiction. However, the role of miR-218 in other drug induced plasticity and the downstream signaling pathways regulated by miR-218 in addiction are remained to be elucidated.Mecp2308/y mice and littermates were maintained by mating heterozygotes with C57BL/6 J. All animal treatments were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Committee of Shanghai Medical College of Fudan University.Male Sprague-Dawley rats were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. and housed with a 12 h reverse dark/light cycle at 23 °C. All subjects were allowed free access to food and water. Rats used for experiments were 8–10 weeks old. 8 IU/ml.The lentivirus construct and packaging were performed as we described previouslyTo facilitate subsequent heroin self-administration, rats were trained to press an active lever for food pellets until they self-administered 100 pellets for 3 consecutive days on fixed-ratio 1 program. Then the rats were anesthetized, and a silastic catheter (0.04 cm interior diameter) was inserted 3 cm into the jugular vein, and the other end of the catheter was attached to a stainless steel pedestal and mounted on the skull. Bilateral 26-gauge guide cannulae were implanted in NAc . After surgery, the rats were allowed to recover for 7 days and the catheters were flushed daily with saline supplemented with heparin (30 IU/ml) and gentamycin (5 mg/ml). Next the rats were allowed to self-administer heroin  during a daily 4 h session. After the 8th session, the rats were anesthetized and subjected to lentivirus infusion with needle  that extend 1.5 mm beyond the guide cannulae. The self-administration procedure was resumed 3 days after lentivirus injection and tested on FR1 schedule for six consecutive days.Mecp2308/y and Mecp2+/y mice followed the same procedure.The apparatus was three-chambered (Med Associates), with two large side chambers with distinct floors and decorations and one small center chamber. In pretest, the rat was first confined in the center chamber for adaption for 5 min, and then allowed to freely explore the chambers for 15 min. Unbiased rats were subjected to lentiviral injection into NAc . The rats were allowed to recover for 7 days. Then the rats were alternatively confined in each large chamber for 30 min after injection of heroin  or saline . On the test session (day 8), the rats were adapted in center chamber for 5 min and allowed to explore all chambers unrestrictedly for 15 min. The time which the rat spent in each large chamber was recorded and CPP score was defined as the time spent in the heroin-paired chamber minus the time spent in the saline-paired chamber during the CPP test. The CPP pretest, conditioning and test procedure of Total RNA are harvested using TRIzol (Invitrogen) and RNeasy mini kit (QIAGEN) according to manufacturer’s instructions. The RNA samples are labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.11.0). Scanning was performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 is used to read the raw intensity of the image. Median normalized signal of each miRNA was used to represent expression level. MiRNAs that exhibit 1.25-fold up-regulation or 0.75-fold down-regulation was chosen for further analysis.Network propagation based model was generated as described−ΔΔCt method.TaqMan small RNA assay (Applied Biosystems) for miRNAs of interest were performed. U6 was used for normalization. Primers used in RT-PCR were Mecp2 , Gabrb3 , GluR2 , Dnmt3a , Ube3a , Nrxn1 , Sema6b , Gng3 , β-actin . All samples were run in duplicate. The relative expression level of mRNA was calculated by 2Renilla luciferase of psiCHECK-2 vector. The target sequences were, Gabrb3 [CCTTTATTTCTGTACTAACTTATCTCATAAGCACACCCAATTCCTCCTAG], GluR2 [TATTGTTAGTCTCTTGATTCATAATGACTTAAGCACACTTGACATCAACT], Dnmt3a [TTGGTTGTCTCTAGCCTGATCAGATAGGAGCACAAACAGGAACAGAATAG], Ube3a [TCTTTGTAGCTGGACAGCACAATGTTTATGATTTATTTAATCTGTAGTTT], Nrxn1 [AAACTTATTTACTTTCCTTTTTATGAAGCACATACAAAAGAAGACAGGGA], Sema6b [CGGGTGGGGATCTCCTCGCCACAGGGAAGCACAAGAGCCCCCTCCATCCC], Gng3 [CGCACTTATCCTGAGATTATCTGAAGCACAAGGCCCTCCTTACCCACCTC]. Mecp2 [TTGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA]. We transfected 500 ng recombinant vector with 10 pmol miRNA mimic or scramble  into HEK293T cells with Lipofectamine 2000 (Invitrogen). After 24 h, dual-luciferase report assay system (Promega) was performed to measure Renilla and Firefly luciferase activity, then Renilla activity was normalized to the Firefly luciferase activity. All the experiments were repeated for at least three times.Predicted target sequences of miR-218 were cloned to the 3′ UTR of The rats were anesthetized and transcardially perfused with saline, followed by 4% paraformaldehyde in PBS. Then the brains were quickly removed and post fixed with 4% paraformaldehyde at 4 °C for about 4 h and stored in 30% PBS-buffered sucrose solution for 72 h. The brains were sectioned (40 μm) with a cryostat (Leica) and the sections were washed in PBS, blocked with blocking buffer (10% donkey serum in PBS containing 0.3% Triton X-100) for 1 h, then incubated with NeuN antibody  at 4 °C overnight. Sections were subsequently rinsed with PBS and incubated for 1 h at room temperature with CY3 goat anti-rabbit antibody . The images were captured with a microscopic CDD camera (Olympus).t-test or one-way analysis of variance (ANOVA). For all the behavior results, data were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s post hoc test. Luciferase activity data were analyzed by student’s t-test or two-way ANOVA followed by Bonferroni’s post hoc test. P < 0.05 are defined as statistically significant.Data are presented as mean ± s.e.m. For quantitative PCR results, data were analyzed by student’s How to cite this article: Yan, B. et al. MiR-218 targets MeCP2 and inhibits heroin seeking behavior. Sci. Rep.7, 40413; doi: 10.1038/srep40413 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


APOBEC3G (A3G) and APOBEC3F (A3F) are DNA-mutating enzymes expressed in T cells, dendritic cells and macrophages. A3G/F have been considered innate immune host factors, based on reports that they lethally mutate the HIV genome in vitro. In vivo, A3G/F effectiveness is limited by viral proteins, entrapment in inactive complexes and filtration of mutations during viral life cycle. We hypothesized that the impact of sub-lethal A3G/F action could extend beyond the realm of innate immunity confined to the cytoplasm of infected cells. We measured recognition of wild type and A3G/F-mutated epitopes by cytotoxic T lymphocytes (CTL) from HIV-infected individuals and found that A3G/F-induced mutations overwhelmingly diminished CTL recognition of HIV peptides, in a human histocompatibility-linked leukocyte antigen (HLA)-dependent manner. Furthermore, we found corresponding enrichment of A3G/F-favored motifs in CTL epitope-encoding sequences within the HIV genome. These findings illustrate that A3G/F‐mediated mutations mediate immune evasion by HIV in vivo. Therefore, we suggest that vaccine strategies target T cell or antibody epitopes that are not poised for mutation into escape variants by A3G/F action. APOBEC3G (A3G) and APOBEC3F (A3F) are members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex (APOBEC) family of cytidine deaminase enzymes. This family includes activation-induced cytidine deaminase (AID), APOBEC1, APOBEC2, APOBEC3A-H, and APOBEC4. All APOBEC3 (A3) sub-branch members  exhibit varying degrees of anti-viral restriction activity, though A3G and A3F are the most potent restrictors of HIV. For this reason, the mechanisms of A3G/F have been the subject of intense study over the last decade. From these studies, it is appreciated that A3G/F mutate cytidine (C) to uridine (U) in trinucleotide mutational hotspots  in the reverse transcribed DNA copy of HIV, registering as guanine (G) to adenine (A) substitutions in the plus strand DNA . These mSince their discovery early in the last decade, the prevalent view of A3G/F has been as anti-retroviral restriction factors. This is because, first A3G/F significantly diminishes viral propagation in several in vitro or ex vivo reporter systems of HIV infection , 10. SecA3G/F have been considered as innate immune agents, because they are constitutively expressed in CD4+ T lymphocytes, macrophages, and dendritic cells regardless of infection status, though inflammatory cytokines can upregulate their expression post-infection . Second,In contrast to lethal mutation of HIV’s genome and its effective restriction by A3G/F in various in vitro cell culture-based assays of HIV infectivity, A3G/F activity in vivo only leads to low level non-lethal mutations of the viral genome. There are several reasons for this: first, the Virion infectivity factor (Vif) protein of HIV binds and targets A3G/F for degradation via a ubiquitin-dependent proteosomal pathway. Thus, when Vif is present, A3G/F effectiveness in viral restriction is severely diminished, and experimental systems where A3G/F can functionally restrict HIV use Vif-deficient viruses F –27. SecoSince most HIV genomes would only suffer low level A3G/F mutations levels, we and others proposed that non-lethal levels of A3G/F-induced mutations could impact adaptive immunity in HIV-infected individuals through modification of specific epitopes . Two earWe sought to determine if A3G/F mutations play a role in adaptive immunity in HIV-infection. To this end, we measured CTL recognition of wild type and A3G/F-mutated epitopes in a cohort of HIV-infected individuals. First, we cataloged all CTL epitopes in HIV proteins using the experimentally-verified HIV molecular immunology database, and identified CTL epitopes whose encoding sequences contain A3G/F-mutable hotspots. Next, we synthesized wild type and A3G/F-mutated peptide CTL epitopes restricted to various Class I HLA molecules and evaluated CTL recognition of wild type and A3G/F-mutated CTL epitopes by CTL from HLA-matched subjects. We found that in contrast to control peptides mutated in sequences outside A3G/F hotspots, A3G/F-induced mutations overwhelmingly diminished CTL recognition of HIV peptides .Two further key observations can be taken as evidence for the physiological significance of A3G/F-mediated CTL evasion by HIV. First, we found that HIV’s genome has enriched for A3G/F mutational hotspots in sequences encoding CTL epitopes, either located within more immunogenic polypeptides , or restricted to class I HLA molecules that present highly immunogenic epitopes (e.g. HLA-B57) . Second,It is likely that A3G/F do indeed impact adaptive immunity in HIV-infected individuals, and that A3G/F-mediated CTL escape is a significant phenomenon in HIV infection. We note that a parallel role for A3G/F in mediating drug resistance mutations of the viral genome has already been demonstrated. Together, these results indicate that A3G/F carry significant pro-retroviral activities.It will be important to understand the extent of inter-individual variation in A3G/F-mediated CTL escape during HIV infection, to gain mechanistic insights into the molecular and cellular pathways through which A3G/F-mediated mutations drive CTL escape, and compare these to known pathways of immune evasion for HIV and other viruses.In the future, it will also be important to fully define the rare set of CTL epitopes that we found are either not transformed into escape variants, or conversely, are made more immunogenic by A3G/F-mediated mutations. Our work suggests that any vaccination strategy, whether eliciting CTL or antibody responses, can be rendered more effective if these “A3G/F-resistant” or “A3G-enhanced” epitopes are taken into consideration.


A315T mice. Similar to our recent results in SOD1G93A mice, TDP-43A315T mice fed a standard pellet diet showed increased 5′ adenosine monophosphate-activated protein kinase (AMPK) activation at postnatal day (P)80, indicating elevated energetic stress during disease progression. We therefore investigated the effects of a high-fat jelly diet on bioenergetic status and lifespan in TDP-43A315T mice. In contrast to standard pellet-fed mice, mice fed high-fat jelly showed no difference in AMPK activation up to P120 and decreased phosphorylation of acetly-CoA carboxylase (ACC) at early-stage time points. Exposure to a high-fat jelly diet prevented sudden death and extended survival, allowing development of a motor neuron disease phenotype with significantly decreased body weight from P80 onward that was characterised by deficits in Rotarod abilities and stride length measurements. Development of this phenotype was associated with a significant motor neuron loss as assessed by Nissl staining in the lumbar spinal cord. Our work suggests that a high-fat jelly diet improves the pre-clinical utility of the TDP-43A315T model by extending lifespan and allowing the motor neuron disease phenotype to progress, and indicates the potential benefit of this diet in TDP-43-associated ALS.Transgenic transactivation response DNA-binding protein 43 (TDP-43) mice expressing the A315T mutation under control of the murine prion promoter progressively develop motor function deficits and are considered a new model for the study of amyotrophic lateral sclerosis (ALS); however, premature sudden death resulting from intestinal obstruction halts disease phenotype progression in 100% of C57BL6/J congenic TDP-43 Summary: A high-fat diet extends lifespan in a TDP-43 mutant mouse model of motor neuron disease and might be a possible treatment strategy for individuals with amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterised by the selective weakening and loss of motor neurons eventually leading to paralysis and death. The prevalence of ALS worldwide is 2.6/100,000 and affected individuals have a median survival time of 22-48 months from diagnosis . The majG93A mice from postnatal day (P)90 and P120 onwards, respectively (G93A mice (5′ adenosine monophosphate-activated protein kinase (AMPK) is a master regulator and sensor of energy levels in the body and functions to promote energy homeostasis . When adectively . Further93A mice .Many pre-clinical ALS studies have been carried out in SOD1 mouse models; however, there is a great need to examine other genetic variants of ALS pathology. Recently, a novel TDP-43 mouse model of ALS expressing human transgene cDNA under the control of the murine prion promoter (mPrP) was created on a mixed C57BL/6;CBA genetic background . These mG93A mice and individuals with ALS, whereas calorie restriction consistently worsens disease outcome  difficulties. High-fat jelly diets have been shown to improve survival outcome in SOD1 outcome . TherefoG93A mice throughout disease progression and observed increased AMPK activation from disease onset (P90) onwards  and non-tg specimens at each time point were collected and probed for threonine-172 phosphorylated and total AMPK protein levels via western blotting. We found significantly increased AMPK activity at P80, whereas no significant change in AMPK activation was noted at the earlier time points P40 and P60 , we next trialled various diet regimes and examined survival rates in male mice  in comparison with standard pellet-fed mice living for an average of 102±19 days . As these mice lived for an average of 147 days on a high-fat jelly diet we examined P60, P90 and P120 time points and probed for active, threonine-172 phosphorylated and total AMPK protein levels via western blotting. We found no significant difference in AMPK activation at any time point examined; however, there was a trend towards elevated AMPK activity at the late time point P120 , P140 (P=0.017), P150 (P=0.006) (Progressive motor dysfunction owing to lifespan extension is evident in TDP-43let diet C-F. The  onwards B when suP=0.006) D compareP=0.006) F that waP=0.006) E.Fig. 6A315T mouse specimens. Motor neurons were identified as large (width >30 µm), multi-polar structures with dark blue or purple staining. No difference in motor neuron survival was noted between non-tg and tg mice fed a standard pellet diet in lumbar or thoracic sections (P85)  and thoracic  spinal cord sections at P85 in standard pellet-fed mice and P120 (late stage disease progression) and P150 (approximate end stage) time points in high-fat jelly fed non-tg and TDP-43ns (P85) A-C. We ons (P85) D,E, wherns (P85) D,F or luA315T mice via a high-fat jelly diet alleviated high energetic demands as evidenced by delayed AMPK activation and decreased ACC phosphorylation. In turn, the high-fat jelly diet regime had a pro-survival effect in TDP-43A315T mice with lifespan extension to 147 days on average. This prolonged survival rate allowed a progressive motor neuron disease phenotype to develop as evidenced by weight loss at P80 and onwards, diminished Rotarod abilities observed at P130, 140 and 150 and decreased gait abilities recorded at P100 and onwards. The weakness and loss of limb function identified was associated with lumbar motor neuron loss. The high-fat jelly diet regimen did not rescue the previously documented enteric dysfunction in this model, however. A high-fat jelly diet therefore significantly improves the pre-clinical utility of the TDP-43A315T model, and suggests a beneficial role for high-fat jelly diet in TDP-43-associated ALS.Our results show that targeting bioenergetic stress in TDP-43A315T mice in the same way as we previously documented bioenergetic imbalance in SOD1G93A mice , which overall led to a prolonged lifespan. Delayed AMPK activation and a pro-survival effect with a high-fat jelly diet has previously also been observed in SOD1G93A and SOD1G68R mice , indicating that a high-calorie and high-fat diet was the key element necessary to relieve bioenergetic stress and to incur a pro-survival effect in TDP-43ne model . We heren-3 polyunsaturated fatty acid (PUFA) intake  intake . Similar) intake . Dorst a) intake . A low-f) intake . Further) intake . Interes) intake . Evidenc) intake . Our pread libitum food and water available. Only male mice were used in this study to avoid sex variation. All experiments were carried out under license (no. B100/4414) from the Department of Health and Children, Ireland, with ethical approval from the Royal College of Surgeons in Ireland Research Ethics Committee (REC625b).Prp-hTDP-43 (A315T) transgenic mice were purchased from Jackson Laboratory . Of note, Jackson Laboratory reports that the background of these mice is now fully congenic on a C57BL6/J background. Mice were housed in cages containing between three and five mice and kept at constant temperature (22°C) on a 12 h light/dark cycle , with A315T mice (sustained on a standard pellet diet) were administered latrepirdine  or vehicle from P40 until P80, and survival was assessed. Animals were randomly assigned to either vehicle or latrepirdine groups.Latrepirdine  stock was dissolved in 1× PBS (vehicle). Non-tg and tg TDP-43Table S1 displays the macronutrient breakdown of the three diets and caloric content of each.Three sets of diets were trialled in this study; a standard rodent pellet diet , a low-fat jelly diet  and high-fat jelly diet . Mice were weaned at P30 to P35 and placed on the respective diet for their study group. The standard pellet diet contained 24% protein, 18% fat and 58% carbohydrate but contains no moisture. The low-fat jelly diet is composed of 4.7% protein, 1.5% fat, 18% carbohydrate and 73% moisture. The high-fat jelly diet nutritional breakdown is 9.9% protein, 21.6% fat, 37.8% carbohydrate and 30% moisture. . End stage of ALS disease progression was determined by a number of factors: a ‘swimming’ or ‘waddling’ gait (documented in Movie 1), a hunched posture, piloerection, lack of grooming, anti-social behaviour, noticeable weight loss, and intestinal enlargement and/or discomfort.For lifespan and motor function assessment, animals were age-, sex- and litter-matched in accordance with recent ALS guidelines for the generation of preclinical data . Mice weCryoprotected thoracic and lumbar spinal cord samples were sectioned (20 µm) on the cryostat from T5-T10 and L1-L5 and Nissl stained with Cresyl Violet (0.1%). Nissl-positive motor neuron cells were counted  in every third section of the ventral horn region of spinal cords and motor neuron survival assessed.Fixed-paraffin embedded intestinal tissue was sectioned (10 µm) using the microtome with a specific focus on the ileocaecal junction and small intestine regions owing to identified pathological changes in these areas of the gastro-intestinal tract in previous publications . IntestiSamples were homogenised in RIPA buffer  supplemented with protease inhibitor mixture  and phosphatase inhibitors . Equal amounts of protein were diluted in Laemmli buffer, separated by SDS-PAGE and transferred to a nitrocellulose membrane using semi-dry transfer apparatus. Membranes were blocked using 4% bovine serum albumin (BSA) in TBS-Tween 20  for 1 h at room temperature. Membranes were then incubated with the following antibodies: anti-threonine-172-phospho-AMPK , anti-AMPK , anti-phospho-ACC , anti-ACC , anti-β-actin , made up in 1% BSA, overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  for 1 h at room temperature. Membranes were washed and incubated with enhanced chemiluminescence substrate (Millipore) and imaged using the LAS 4000 Reader (Fujifilm). Densitometric analysis was performed in ImageJ software (NIH) on all samples and values were normalised to loading control (β-actin) and standardised to the corresponding non-tg control.t-test was used . Otherwise, the two-tailed Mann–Whitney U-test was used.All data are presented as mean±s.e.m. Determining whether data was parametric or non-parametric was subject to the power (i.e. depending on the sample number) and the distribution of the data. Therefore, if sample numbers were greater than five per treatment or genotype group a histogram representation of the data was used to determine whether the data was normally distributed (bell-shaped curve) or abnormally distributed. As low sample numbers (less than five per group) were too small to test for normality, the standard tests assuming normal distribution were employed. For normally distributed data i.e. parametric data, when only two groups were analysed, a two-tailed Student Survival results were assessed by the Kaplan–Meier estimate test. The Kaplan–Meier test assumes normal distribution and is one of the best methods to analyse the fraction of subjects in a group living post treatment . As the P<0.05.Statistical analysis was performed by PASW statistics version 22 Software  or GraphPad Prism  and significance was incurred at