Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3′half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression.We have cloned the  TPS1 gene TPS2Saccharomyces cerevisiae or Kluyveromyces lactis mutations in the gene TPS1 cause inability to grow in glucose S. cerevisiaeYarrowia lipolytica with a Ki of 3.5 µM Y. lipolytica is a dimorphic yeast that separated early from the yeast evolutionary trunk Y. lipolytica is also being used as model to study physiological processes like lipid accumulation Y. lipolytica enzymes S. cerevisiaeY. lipolytica hexokinase to T6P it appeared worthwhile to isolate the TPS1 gene of this yeast and to analyze the effects of its disruption. The isolation of this gene presents also a potential technological interest as in Aspergillus niger the degree of expression of the tpsA gene that encodes T6P synthase, influences the rate of citric acid production Y. lipolytica excretes this acid in some conditions Y. lipolytica has a single gene encoding T6P synthase, that its disruption does not preclude growth in glucose but decreases sporulation efficiency and slows down growth at 35°C. In addition we report that disruption of YlTPS3 abolishes the increase of trehalose observed during heat shock.Trehalose, a non-reducing disaccharide formed by two glucose units, has important and varied functions in different organisms Y. lipolytica was cultured in a synthetic medium with 0.17% yeast nitrogen base without amino acids and ammonium sulfate  and 0.1% glutamate pH 6. S. cerevisiae was cultured similarly but using ammonium sulfate as nitrogen source. Auxotrophic requirements were added at a final concentration of 20 µg/ml and 2% glucose was generally used as carbon source. Liquid cultures were shaken at 30°C. Sporulation medium was based in commercial V8 drink essentially as The yeasts strains used are shown in Y. lipolytica cDNA library under the control of the S. cerevisiae PGK1 promoter in plasmid pFL61 Y. lipolyticaY. lipolytica and in S. cerevisiae. Primers used in PCR reactions are shown in A S. cerevisiae were constructed:The following plasmids for YlTPS1 gene was isolated from a cDNA library S. cerevisiae tps1 strain.pCLF1 carrying the YlTPS1, was constructed as follows. The BamHI fragment from plasmid pAN10 S.cerevisiae ADH1 was inserted into pRS316 BamHI. A 1.5 kb blunt-ended NotI fragment with YlTPS1 from pCLF1 was inserted in the blunt-ended HindIII site of this plasmid.pCLF2, a centromeric plasmid that carries YlTPS3 under the control of the ScADH1 promoter. YlTPS3 (YALI0E31086) is annotated as an intron containing gene. Using the FirstChoice RLM-RACE Kit (Ambion) we checked the correctness of the ATG and the cDNA predicted sequence. Primer design to amplify the cDNA from genomic DNA was based on the fact that the first exon is only 23 bp long. Primer 1006 covers the first exon and the first 19 bp of the second exon; together with primer 1007 produced a PCR product of 3168 bp containing the cDNA of YlTPS3. This product was cloned in the pCR-Blunt vector (Invitrogen) and the resulting plasmid was digested with NotI and SpeI blunt-ended and cloned in pDB20 URA3 marker had been substituted by LEU2. The cDNA of YlTPS3 was sequenced again when introduced in this plasmid.pCLF7 expresses Y. lipolytica were constructedThe following plasmids for Y. lipolytica TPS1 gene and was isolated by screening a Y. lipolytica genomic library YlTPS1 probe.pCLF3 carries a fragment of 5.4 kb that contains the YlTPS1 under the control of its own promoter. It was constructed as follows: the YlURA3 marker in plasmid pCL49 YlLEU2 to give pCL49L. The SphI-BamHI fragment of pCL49L was replaced by a 2.8 kb HpaI-BamHI fragment from pCLF3 bearing the YlTPS1 ORF and 1 Kb of upstream sequence.pCLF4 expresses YlTPS1 (a 1.5 Kb NotI fragment) from plasmid pCLF1 under the control of the YlTEF1 promoter in plasmid pCL49L.pCLF5 carries the coding region of YlTPS1 promoter to lacZ. A 1214 bp DNA fragment that includes the 14 initial amino acids of YlTPS1 was obtained by PCR, using oligonucleotides 1010 and 1011 cloned into pGEM-T easy, digested with NotI and BamHI and inserted into plasmid pINA354B ApaI to direct integration into the YlLEU2 locus. Correct integration was checked by PCR and Southern analysis.pCLF8 carries a fusion of the ScTPS1 obtained by PCR using primers 1012 and 1013 in plasmid pCL49L.pCLF9 carries the coding region of SceI restriction site and a deletion of 658 bp were created. Primers 1001 and 1002 with complementary ends including the recognition site of meganuclease I-SceI, 1000 and 1003 were used. With pCLF3 as template and the mentioned primers two fragments corresponding to the 5′ and 3′regions of the disruption cassette were obtained. These products were used as template with primers 1000 and 1003 in a PCR reaction to obtain a disrupted YlTPS1 copy. The product was cloned into pGEM-T easy, digested with I-SceI and ligated to a 1.2 kb YlURA3 fragment flanked by I-SceI sequences from plasmid pINA-URA3-I-SceI NotI was used to disrupt the chromosomal copy of YlTPS1 gene.In two consecutive PCR reactions a I-YlTPS3 gene was obtained by PCR from genomic DNA and primers 1004 and 1005 and cloned in pGEM-T easy (Promega). The resulting plasmid was digested with XhoI, to remove a fragment of 758 bp, and ligated to a 2.1 kb band containing the YlLEU2 gene from a NcoI digestion of the plasmid pINA62 YlTPS3 was disrupted using a 4996 bp NotI fragment from the previous plasmid. Correct disruption was checked by PCR.A piece of 3611 bp containing the YlNTH1) was obtained by PCR using primers 1008 and 1009 and cloned in pGEM-T easy. The resulting plasmid was digested with NheI and XhoI to substitute an internal 816 bp fragment of the NTH1 gene with a 2.1 kb fragment containing the YlLEU2 gene from plasmid pINA62 NcoI. A 3342 bp NotI fragment from this construction was used to disrupt the chromosomal copy. Correct disruption was checked by PCR and Southern analysis.A fragment of 2049 bp containing YALI0D15598 Y. lipolytica was extracted from flash-frozen cells Y. lipolytica CLIB122 genomic sequence. Total RNA was reverse-transcribed into cDNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). The cDNA levels were then analyzed using a LightCycler 480 from ROCHE and the LightCycler 480 SYBR Green I Master mix (Roche) with each primer at 250 nM. Each sample was tested in triplicate. After completion of the RT-qPCR melting-curve data were collected to verify PCR specificity, the absence of contamination and primer dimers. The gene YALI0F27533 (ARP4) was used to normalize the data.Total RNA from 2, 1 mM EDTA, 10 mM glucose 6-P and 5 mM UDP-glucose. Samples were taken at different time intervals and boiled for 3 minutes to stop the reaction. The UDP formed was measured spectrophotometrically in a coupled assay with NADH, pyruvate kinase and lactate dehydrogenase. A blank without added glucose-6P was run in parallel for each sample. Hexokinase and glucokinase were assayed with glucose and fructose as in 4, 40 mM β mercaptoethanol, and 2.6 mM 2-nitrophenyl β-D- galactopyranoside as described by Wallenfels Cell free extracts were prepared by breaking the yeasts in buffer with glass beads in six cycles of 1 min of vortexing and 1 min on ice. The buffer was 20 mM imidazole pH 7, with the addition of 1 mM DTT and 1 mM EDTA when T6P synthase was assayed. The extract was centrifuged in the cold for 15 min at 13000 rpm in an Eppendorf table top centrifuge and the supernatant used for determination of enzyme activities. T6P synthase activity was determined by a two step method The cultures were harvested by centrifugation, washed with water and frozen until use. Trehalose was extracted with boiling water as described in Yeast cells were quickly filtered through a Millipore AAWPO4700 and snap frozen in liquid nitrogen. The frozen pellets were dropped in boiling ethanol and treated as in Nucleotide accession number.- The sequence of the YlTPS1 cDNA was deposited at the GenBank database with the accession number AJ011032.S. cerevisiae tps1 mutant TPS1 gene from Y. lipolytica. We transformed a S. cerevisiae tps1 mutant strain with a cDNA library from Y. lipolytica under the control of the S. cerevisiae PGK1 promoter  TPS1 in this yeast. Attempts to disrupt YlTPS1 placing the disruption cassette after nucleotides 188 or 406 after the ATG failed, only when it was displaced to nucleotide 710 a correct disruption was obtained  exceeded that of the gene encoding hexokinase  (Lack of growth in glucose of obtained . We attrFC1 gene . Lack ofxokinase . Also enpolytica . Since gactivity . Concentactivity  in contrriments) . No immeY. lipolytica grown in glucose up to stationary phase or in glycerol were below 1 nmol/mg dry weight. A possible explanation for this result could be that the genes encoding the trehalose biosynthetic pathway enzymes were not expressed during growth in glucose, therefore we measured the expression of those genes in Y.lipolytica. In the Génolevuress database S. cerevisiae TPS2 and YALI0E31086 shows the highest homology with S. cerevisiae TPS3/TSL1. All these genes were expressed during growth in glucose although the levels of YlTPS2 and YlTPS3 were low when compared to that of YlTPS1 . This treatment was considered a strong one as this yeast does not grow at temperatures over 35°C. We measured the levels of mRNA corresponding to the genes related with trehalose metabolism after heat treatment . Relative abundance of mRNAs corresponding to YlTPS2 and to YlTPS3 increased 4 and 6 times respectively with the heat treatment. The higher increase of YlTPS3 suggested an important role for this gene in the heat response. A disruption of YlTPS3 abolished trehalose accumulation upon heat treatment raising the question that Tps3 could be another T6P synthase an hypothesis that cannot be ruled out due to the similarity between the YlTPS1 and YlTPS3 sequences. This possibility was made unlikely by the absence of trehalose in a Yltps1 mutant after heat shock and by the lack of complementation of the glucose negative phenotype of a S. cerevisiae tps1 mutant by the YlTPS3 cDNA  shows high similarity in its zinc finger domain to that of Msn2/4 and also binds STRE sequences Y. lipolytica database for genes encoding homologues of ScHSF1 yielded gene YALI0E13948. Levels of mRNA corresponding to those genes increased about 3 times after heat shock (In at shock  consisteY. lipolytica diploids homozygous for the tps1 mutation (CJM 724) were placed in sporulation conditions the sporulation frequency was reduced with respect to that of wild type (CJM 722) or heterozygous TPS1/tps1 (CJM 723) diploids. A similar behaviour in tps1/tps1 diploids in S. cerevisiaeMCK1, a gene that stimulates expression of IME1 which encodes a transcriptional activator of sporulation S. cerevisiae MCK1. We have measured the levels of mRNA corresponding to YALI0D20966 and to the genes implicated in trehalose metabolism both in a wild type diploid and in one homozygous for the tps1 mutation in sporulation conditions. After 8 days on sporulation medium the level of YALI0D20966 mRNA in a wild type was increased about 15 times while in a homozygous tps1/tps1 strain it reached a maximum of 3 fold  in their promoters S. cerevisiae requires the Msn2/Msn4 proteins Y. lipolytica. Hurtado and Rachubinsky in vitro. These authors reported that the levels of MHY1 mRNA were not increased after a heat shock at 35°C. Our results show that upon a heat shock at 40°C the levels of MHY1 mRNA increase suggesting that MHY1 may play a role in the regulatory response to this stress. It should be noticed that the high GC content in the Y. lipolytica DNA Heat shock increased the levels of trehalose and changed the levels of mRNA corresponding to Y. lipolytica does not grow at temperatures over 35°C. The finding that disruption of YlTPS1 impairs growth at this limit temperature suggests that trehalose plays a protective role against the changes produced under this condition C. neoformans and C. gattii show important variations in the effects caused by perturbations of that pathway Y. lipolytica that separated early in evolution from other yeasts Many evidences show that in different organisms the trehalose biosynthetic pathway, in addition to its primary role, has an influence in a variety of processes that range from growth on certain substrates or temperatures, to differences in virulence in pathogens. The targets of the pathway are different depending on the organism and even closely related yeast species like Figure S1Growth of wild type and Yltps1 strains. The strains were grown as described in Yltps1) and CLF279 (Yltps1/pCLF4). A representative curve is shown for each strain.(TIF)Click here for additional data file.Table S1Primers used for DNA cloning.(DOC)Click here for additional data file.Table S2Primers used in RT-qPCR.(DOC)Click here for additional data file.


EYA1, SIX1 and SIX5 genes have been associated with BOR syndrome. In this study, clinical and genetic analyses were performed in patients with BOR/BO syndrome focusing on auditory manifestations and rehabilitation.Branchio-oto-renal (BOR) or branchio-otic (BO) syndrome is one of the most common forms of autosomal dominant syndromic hearing loss. Mutations in EYA1, SIX1, and SIX5 genes.The audiologic manifestations were reviewed in 10 patients with BOR/BO syndrome. The operative findings and hearing outcome were analyzed in patients who underwent middle ear surgeries. The modality and outcome of auditory rehabilitation were evaluated. Genetic analysis was performed for EYA1 mutations and a large deletion encompassing the EYA1 gene.All patients presented with mixed hearing loss. Five patients underwent middle ear surgeries without successful hearing gain. Cochlear implantation performed in two patients resulted in significant hearing improvement. Genetic analysis revealed four novel EYA1 mutations may add to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population.Auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate after middle ear surgeries. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel  Mutations of the EYA1 gene are found in approximately 40% of patients with BOR syndrome EYA1 gene, mutations in the SIX1 and SIX5 genes have been reported to cause BOR phenotypes, although the pathogenic role of the SIX5 gene has been questioned recently SIX1 mutations have been shown to disrupt the EYA1-SIX1-DNA complexes Branchio-oto-renal (BOR) syndrome (OMIM 113650) or branchio-otic (BO) syndrome (OMIM 602588) is one of the most common forms of autosomal dominant syndromic hearing loss with an incidence of 1∶40,000 and is responsible for causing 2% of profoundly deaf children EYA1 gene located on chromosome 8q13.3 encodes a transcriptional co-activator required for eye morphogenesis which consists of three isoforms  and four transcript variants (EYA1A–1D) as a result of alternative splicing The EYA1 gene have been associated with BOR/BO syndrome, and frameshift or nonsense mutations are the most commonly detected mutations, followed by splice-site and missense mutations EYA1 gene that are not detected by direct sequencing of the coding region EYA1 mutations causing BOR syndrome and most of the mutations are unique to individual families To date, approximately 160 mutations of the et al.et al.Hearing impairment, the most common phenotypic feature of BOR syndrome, is found in various forms among which mixed type of hearing loss is most frequently reported (50%), followed by conductive (30%) and sensorineural type (20%) EYA1, SIX1, and SIX5 genes are reported.Because of the conductive component of hearing loss identified in most cases of patients with BOR syndrome, attempts have been made to improve hearing through middle ear exploratory tympanotomy and ossicular reconstruction. However, there are only several studies dealing with hearing outcome of exploratory tympanotomy in patients with BOR syndrome mostly reported before the identification of genetic causes of BOR syndrome, which demonstrated unsatisfactory results et al.Seven families (10 patients) including one multiplex family showing hearing loss and one or more of the typical features of BOR syndrome were included in this study for clinical and genetic analyses. The clinical diagnosis as typical BOR syndrome was made when the clinical criteria proposed by Chang Serial pure tone audiometries were performed in all patients and the various clinical manifestations regarding hearing loss were carefully reviewed. The type, degree, onset, progressiveness and/or fluctuation of hearing loss were evaluated in each patient. The threshold of pure tone audiometry was defined as the average of thresholds at 500, 1000, 2000, and 3000 Hz. The follow-up period of audiologic evaluations ranged from 3 months to 7.5 years. Speech audiometry and language evaluations were carried out in some of the patients whenever possible.The temporal bone CT scan was performed in all of 10 patients and temporal magnetic resonance imaging (MRI) was available for analysis in 4 patients. The temporal bone CT scan was performed with a 16 multidetector row CT scanner  using a standard temporal bone protocol. Contiguous 0.7-mm scans of the temporal bone were acquired in the axial plane and reformatted coronally with 1.0-mm increments. CT images were performed, digitally stored, and displayed by using the Picture Archiving Communication System (PACS) .MRI was acquired by using a 3.0-T  or 1.5-T system  with a six-channel sensitivity encoding (SENSE) head coil. The targeted parasagittal scan perpendicular to the long axis of the internal auditory canal was obtained with T2-weighted three-dimensional (3-D) turbo spin-echo (TSE) sequence with driven equilibrium RF reset pulse (DRIVE), following routine MR sequences with spin-echo T1- and T2-weighted images. The sequence parameters for the T2-weighted 3-D FSE sequence with DRIVE were as follows: repetition time (TR)/echo time (TE) = 1500/200 ms, 256 acquisition/256 reconstruction, 15-cm field of view, 1.5-mm section thickness with a 0.75-mm overlap, number of acquisitions = 2, and the scan time was less than 5 minutes.The morphologies of the cochlea, vestibule, semicircular canals, internal auditory canals, vestibular aqueduct, and middle ear structures were analyzed on temporal bone CT scans. On temporal MRI, abnormalities of the brain, the cochleovestibular and facial nerves, as well as the endolymphatic duct and sac were evaluated.EYA1, SIX1 and SIX5 gene were amplified by Polymerase Chain Reaction (PCR) with specific primers (http://frodo.wi.mit.edu/). PCR with H taq polymerase  proceeded as following cycles: 15 minutes at 95°C, repeated of 30–40 cycles of denaturation at 94°C for 20 seconds; annealing at * °C for 40 seconds; extension at 72°C for 30 seconds (* is depended by melting temperature of primers). Last extension step was performed at 72°C for 5 minutes. Particularly, exons 1 and 2 of SIX5 were amplified using LA taq , because these regions have high GC contents. PCR products were separated on 1.5% agarose gel. The PCR products were purified using Shrimp alkaline phosphatase  and exonuclease I at 37°C for 70 minutes and directly sequenced using the Bigdye Terminator v3.1 Cycle Sequencing Kit . Ethanol precipitation was used for purification of sequencing reaction products before running the samples on the 3130xl Genetic Analyzer . The data was analyzed utilizing Sequencing analysis v5.2  and Chromas Pro v1.5 software . Multiple alignments of the analyzed sequences were performed using CLC sequence Viewer v.6.0 software .All exons and exons-intron boundaries of  primers  designedBamHI and EcoRI were inserted into multiple cloning sites between exon A and exon B in pSPL3 or pSPL3b vectors. For in vitro splicing assay, HeLa cells were cultured in DMEM  at 37°C in 5% CO2 concentration. Prior to transfection, HeLa cells were seeded at a density of 2.7×106 on a 60 mm culture dish. Hybrid minigenes in pSPL3 or pSPL3b vector were transiently transfected into HeLa cells using Fugene 6 Transfection reagent  at 3 µL per µg of DNA, and the transfected cells were harvested 24 hours after transfection. Total RNA was extracted from the transfected HeLa cells using an RNeasy Mini Kit  according to the manufacturer’s protocol. Approximately 1 µg of total RNA was reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit . The cDNA was used as a template for PCR amplification of pSPL3 or pSPL3b vector-specific primers SD6 and SA2. The size of amplified normal and mutant fragments was confirmed on 1.5% agarose gel by electrophoresis.To analyze the splicing pattern, minigene vector was manufactured to include each exon along with about 300 bp of 5′- and 300 bp of 3′- intronic flanking regions . Amplified wild or mutant type products digested with EYA1 kit  that was used includes 17 probes for 14 of the 18 EYA1 exons  and 14 control probes. MLPA was performed according to the manufacturer’s instructions: denaturation at 98°C for 5 minutes; stabilization at 25°C; hybridization at 95°C for 1 minute and at 60°C for 16–20 hours, stabilization at 54°C; ligation at 54°C for 15 minutes, inactivation at 98°C for 5 minutes, and stabilization at 20°C. PCR was carried out as follows: 35 cycles of denaturation at 95°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 72°C for 1 minute. Final extension step was performed at 72°C for 20 minutes. Amplified products containing GeneScan™-500 LIZ® Size Standard  and Hi-Di Formamide  were separated and quantified by capillary electrophoresis on the 3130xl Genetic Analyzer , and the data were analyzed using GeneMarker software v1.6 .Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to detect copy number variations such as deletions or duplications. The SALSA MLPA probemix P153-A2 EYA1 gene were selected from the NCBI database (www.ncbi.nlm.gov) considering their heterozygosity. PCR with H taq polymerase was performed using fluorescently tagged primers. Two PCR products of different fragment size and 0.1 µL of GeneScan™-500 LIZ® Size Standard  were mixed and diluted with 8.9 µL of Hi-Di Formamide . Final diluted products were separated and detected by using ABI 3130x genetic analyzer. GeneMapper v4.0 software  was used to analyze genotypes for each marker.Four microsatellite markers, D8S1795, D8S1060, D8S1807 and D8S570, in the region of 2 Mb including the et al.Seven families including 10 patients were analyzed. All of the patients were females and their age at the time of diagnosis ranged from 1 to 43 years . The cliResults of renal manifestations were evaluated in 4 of 10 patients. In one patient (patient 9), a tiny cyst was seen in the right renal cortex and mild pelvic dilatation of the right kidney was identified by renal ultrasonography without any evidence of renal dysfunction. The other three patients did not reveal any abnormality on renal ultrasonography or blood testing.SLC26A4 mutations  on normal splicing, each exon and the flanking intronic sequences sufficient to allow splicing were inserted into a pSPL3 or pSPL3b vector. Each vector was transfected into the HeLa cells, and transcribed to mRNA in the cells. All three splice site mutations were found to disrupt the normal splicing . Exons 1EYA1, SIX1 and SIX5) by direct sequencing, and the results were compared to the data acquired in one normal control with normal hearing confirmed by pure tone audiometry. The entire EYA1 gene was suspected to be deleted in patient 8 . The results of the MLPA indicated that patient 8 had a heterozygous deletion encompassing the whole EYA1 gene. To investigate the deleted region including EYA1 in patient 8, genotype analysis using microsatellite markers was performed. Only patient 8 revealed homozygous alleles for all 4 markers, while all 12 normal controls showed heterozygous alleles for at least one of the microsatellite markers  who were found not to carry any mutations in the three genes  which is higher than 40% previously reported by Chang et al.EYA1 gene. In the Korean population, there have been only 3 case reports of patients with BOR/BO syndrome carrying EYA1 mutations, and this study adds to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population SIX1 mutations associated with BOR/BO syndrome, more than 10 mutations have been identified worldwide, while there is only one missense mutation reported in the East Asian population SIX1 or SIX5 mutation was found in this study suggesting the limited role of SIX genes as the cause of BOR/BO syndrome in the East Asian population. Since a large deletion was identified by MLPA in one patient with BOR/BO syndrome, we suggest that additional studies should be performed to identify complex rearrangements or large deletions when no mutation is found by conventional sequencing techniques.In contrast to the western population where large cohort studies have been performed on BOR/BO syndrome, limited information is available concerning genetic mutations of this syndrome in the East Asian population, and a total of 16 mutations in the EYA1 gene was identified in a patient presenting with only mixed hearing loss and enlarged vestibular aqueduct (patient 7) EYA1 gene (patient 8) compared to the patients with intragenic EYA1 mutations.No clear genotype-phenotype correlation could be demonstrated in this study in accordance with previous reports, in which no mutation was found in a patient with all major features of BOR syndrome (patient 9) whereas a splice site mutation of the et al.et al.eya1 in the sensory hair cells continued after birth and after maturation (P16 in mouse), suggesting an additional role for eya1 in the differentiation and/or survival of the inner ear cell populations in particular the sensory cells EYA1 mutations considering the possible role of EYA1 in the maintenance and survival of the hair cells and the supporting cells. Therefore, we believe that the possibility of hearing progression should be explained to the patients with BOR/BO syndrome, and regular auditory tests should be performed in order to treat these patients with proper modality of auditory rehabilitation at an appropriate timing.The progression of hearing loss was variable in patients with BOR/BO syndrome included in this study. Cremers eya1 has been reported to be expressed in the mesenchyme surrounding the cartilage premordia of all three ossicles and also in the epithelium of the tubotympanic recess which later develops into the tympanic cavity at E13.5, meaning that mutation in the EYA1 gene can disrupt normal development of the middle ear in various aspects This study clearly demonstrated the limitation of middle ear surgeries for hearing improvement in patients with BOR/BO syndrome consistent with previous reports EYA1 gene is involved in the development of the spiral ganglion and cochlear hypoplasia often seen in patients with BOR/BO syndrome has been known to be a poor prognostic factor of cochlear implantation, successful outcome in terms of speech and auditory performances could be achieved in our patients after cochlear implantation Since patients failed to gain hearing after one or more middle ear surgeries, most of the patients in this study used bilateral hearing aids. In two of the patients with severe to profound hearing loss who could not benefit from hearing aids, cochlear implantation was performed. In patients with syndromic hearing loss, multiple factors have to be considered regarding auditory rehabilitation, including combined mental retardation and developmental delay in addition to various inner ear malformations. Cochlear implantation in patients with syndromic hearing loss is challenging in both surgical and audiologic aspects. Although good results of cochlear implantation have been reported in these patients, some of the syndromes associated with cochlear nerve deficiency or narrow internal auditory canals such as CHARGE syndrome have shown limited outcome EYA1 gene in Korean patients with BOR/BO syndrome, the mutational analysis of EYA1 should be an integral part of the diagnosis of BOR/BO syndrome in the East Asian population. The characteristic inner ear and middle ear anomalies and mixed type hearing loss may also provide clinical clues to suspect BOR/BO syndrome even in the absence of other typical clinical features. The management of hearing loss and auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate of hearing gain achieved by middle ear explorations in patients with mixed hearing loss. Hearing aids are good options in patients with mild to severe hearing loss, but regular hearing evaluations are needed considering the possibility of progression of hearing loss in order to treat these patients with proper modality of auditory rehabilitation at an appropriate timing. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel EYA1 mutations identified in this study adds to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population and the clinical results of this study may provide evidence for recommending proper means of auditory rehabilitation in patients with BOR/BO syndrome.Considering the high mutation rate of the Figure S1Temporal bone CT and temporal MRI findings of patient 10 demonstrating enlarged vestibular aqueduct in a circular shape. (A–F) Axial view of temporal bone CT shows enlarged vestibular aqueduct (black arrows) observed as a circular shape with a diameter significantly larger than that of the posterior semicircular canal (black arrowhead in (TIF)Click here for additional data file.Table S1Primer information of EYA1, SIX1 and SIX5.(DOC)Click here for additional data file.Table S2Identification of EYA1 deletion by microsatellite marker.(DOC)Click here for additional data file.


These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca LRRK2 is a ∼280 kDa protein with two enzymatic domains (Ras of complex GTPase domain and kinase domain) and several protein-protein interaction domains such as an amino terminal leucine-rich repeat domain and a carboxy terminal WD40 domain Despite the importance of LRRK2 for the pathogenesis in various diseases little is known about the cellular function of LRRK2. LRRK2 has been implicated in many different signaling pathways such as membrane trafficking 2+ concentration ([Ca2+]c) being at center stage 2+]c2+ influx via vesicular P2X4 receptors which promotes fusion pore expansion and subsequently facilitates surfactant release During LB exocytosis a sequence of highly regulated steps leads to fusion of exocytic vesicles with the plasma membrane, subsequent opening of a fusion pore and finally content release. Several intracellular signalling cascades stimulate LB fusion with the plasma membrane during the exocytic pre-fusion phase 2+ release from intracellular stores upon stimulation with ATP, which promotes triggering of LB exocytosis. However, using a biochemical approach to quantify surfactant secretion we found that surfactant release to the extracellular space was decreased after stimulation. This was not due to failure of actin coat formation and active extrusion of vesicle contents during the exocytic post fusion phase. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signalling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Within this study we investigated whether morphological changes of LBs observed in LRRK2 -/- mice are also present in LRRK2 -/- rats and whether they affect LB exocytosis and surfactant secretion. We measured single LB fusion events with the plasma membrane using high resolution, real-time fluorescent microscopy. ATII cells are particularly suitable for analysing single exocytotic events due to large size of secretory vesicles and slow, sequential vesicle fusion with plasma membrane. LB exocytosis has been intensively investigated in the past decade and there are several established microscopy methods for detection of single vesicle fusion events Rats were maintained at the central animal facility of the Ulm University according to institutional guidelines for ethical care of animals. All experiments in this study were approved by Regierungspräsidium Tübingen, grant Nr. 833.tm1sage, product number: TGRL4620) as well as age- and gender-matched wildtypes (Long-Evans). The age of animals ranged between 5 and 9 weeks.LRRK2 -/- rats were purchased from SAGE Labs , seeded on chamber slides , and used for experiments for up to 48 h after seeding.Alveolar type II cells were isolated from wt and LRRK2 -/- rats according to the procedure of Dobbs et al. Cells were washed twice in ice-cold DPBS  fixed for 20 min in 4% paraformaldehyde  in DPBS and permeabilised for 10 min with 0.2% saponin and 10% FBS  in DPBS. Cells were subsequently stained with primary (1∶300) and secondary (1∶400) antibodies in PBS, 0.2% saponin and 10% FBS for 30 min. Images were taken on an inverted confocal microscope  using a 63× lens (Leica HCX PL APO lambda blue 63.0×1.40 OIL UV). Images for the blue (DAPI), green (AlexaFluor 488), red (AlexaFluor 568) and far red (AlexaFluor 633) channel were taken in sequential mode using appropriate excitation and emission settings. All used primary antibodies were from Abcam  apart from antibodies against surfactant proteins B and C which were a gift from T. Haller . AlexaFluor phalloidin 568 and all conjugated secondary antibodies were from Invitrogen.2+]c and detection of individual LB fusion events was performed as described previously 2, 2 CaCl2, 5 glucose, 10 Hepes; pH 7.4) and kept in bath solution with 0.5 – 1 μM of FM 1–43. To efficiently induce LB exocytosis ATII cells were treated with various known and potent agonists for surfactant secretion: ATP (100 μM), PMA (300 nM), a combination of ATP and PMA, or ionomycin (1 μM) . The combined application of ATP and PMA was used because the previous report showed that the combined application can further increase LB fusion probability 2+]c as described in detail earlier The fura-2/FM1-43 assay for combined measurement of changes in the c = Kd×[(R−Rmin)/(Rmax−R)]×(Sf2/Sb2).We estimated the intracellular free Camin was measured after Ca2+ ionophore ionomycin (20 µM) was added to a Ca2+ free perfusion solution . Rmax was measured after ionomycin (20 µM) addition to Ca2+-containing perfusion solution . The proportionality coefficient Sf2 was measured as the maximal 380 nm fluorescence intensity after ionomycin addition to Ca2+-free solution. The second proportionality coefficient, Sb2, was measured as the minimal 380 nm fluorescence intensity after ionomycin addition to Ca2+-containing solution. We corrected both coefficients for the background fluorescence which was measured after MnCl2 quench  at the end of the experiment. We used 224 nM as a Kd value for fura-2 ATII cells were seeded in perfusion chambers  and stained with fura-2 AM (3 µM for 20 min). Ratio (R) was calculated from 340/380 nm excitation intensities in unstimulated cells in normal experimental bath solution. RATII cells were incubated with 100 nM LysoSensor Yellow/Blue (Invitrogen) at 37°C for 30 min. Cells were washed with experimental bath solution and imaged on the Zeiss Observer microscope  and 40× fluar oil objective using Metafluor software. After background subtraction, LB intensities at 340 and 380 nm excitation were measured in ImageJ and the 340/380 nm ratio was used for pH calculation.10((a−b)/(ratio−b)−1), where a, b and c are constants.Calibration of the ratio to pH relation was done as described previously We used Microsoft Excel and GraphPad Prism  for statistical analysis. Unless stated otherwise data are expressed as mean +/− SEM. n denotes true biological replicates  and non-paired student's t-test was used to assess the statistical differences between wt and LRRK2 -/- cell populations. Unless stated otherwise the number of experiments performed/cells analysed in each biological replicate was kept constant to account for correct weighting of individual biological replicates when generating means. Due to the low number of fusions/cell (on average 1–2 per responding cell) and within individual experiments (6–8 responding cells) fusion delay histograms were derived from pooled data from 3 to 6 animals. A non-parametric Mann-Whitney test was used to compare the medians of fusion delays after stimulation.The previous histological study of the lungs from LRRK2 -/- mice showed enlarged size and number of LBs in ATII cells Expression and localization of LB markers were not changed. LB membrane marker proteins Lamp1 and ABCa3 transporter were localized to the LB membrane in LRRK2 -/- as well as in wt cells  [38]. SuTo further investigate whether the observed increase in LB size is linked to changes in lamellar body homeostasis we also performed experiments to assess the pH in LBs from wt and LRRK2 -/- cells using the dye LysoSensor. No significant difference (p = 0.45) between the vesicular pH in LBs from wt animals  and LBs from LRRK2 -/- animals  could be observed. Approx. 150 to 250 LBs were analyzed per animal.3 mediated calcium release from internal calcium stores resulting in a “triggered” fusion response. PMA on the other hand acts via activation of protein kinase C without affecting intracellular Ca2+ concentrations and enables a more prolonged response to stimulation whereas ionomycin acts as an ionophore and causes massive Ca2+-entry from the extracellular space 2+ (ionomycin) signaling pathways and result in comparable activity responses  increased fusion response compared to wt cells   . When anctively) . These d2+]c using fura-2 while monitoring individual LB fusion events  in LRRK2 -/- cells and 0.49±0.19 µM (n = 2) in wt cells, respectively (p = 0.08) . However animals . Mean pe = 0.08) .Based on our finding that knock-out of LRRK2 results in a significantly enhanced LB fusion response in ATII cells when stimulated with 100 µM ATP, we next examined whether this also results in increased surfactant secretion. We analyzed the amount of secreted phospholipids (DPPC is the main component of surfactant) using a recently described enzymatic protocol To further elucidate these conflicting results – increased number of responding cells but reduced surfactant secretion in LRRK2 -/- cells – we investigated the potential impact of altered LRRK2 expression on the regulation of surfactant secretion during the post-fusion phase of LB exocytosis. The lipophilic nature of surfactant impedes rapid dispersal in aqueous solution and therefore surfactant does not readily diffuse out of fused LBs following opening of the exocytic fusion pore. We already showed that actin coating of fused LBs and subsequent actin coat compression play a pivotal role in surfactant expulsion from the vesicles Although LRRK2 was suggested to play a role in variety of diseases its cellular role remains elusive. Recent evidence implicated LRRK2 in regulation of secretory vesicle trafficking The original observation on increased LB size was made in LRRK2 -/- mice 2+]c2+ was described as a final trigger for LB fusion in ATII cells, the threshold necessary for fusion in ATII cells is very low (∼320 nM), and fusion kinetics correlate with [Ca2+]c kinetics 2+]c is not significantly elevated in LRRK2 -/- cells suggests that the increased rise in [Ca2+]c upon stimulation with ATP is the main effector of the increased fusion response in these cells. It is easily conceivable that in LRRK2 -/- cells the threshold for LB fusion is reached more readily and exceeded for prolonged times and hence more cells exhibit LB fusions upon stimulation with ATP. Such a model is also supported by the observation that in LRRK2 -/- cells the exocytic response following ATP treatment is similar to the response following stimulation that causes maximum elevation of [Ca2+]c and activation of Ca2+-dependent fusion activity (ionomycin). The increase in the rise of [Ca2+]c upon ATP treatment in LRRK2 -/- cells could be due to altered Ca2+ levels in intracellular Ca2+ stores or increased Ca2+ release kinetics. It is unlikely to result from altered Ca2+ extrusion mechanisms as the time-course of the [Ca2+]c decay is unchanged. Effects of LRRK2 on Ca2+ homeostasis and Ca2+ signaling have already been reported in other cell types 2+ homeostasis 2+ release from lysosomal Ca2+ stores 2+ release from intracellular stores. This difference could be due to the different signaling mechanism underlying Ca2+ mobilization in the different systems. In ATII cells ATP elicits Ca2+ release via activation of IP3 receptors 2+ mobilization 2+ release in LRRK2 -/- cells following stimulation with ATP likely facilitates or accelerates fusion of LBs that are already localized close to the plasma membrane 2+]c it barely affects recruitment or trafficking of additional LBs to the plasma membrane for fusion .Stimulation of primary ATII cells with 100 µM ATP resulted in a significantly increased percentage of cells that responded to stimulation with exocytic activity when LRRK2 expression was ablated. LB exocytosis is exceptionally sensitive to [Ca2+ independent LB fusion activity as PMA induced LB fusion response is unchanged in LRRK2 -/- cells. However, we cannot fully exclude the possibility that PMA already causes a maximum stimulatory effect that cannot be increased upon. It is well established that PMA causes a strong fusion response (i.e. more cells responding within a defined period of time after stimulation) due to the long lasting stimulatory effect. Hence the combined application of ATP and PMA, evoking a Ca2+ increase as well as direct stimulation of protein kinase C, does not necessarily need to exceed the PMA response LRRK2 -/- does not seem to affect Ca2+ signaling remains to be answered.The observed increase in exocytic activity in LRRK2 -/- cells is also in line with observations from cortical neurons where LRRK2 silencing increased fusion kinetics Shin et al showed that LRRK2 silencing in neurons leads to impaired compensatory endocytosis Although ATII cells from LRKK2 -/- rats contain larger LBs and these LBs had an increased propensity for fusion with plasma membrane upon physiological stimulation, the amount of secreted phospholipids following stimulation was lower than in wt cells. Therefore, we tested the possibility that the extrusion of secretory material might be impaired in LRKK2 -/- cells. Surfactant is a poorly soluble substance and the opening of the exocytic fusion pore is not sufficient for efficient surfactant release from the fused vesicle. Actin coating of fused LBs and myosin driven compression of this coat is necessary for active surfactant extrusion 2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP. However, further studies are required to fully elucidate the molecular mechanism how LRRK2 regulates intracellular Ca2+ release in these cells and whether LRRK2 affects surfactant loading of LBs. Results of these studies will also shed some more light on whether the observed effects of LRRK2 on exocytosis and secretion in other cell types are linked to changes in intracellular Ca2+ signaling.In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca


Ornithodoros tholozani specimens were tested with a Borrelia flaB-PCR. Results were positive for all patients and 2%–40% of ticks. A 7–amino acid gap characterized all 9 sequenced flagellin gene amplicons. By phylogenetic analysis, Israel TBRF Borrelia sequences clustered separately from American and African groups.Blood samples from 18 tickborne relapsing fever (TBRF) patients and  Tickborne relapsing fever (TBRF) is caused by Borrelia species and is transmitted to humans by Ornithodoros soft ticks. Worldwide, a dozen Borrelia species are known to cause this disease , and 88% of the case-patients were infected in caves (TBRF in Israel was first reported by Nicholson (Although American (2 traps in caves and were identified as Ornithodoros tholozani (We designed a genus-specific set of primers (BOR1: 5´ TAA TAC GTC AGC CAT AAA TGC 3´ and BOR2: 5´ GCT CTT TGA TCA GTT ATC ATT C 3´) that targeted the Borrelia flaB flagellin gene . Plasmids containing inserts were purified and sent for 2-strand sequencing. Direct sequencing of DNA amplified by the BOR1 and BOR2 primers was performed later.Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version 3.1 (Three PCR amplicons (from 1 tick and 2 human samples) were sequenced after cloning, whereas 6 amplicons (from 2 ticks and 4 human samples) were analyzed by direct sequencing. These 9 sequenced samples showed 98%–100% homology between them and could be divided into 3 groups. The same DNA sequence was found in tick TG52 and in blood from 2 patients, HumanBlood2 and HumanBlood4. These 3 sequences had an additional triplet at the position 627. The second group of sequences, which consisted of tick samples TGd1 and CBkc7 and blood samples C1025B, FL1, and HumanBlood3, were identical, with only 3 minor substitutions between them. The third group consisted of the HumanBlood1 sample.All the translated sequenced amplicons showed a very specific signature by the presence of a 7–amino acid (aa) gap at position 216  when comComparison with published flaB protein sequences of TBRF Borrelia showed 88%–90% homology with B. duttonii and B. recurrentis, 85%–90% with B. crocidurae, 86%–88% with B. turicatae, 87%–89% with B. hermsii, and 85%–88% with B. parkeri. The sequences of the Israel TBRF Borrelia isolated from different samples clearly clustered in a separate group from the American and the African TBRF species .Our results suggest that infection rates differ according to location, despite the small number of ticks tested and the use of pools. BOR1-BOR2 PCR was more sensitive than blood smear examination (100% vs 83%). An identical DNA sequence was found in both tick and patient samples and thus confirms, at the molecular level, the role of O. tholozani as the vector of TBRF in Israel.A signature (7-aa gap) of the flaB flagellin defined the Israeli TBRF sequences as a homologous group different from other TBRF species. Despite the small number of samples studied, a clear polymorphism existed also at the protein level, resulting in 3 local types. This diversity can be explained by the use of direct sequencing of samples rather than through cultivation that reduces the biodiversity of isolates by selecting the most successful in vitro clone.This study opens a new avenue in TBRF Borrelia studies by demonstrating a Middle East cluster in addition to the American and African groups. These results open encouraging perspectives for the better understanding of entomologic, epidemiologic, and bacteriologic aspects of this disease and may contribute to better diagnosis and treatment.


Despite various immunosuppressive agents, juvenile idiopathic arthritis (JIA) related uveitis often take a serious course that leads to sight threatening complications.To evaluate efficacy of Abatacept in combined treatment of JIA related uveitis.10 children aged 4 – 12 years with anterior uveitis associated with JIA were treated by Abatacept in standard infusions pattern and doses. Indication for using Abataceptwas ineffectiveness of standard therapy of arthritis and/or uveitis. 6 patients were suffering from polyarticular variants, 4 – oligoarticular JIA. Abatacept was combined with Methotrexate in 9, Sulfasalazin in 1 and low-dose steroids in 4 children. 3 patients were switched to Abatacept from Infliximab due to non-efficacy, 7 received abatacept as a first line biologic. 9 patients had bilateral eye involvement. At the administration of Abatacept severe inflammation was seen in 1, moderate in 7, mild in 2 of the cases. Follow up period ranged from 3 to 11 month (mean 7.6). The main outcome measure was the degree of inflammation.nd – 4th injection. From patients switched from Infliximab to Abatacept 1 achieved remission of uveitis, 1 improved, 1 remained stable. Patients with remission of uveitis diminished or discontinued topical medications. No ocular or systemic adverse effects were observed. Glaucoma or cataract surgery was uncomplicated in all 4 cases.Remission of uveitis in current treatment was achieved in 6 (60%), improvement in the degree of inflammation in 2 (20%) of the cases. The initial response was seen after the 2Administration of Abatacept was effective in 80% of children with JIA related uveitis. Further investigations are required to define clear indications for this treatment in severe uveitis.


Hox6 paralog group genes have been implicated as key determinants of LMC fate at forelimb levels of the spinal cord, through their ability to promote expression of the LMC-restricted genes Foxp1 and Raldh2 and to suppress thoracic fates through exclusion of Hoxc9. The specific roles and mechanisms of Hox6 gene function in LMC neurons, however, are not known. We show that Hox6 genes are critical for diverse facets of LMC identity and define motifs required for their in vivo specificities. Although Hox6 genes are necessary for generating the appropriate number of LMC neurons, they are not absolutely required for the induction of forelimb LMC molecular determinants. In the absence of Hox6 activity, LMC identity appears to be preserved through a diverse array of Hox5–Hox8 paralogs, which are sufficient to reprogram thoracic motor neurons to an LMC fate. In contrast to the apparently permissive Hox inputs to early LMC gene programs, individual Hox genes, such as Hoxc6, have specific roles in promoting motor neuron pool diversity within the LMC. Dissection of motifs required for Hox in vivo specificities reveals that either cross-repressive interactions or cooperativity with Pbx cofactors are sufficient to induce LMC identity, with the N-terminus capable of promoting columnar, but not pool, identity when transferred to a heterologous homeodomain. These results indicate that Hox proteins orchestrate diverse aspects of cell fate specification through both the convergent regulation of gene programs regulated by many paralogs and also more restricted actions encoded through specificity determinants in the N-terminus.A critical step in the assembly of the neural circuits that control tetrapod locomotion is the specification of the lateral motor column (LMC), a diverse motor neuron population targeting limb musculature.  Hox genes in motor neuron specification and patterns of muscle connectivity are poorly understood. We have found that members of the Hox6 gene paralog group  contribute to diverse aspects of motor neuron subtype differentiation. Hox6 gene activity is required during two critical phases of motor neuron development: first as motor axons select a trajectory toward the forelimb and second as they choose specific muscles to innervate. At the molecular level, these two functions are encoded by distinct peptide domains within Hox proteins. This work indicates that Hox proteins execute their critical functions in motor neurons through intrinsic modules that confer distinct specificities and that these activities are central in the genetic network required for motor neuron differentiation.Coordinated motor behaviors—as complex as playing a musical instrument or as simple as walking—rely on the ability of motor neurons within the spinal cord to navigate towards and establish specific connections with muscles in the limbs. The establishment of connections between motor neurons and limb muscles is mediated through the actions of genes encoding Hox proteins, a large family of transcription factors conserved amongst all metazoans. However, the specific requirements for  The neural circuits that govern locomotor behaviors rely on the establishment of orderly sets of connections between motor neurons (MNs) and their peripheral and central synaptic targets. A critical and early step in the emergence of locomotor circuitry is the selection of specific muscle targets by a diverse array of MN subtypes. Three organizational features of MNs emerge during embryonic development that contributes to the specificity of their connections with target cells. First, MNs that project axons to common peripheral targets are organized into columns longitudinally arrayed along the rostrocaudal axis of the spinal cord Hox genes are expressed by MNs, with subsets of related paralogs functioning at distinct levels of the MN differentiation pathway Hox6, Hox9, and Hox10 genes have been implicated in the early columnar organization of MNs and contribute to the specificity of their initial projections into the periphery Hox genes contribute to the specification of MN pools, in part, through the induction of intermediate transcription factors Within the developing spinal cord, Hox proteins exert central roles in the specification of MN columnar and pool subtypes Drosophila indicate two key mechanisms through which Hox proteins regulate target genes Studies in Hox gene, Hoxc9. Hoxc9 is required for the appearance of thoracic-level MN columnar subtypes including preganglionic column (PGC) and hypaxial motor column (HMC) neurons Hoxc9 all brachial Hox genes are derepressed at thoracic levels, and MNs acquire an LMC fate. This broad repressive activity appears to be mediated by direct interactions of Hoxc9 with multiple sites in the HoxA and HoxC loci. Genome-wide analysis of Hoxc9 binding revealed a consensus binding motif which matches a high affinity Hox/Pbx site (TGATTTAT) identified by several groups through in vitro site selection Some insights into the mechanisms by which Hox proteins regulate target genes in MNs have emerged through analysis of mice mutant for a single thoracically expressed Hox6 and Hox10 genes have been implicated in the initiation of the LMC program at brachial and lumbar levels respectively, through activation of the gene encoding the transcription factor FoxP1 Raldh2The problem of Hox specificity in MNs is particularly relevant at limb levels of the spinal cord, where individual neurons express multiple Hox proteins at the time of their differentiation Hox5 genes (Hoxa5 and Hoxc5) and Hoxc8, respectively; and the actions of these Hox genes are necessary for delineating the rostrocaudal position of MN pools Hox4–Hox8 proteins are thought to promote the intrasegmental diversity of MNs, by defining specific molecular codes for each pool subtype. For example, misexpression studies in chick have provided evidence that Hoxc6 is selectively required for the intrasegmental differentiation of pools within the caudal (Hoxc8+) half of the LMC Hox6 paralog group that determines the early columnar identity of forelimb-innervating MNs contains members that promote motor pool fates.While Hox proteins seem to be critical for LMC specification, it is less clear how they contribute to MN pool diversity. At brachial levels the LMC is broadly divided into rostral and caudal domains by expression of Hox6 genes during MN columnar and pool specification programs. First, what are the specific contributions of the three murine Hox6 genes to MN fate specification? Second, to what extent are the diverse activities of a Hox protein unique, or are they shared amongst gene paralogs within a cluster? Third, are there motifs intrinsic to Hox proteins that subfunctionalize in vivo specificities? To address these questions we analyzed mice in which all Hox6 genes are mutated, as well as employed an in vivo approach to dissect functional domains required for Hox specificity in MNs. We find that although LMC specification is retained in mice lacking Hox6 genes, Hoxc6 has a specific role in promoting MN pool identity and appropriate patterns of limb connectivity. The preservation of LMC fate in Hox6 mutants appears to be mediated by a diverse group of Hox5–Hox8 genes expressed at brachial levels. Dissection of a single Hox protein reveals in vivo specificity relies on motifs that ensure deployment of programs common to all LMC neurons, as well as distinct modules that contribute to MN pool identity.In this study we sought to address several unresolved issues concerning the function and specificity of Hox6 genes in the specification of LMC neurons at brachial levels of the spinal cord. Two Hox6 genes, Hoxa6 and Hoxc6, are selectively expressed by brachial MNs in chick, and can convert HMC and PGC neurons to an LMC fate when misexpressed at thoracic levels Hox6 activities are absolutely required for LMC specification in mice is not known. To begin to answer this question we first analyzed the expression of Hox6 paralogs  at brachial levels near the time of LMC differentiation at embryonic day (e) 11.5. Hoxa6 and Hoxc6 are expressed throughout the brachial LMC, while Hoxb6 is expressed by MN progenitors . Hox6 genes are therefore not required for the generation of MNs as a class or in maintaining Hox expression patterns.To assess the function of and Lhx3 . In addi mutants , likely Hoxa6/c6 mutants. We assessed the expression of Foxp1 and Raldh2, two genes that are induced downstream of Hox proteins Hoxa6−/−Hoxc6+/+, Hoxa6+/−Hoxc6+/−, and Hoxa6−/− Hoxc6+/− embryos the number of LMC neurons was similar to wildtype embryos, while both Hoxc6−/− and Hoxa6−/−Hoxc6−/− mutants displayed significant LMC losses  subtype, a motor neuron column normally present at thoracic levels Hoxa6/Hoxc6, MNs that fail to acquire an LMC fate revert to an HMC-like identity.We next determined the fate of the LMC neurons that are lost in Hoxb6 in MN progenitors could account for the maintenance of LMC identity in Hoxa6/Hoxc6 mutants we also analyzed mice in which all three murine Hox6 alleles are deleted. We found that in Hoxa6/Hoxb6/Hoxc6 triple mutants FoxP1+/Raldh2+ MNs were present, and LMC numbers were grossly similar to Hoxa6/Hoxc6 double mutants  the most prominently expressed by brachial MNs  Smad1/5/8  Pea3 mutants, motor axons project to the CM but fail to branch and arborize the muscle Hoxc6 mutants  muscle, whereas Scip+ MN pools project along the median and ulnar nerve  mutants –S6D. In  mutants –S6D, sugHoxc6 mutants many RhD+ Scip− neurons were observed  and the homeodomains of heterologous Hox proteins. Fusion of the Hoxc6 N-terminus to the homeodomain (HD) of the “LMC-neutral” Hox protein, Hoxc4 , activatc levels . Thus thss Hoxc9 . Similarpression . These opression .Hoxc9.Hox proteins are known to contain peptide motifs that confer activation and repression of target genes independent of the homeodomain Foxp1 is regulated by Hox proteins we searched for potential Hox sites within the Foxp1 locus. In silico analysis using the Vista enhancer browser Foxp1 transcription start site. This enhancer (Foxp1/hs1149) is highly conserved amongst vertebrates and drives high levels of expression at limb levels of the spinal cord, and lower levels thoracically , which generally conforms to the Hox/Pbx consensus hs1149 element is bound by Hoxc6 in vivo.We next used gel mobility shift assays to determine if Hox proteins could bind the hs1149 element, as well as an optimized Hox consensus binding site (Fkh250con*) Foxp1/hs1149 and Fkh250con*, although with slightly reduced affinity , possibly because Hoxc9-mediated repression is established prior to the time Hoxc6IM is expressed. These observations suggest that either Pbx interactions are dispensable for LMC specification, or that the LMC neurons produced under these conditions reflect the actions of Hox proteins resident to the thoracic spinal cord whose functions are unmasked through suppression of Hoxc9.Because Hox proteins often require cooperative interactions with Pbx proteins to bind DNA, we also examined the consequences of mutating the YPWM motif (Hoxc6IM: YPWM->AAAM). Surprisingly expression of Hoxc6IM did not alter the capacity of Hoxc6 to repress c levels . BecauseHoxc9 repression domain (NΔ91) and YPWM motif were mutated, but retained the intrinsic activation domain (aa 105–111). While Hoxc6 NΔ91 cooperatively bound with Pbx3 to the hs1149 element and Fkh250con* and activated LMC genes (Hoxc9 repression domain and Pbx interaction motif is the ability of Hoxc6 to promote LMC fate lost. These results suggest that Hoxc6 can promote LMC identity through two distinct mechanisms 1) Pbx-independent restriction of Hoxc9 and 2) Pbx-dependent interactions with Foxp1, activities that are separable  presumably acts by overriding the dampening influence of Hoxc9, perhaps by competing for binding sites. This idea is supported by the observation that Hox5–Hox8 proteins can induce aspects of LMC differentiation without suppressing Hoxc9. During normal development this process is likely played out in two distinct contexts: at the border between brachial and thoracic spinal cord, where many LMC MNs express low levels of Hoxc9 While regions outside the homeodomain that contribute to the specificities of Hox proteins in Hoxc9 is a condition sufficient to promote LMC fates even in the absence of a functional LMC Hox protein. When the Pbx interaction motif is deleted from Hoxc6, Hoxc9 is still repressed and LMC neurons are generated, although Hoxc6 presumably will no longer have a direct impact on activating the LMC program. One plausible explanation for this result is that the suppression of Hoxc9 unmasks the activities of Hox proteins that are endogenous to the thoracic spinal cord that have the capacity to activate high levels of Foxp1 , where ΔΔCt = (CtIP−CtInput)−(CtIgG−CtInput).Chromatin immunoprecipitation was performed as previously described pET-14b plasmids carrying His-tagged constructs were used to transform BL21 pLys bacterial strain and protein expression was induced by 0.5 mM IPTG at room temperature overnight. Bacteria were lysed under native conditions in lysis buffer (50 mM Tris pH 7.5 and 100 mM NaCl) followed by sonication. The supernatant was incubated with Ni-NTA agarose beads at 4°C for 1.5 hours and washed three times in buffer containing 50 mM Tris pH 7.5, 300 mM NaCl, 20 mM imidazole and 0.5% Igepal CA-630. Recombinant proteins were eluted in 50 mM Tris pH 7.5, 300 mM NaCl, 250 mM imidazole and dialyzed overnight in 50 mM Tris-HCl pH 8 and 150 mM NaCl.AGCTGTGGGACGAGG). Double stranded probes were synthesized using Klenow DNA polymerase. Binding between recombinant proteins and DNA probes was performed in binding buffer containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl2, 20% Glycerol, 2.5 mM DTT, 2.5 mM EDTA pH 8, 250 ng/µL poly dIdC and 0.1% BSA for 20 minutes at room temperature. For each binding assay equivalent molar amounts (0.5–2 pmol/reaction) of recombinant protein were used. Binding reactions were resolved on a non-denaturing acrylamide gel and the IRDye-800 was detected using the Odyssey system (Li-Cor).Oligonucleotides containing putative Hox binding sites were annealed to an IRDye-800 labeled linker . We do not work with a species or procedure, including euthanasia, with which I and those members of my research staff involved in this project are not experienced, without first seeking the advice and instruction of a veterinarian from the Division of Laboratory Animal Resources, consult the Division of Laboratory Animal Resources as circumstances require. To the best of my knowledge, the research does not unnecessarily duplicate previous research with respect to the use of laboratory animals. We comply with all requests for data as may be required by governmental and institutional guidelines. We seek the approval of the Institutional Animal Care and Use Committee on all procedures which involve laboratory animals.Figure S1Hoxa6/c6 mutants at e11.5. (A) Increase in the number of HMC neurons at rostral brachial levels in Hoxa6/Hoxc6 mutants at e11.5. Serial sections from rostral to caudal levels of the LMC are shown left to right. HMC neurons are identified by Hb9+Isl1/2 coexpression, indicated in cyan.  Normal expression of Hoxc4 and Hoxc8 in Hox6 mutants at e11.5. Serial sections along the rostrocaudal axis showing normal expression of Hoxc8 and Hoxc4 in FoxP1+ LMC neurons. HoxA genes are also expressed normally in Hox6 mutants (data not shown).Analysis of MN columnar specification in (PDF)Click here for additional data file.Figure S2Hoxc9 is not derepressed at brachial levels in Hoxa6/Hoxc6 mutants. Serial sections at caudal brachial levels showing that Hoxc9 is normally restricted from FoxP1+ LMC neurons in Hoxc6 and Hoxa6/Hoxc6 mutants. At these levels Hoxc9 is normally expressed in neurons located dorsal to the LMC. Rostral to caudal is shown left to right.(PDF)Click here for additional data file.Figure S3Hox6 mutants. (A) Total number of FoxP1+ LMC neurons in the brachial spinal cord of various Hox6 mutant allele combinations. (B) Levels of FoxP1 protein expression are reduced in rostral brachial regions in Hoxa6/Hoxc6 mutants. Levels were determined by measuring the pixel intensities of FoxP1 nuclear staining.  Decrease in the number of FoxP1+ LMC neurons at brachial levels in Hoxa6/Hoxc6 mutants at e10.5 and e11.5. Images show serial sections along the rostrocaudal axis from left to right. Loss of FoxP1 is prominent at rostral brachial levels (Hoxa5/Hoxc5+ region) of the spinal cord. Approximate position of the Hox5/Hoxc8 boundary is indicated.Analysis of LMC specification in (PDF)Click here for additional data file.Figure S4Hox6 triple mutants. In mice lacking all three Hox6 genes  LMC neurons are still generated in caudal brachial spinal cord, as assessed by FoxP1 and Raldh2 expression. In rostral brachial spinal cord, there is an additional loss in LMC neurons in triple mutants when compared to Hoxb6/c6 double mutants, but essentially phenocopies the LMC loss in Hoxa6/c6 double mutants Click here for additional data file.Figure S5Hox gene. Error bars show s.e.m.Efficiency of LMC induction by Hox4–Hox8 proteins. (A) Examples of Hox electroporations in chick showing similar levels of protein expression to endogenous brachial levels. (B) Quantification of mean pixel intensities of Hox staining in n>40 nuclei of electroporated neurons at brachial and thoracic levels. (C) Quantification of the percentage of electroporated MNs (defined by Isl1/2 expression) that express high levels of FoxP1 at thoracic levels after misexpression of the indicated (PDF)Click here for additional data file.Figure S6Hoxc6 mutants. (A–D) Additional examples of whole mount GFP staining showing defects in motor axon innervation of the cm muscle in Hoxc6 mutants at e12.5 and e13.5. (E) Loss of Pea3+ and retention of Scip+ motor neuron pools at e11.5 in Hox6 mutants. There is a marked decrease in the number of Pea3+ MNs at e11.5 in Hoxa6/Hoxc6 mutants.Motor neuron pool defects in (PDF)Click here for additional data file.Figure S7Hoxc6 mutants. (B) Summary of the distribution of labeled MNs after ulnar injection. In control mice only Scip+ MNs are labeled. In Hoxc6 mutant mice Scip− MNs are labeled, the position of these labeled MNs extends rostrally, and overlaps with the position of the former Pea3+ MN pool. (C) Serial sections from rostral to caudal showing distribution of labeled MNs after ulnar injections in control and Hoxc6 mutant mice.Analysis of tracer injections into the ulnar nerve in Hoxc6 mutants. (A) Summary of the position and distribution of the Pea3+ and Scip+ MN pools in the caudal half of the lateral motor column (cLMC). Relative positions of the pools in transverse sections are indicated for both control and (PDF)Click here for additional data file.


MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (tm1aMcph1tm1a/) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that tm1aMcph1tm1a/ mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired tm1aMcph1tm1a/ mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of tm1aMcph1tm1a/ mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive.  Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment in children. As a multifactorial disease, the pathogenesis of OM is complicated. Based on previous research, many factors are thought to contribute to the development and persistence of OM including: environmental factors such as smoking and type of child care; anatomical dysmorphology; Eustachian-tube function; adaptive and innate immune system function; viral and bacterial load; and genetic predisposition. However, the mechanisms underlying OM are still elusive. Heritability estimated from twin studies Mcph1)-deficient (tm1aMcph1tm1a/) mice were found to exhibit hearing impairment as a part of the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the Knockout Mouse Project and the European Conditional Mouse Mutagenesis Program (KOMP/EUCOMM) resource of over 17,000 genes targeted in ES cells In the present study, microcephalin 1  encodes MCPH1, which contained three BRCT domains: one in its N terminus and two in its C terminus. BRCT domains have been found predominantly in proteins involved in cell cycle checkpoint functions and in proteins involved in the DNA damage response tm1aMcph1tm1a/ mouse mutants not only had some expected features such as small skull size and increased micronuclei reflecting genome instability, but also showed some unexpected phenotypes including susceptibility to OM implicating MCPH1 in genetic predisposition to OM. This finding implicates a new molecule in the pathogenesis of OM that is relevant to understanding the underlying mechanisms irrespective of the initial trigger for OM.The broad phenotypic screening of the MGP revealed that All mouse breeding and investigation was carried out with authorization of the UK Home Office. Mice were killed by cervical dislocation and decapitation. All efforts were made to minimize suffering.Mcph1-deficient (tm1a(EUCOMM)WtsiMcph1, abbreviated to tm1aMcph1 in this report) mice carry a knockout-first allele LacZ and neo genes were inserted in intron 3–4 of the Mcph1 gene (http://www.knockoutmouse.org/kb/entry/102/), exon 4 of Mcph1 was chosen as the critical exon. The vectors containing the Mcph1 knockout-first allele were electroporated into embryonic stem cells (JM8F6) derived from C57BL/6N mice. Targeted embryonic stem cell lines were selected using neomycin and screened by long range PCR after homologous recombination. The presence of the LoxP site was confirmed by sequencing. Correct integration of the 5′ arm and 3′ arm was confirmed by long range PCR using a universal primer and two genome-specific primers, and the subsequent PCR amplicon was verified by sequencing. The positive stem cells were injected into host mouse blastocysts and were used to generate chimeras containing the targeted allele. Male chimeras with 80–90% of targeted cells were bred with C57BL/6Brd-c-BrdTyr females and germ line transmission of the Mcph1 knockout-first allele was confirmed by a series of genotyping PCR analyses (http://www.knockoutmouse.org/kb/25/) using the mouse tissue DNA as template. These heterozygous mice were inter-crossed to expand the colony. The mice were maintained in individually-ventilated cages at a standard temperature and humidity and in specific pathogen-free conditions on the mixed C57BL/6N and C57BL/6Brd-c-BrdTyr genetic background. To genotype animals  and exon 14 . Real-time PCR was performed in quadruplicate for each sample using the probe  in an ABI Prism 7000 (Applied Biosystem). Hypoxanthine-guanine phosphoribosyltransferase (Hprt) was amplified simultaneously  as the internal reference. The relative quantity of Mcph1 RNA was calculated using 2−ΔΔCt method RNA was isolated from the tissues of middle ear, inner ear and forebrain. Littermates were used . Total RNA was isolated with QIAshredder columns  and RNAeasy mini kit . cDNA was synthesized with normalization of the same original amount of RNA using oligo dT and SuperScrip II (Invitrogen). Primers were designed to amplify part of exon 9–13  and xylazine hydrochloride  and subcutaneous needle electrodes were inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip  stimuli at a range of intensity levels in 3 or 5 dB steps. Averaged responses to 256 stimuli, presented at 42.2 per second, were analysed and thresholds established as the lowest sound intensity giving a visually detectable ABR response. The peak-peak amplitude of wave 1 of click-evoked ABRs (P1-N1 amplitude) was measured and plotted as a function of sound level above threshold to produce input-output functions (IOFs). The IOF slope from 0–35 dB above threshold was calculated since 0–35 dB above threshold covered the more linear range before the function begins to flatten towards a plateau and also to ensure that a similar dB SL (sensation level) range was covered in both wild type and Mice used for recurrent ABR measurements were sacrificed after the last measurement and the anatomy of their middle ears was examined. Briefly, the external ear canals, tympanic membranes, ossicles and middle ear cavities were carefully dissected, examined and imaged. The inner ears were dissected out, fixed in 4% paraformaldehyde and examined following inner ear clearing in glycerol 2 at room temperature for 3 hours. Cochleae were finely dissected in PBS. This was followed by further processing by an osmium-thiocarbohydrazide-osmium (OTOTO) method Inner ears of six mice at postnatal day 4 (three mice each of homozygote and wild type) were fixed by 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 3 mM CaClImmunohistochemistry staining was performed using the Ventana Discovery machine (Roche) and reagents according to the manufacturer's instructions. Paraffin sections from wild type mice at postnatal day 7 (n = 3) and 4 weeks old (n = 5) were obtained as described above. The expression of Mcph1 was detected by using Mcph1 antibody .Digital X-ray images are acquired using a Faxitron system MX20 (Faxitron X-ray corporation) in 14-week old mice . Mice were anaesthetised and up to five standard images were taken for each mouse. The entire skeleton morphology was assessed using a standardised protocol capturing 41 parameters. To extract the brains, a vertical incision was made through the scalp and both sides were peeled back. Then a vertical cut was made through the midline of the skull using a pair of scissors and the skull was peeled off with a pair of fine forceps. Brains were then removed using a fine spatula and weighed on a Sartorius TE212, accurate to two decimal places.The prevalence of micronucleated normochromatic erythrocytes (MN-NCE) was determined using a flow cytometric assay of micronucleus formation Salmonella enterica serovar Typhimurium M525 Flow analysis was performed on heparinised blood collected from 16-week old mice . The following parameters were analysed: percentages of total T cells, CD4+ and CD8+ T cells, NKT cells, NK cells, B cells, Granulocytes and Monocytes are presented relative to the total CD45+ WBC population. Percentages of memory CD4 and CD4+CD25+ Treg cells are presented relative to the total CD4+ T cell population. Percentages of memory CD8 and mature IgD+ B cells are presented relative to the total CD8+ T cell and B cell populations respectively. All samples are analysed on a BD LSR II.Escherichia coli  adjuvant. Mice were boosted on days 7 and 21. Serum samples were collected on days 28. Detection of TetC-specific antibodies from sera was performed by ELISA. For the measurements of antibody levels, mouse blood was collected by cardiac puncture, and serum was prepared and stored at −20°C. For antigen specific antibody measurements in mouse serum, Nunc MaxiSorp plates were coated overnight at 4°C with 2 mg/ml tetanus toxin fragment C recombinant protein (TetC) in 0.1 M Na2HPO4 (pH 9.0), blocked with 3% (w/v) BSA in PBS for 1 h, and incubated with 5-fold serial dilutions of mouse serum in PBS with 1% BSA for 1 h. The plates were developed with anti-mouse Ig HRP-conjugated Abs (Dako), followed by o-phenylenediamine substrate tablets (Sigma-Aldrich) dissolved in water. Absorbance was read at 490 nm and optical densities represented as titres.Recipient mice  were immunized by intranasal inhalation of 30 µl PBS containing 10 mg TetC  combined with 1 mg heat-labile toxin of Mice underwent ophthalmic screening at 15 weeks of age. They were assessed for gross morphological changes to the eye using a slit lamp (Zeiss SL130) and ophthalmoscope (Heine Omega 500). The eye was examined both undilated and dilated (tropicamide). Images using the slit lamp were collected using a Leica DFC420 camera. The mice were culled under terminal anaesthesia followed by cervical dislocation and six eyes from 3 male homozygous mutants and 3 wild type mice were removed and fixed. Pupil-optic nerve sections were processed with hematoxylin and eosin, and standard images were captured under light microscopy for review Blood was collected at 16 weeks of age into ETDA microvette tubes (Startedt) and analyzed on an analyzer  with ten parameters tested.16-week-old mice were terminally anaesthetized and blood was collected from the retro-orbital sinus into lithium-heparin tubes. The plasma was immediately analyzed on an Olympus AU400 Analyzer with 28 parameters tested.We assessed glucose tolerance in mice fed on a high-fat diet  from 4 weeks of age until 13 weeks of age. At 13 weeks, mice were fasted overnight before a blood sample was taken and glucose was measured using an Accu-Chek Aviva (Roche). A bolus of glucose (2 mg/g) was administered intraperitoneally and blood glucose concentration from the tail tip was measured using Accu-Chek Aviva (Roche) after 15, 30, 60 and 120 min.Mcph1 mutant on a C57BL/6 genetic background. The mutant allele is designated tm1a(EUCOMM)WtsiMcph1, and abbreviated to tm1aMcph1 in this study. The design of the tm1aMcph1 allele and genotyping protocol are illustrated in 2 test, p<0.001). Both male and female homozygous mutants are infertile, similar to other Mcph1 mutants reported previously tm1aMcph1tm1a/ mice (data not shown). Reverse transcription PCR was performed to test the effect of the mutation of Mcph1 on transcription. The homozygous mutants, heterozygous and wild type mice produced bands of expected size and sequencing of the PCR products validated the results. This indicated that there was residual Mcph1 transcript in tm1aMcph1tm1a/ mice. Quantitative real-time PCR revealed the residual transcript of Mcph1 in the homozygous mice is only 1–4% of the level compared to the wild type mice and the residual levels vary in different organs . tm1aMcph1tm1a/ mice showed mild to moderate hearing impairment with increases of 10–50 decibels (dB) compared to the normal thresholds for both click and pure tone stimuli (6–30 kHz)  and +Mcph1+/ (n = 13) mice showed normal ABR thresholds, whereas elevated thresholds can be detected in tm1aMcph1tm1a/ mice as early as 3 weeks of age (n = 13). Thresholds were generally stable over time, although there was progressive or fluctuating hearing impairment over time in some mice  above the normal hearing reference range as affected in this study, and the penetrance of hearing impairment in tm1aMcph1tm1a/ mice is around 70% based on this criterion.Hearing impairment was discovered by ABR measurement in 14 week old –30 kHz) . To furtome mice . Heterozutations . Mcph1tmtm1a/tm1aMcph1 mice  and +Mcph1+/ (n = 13) mice showed a transparent tympanic membrane, air-filled middle ear cavity, and normal morphology of ossicles (apart from one heterozygous mouse that had some white secretion in the hypotympanum in the right middle ear cavity). Dissection of tm1aMcph1tm1a/ mice (n = 13) that had elevated ABR thresholds revealed a range of defects in the middle ear including thickened and white bone forming the bulla instead of the normal thin and transparent bone, retracted tympanic membrane, bubbles present underneath the tympanic membrane, or middle ear cavities filled with clear or cloudy fluid. In addition, two tm1aMcph1tm1a/ mice had an amorphous tissue mass in the middle ear cavity . The effusion was confined within the middle ear cavity and did not appear to extend through the round window. In two out of three 60-week old tm1aMcph1tm1a/ mice, we saw only a few inflammatory cells scattered in the middle ear cavity, but the thickened mucoperiosteum was still present (data not shown). The Eustachian tube had a similar appearance in mutant (n = 4) and control  mice in serial sections through the tube as it exits the middle ear (data not shown).Histological examination was performed to investigate the pathological change . Hearingtm1aMcph1tm1a/ and wild type mice. DNA was extracted from these and the 16S rRNA gene was amplified via PCR with universal bacterial primers (7f and 1510r). From this, 16S rRNA clone libraries were created for a tm1aMcph1tm1a/ and wild type mouse for each tissue sampled. The predominant phylotype found to be present in the nasopharynx of both mutant and wild type mice was matched through BLAST analysis to a previously-uncultured Streptococcus sequence (ERD01G accession number GQ456229.1) tm1aMcph1tm1a/ mouse. We were then able to culture this bacterium from the mutant middle ear by plating it onto a variety of media under micro-aerophilic conditions, thus confirming its presence within the tissue. The identity of this isolate as Streptococcus bacterium (Strep ERD01G) was confirmed by 16S rRNA PCR .We collected tissue from the nasopharynx, middle ears and external ear canal of tm1aMcph1tm1a/ mice have inner ear defects, scanning electron microscopy (SEM) and temporal bone sections were used to examine cochleae in young pups and adult mice respectively. At postnatal day 4, tm1aMcph1tm1a/ mice showed normal morphology of the upper surface of the organ of Corti by SEM  and there was no evidence of any abnormality of the cochlea in tm1aMcph1tm1a/ mice  in rol mice  indicatiSalmonella Typhimurium challenge, tm1aMcph1tm1a/ mice had a similar change of body weight compared to control mice over a 21 day period following the infection . To test if this alteration to the B cell frequency resulted in any changes in antibody production, we performed a prime boost immunisation with Fragment C of tetanus toxin. However, in agreement with the normal response to infection there was no significant change in antibody level in the tm1aMcph1tm1a/ mice . The only significant difference was an increase in the circulating B cell frequency in female m1a mice .tm1aMcph1tm1a/ mice displayed ocular abnormalities including corneal opacity and vascularisation .Mcph1 (tm1a(EUCOMM)WtsiMcph1, knockout-first design Mcph1 in the homozygous mutant mice to less than 4% of the wild type level. During the standardised phenotypic screen of these mutants, we found an unexpected phenotype: mild to moderate hearing impairment. ABR thresholds were raised uniformly across all frequencies tested and the growth of amplitude of the waveform with increasing sound stimulation above threshold was similar in mutants and controls, both features consistent with a conductive hearing impairment. Subsequent ABR measurement, dissection of the middle ear and histopathology indicated otitis media with effusion was present to varying degrees in the mutant middle ears. We found expression of Mcph1 in the epithelia lining the middle ear consistent with its involvement in otitis media. These findings suggest that tm1aMcph1tm1a/ mice are a model for one form of heritable otitis media and reveal a new molecule involved in the pathogenic pathways underlying otitis media that can be used to unravel the underlying mechanisms irrespective of the initial trigger.We report here a new mouse with a targeted mutation of ABR has been used extensively for assessment of mouse auditory function and can detect moderately-raised thresholds due to middle ear inflammation among many other mechanisms underlying hearing impairment tm1aMcph1tm1a/ mice. Furthermore, round windows appeared intact and the exudate was confined within the middle ear cavity. Most tm1aMcph1tm1a/ mice showed ABR threshold elevations no higher than 40 dB, which is what is expected in conductive hearing impairment and all tested frequencies were affected which is another characteristic of conductive hearing impairment. Furthermore, input-output function analysis suggested that there is no evidence for a recruitment-type effect in the hearing-impaired tm1aMcph1tm1a/ mice, where a steeper slope would be seen and be indicative of recruitment of additional auditory nerve fibers into the ABR wave 1 component with increasing sound levels as a result of sensorineural hearing loss. Thus, the features of hearing impairment seen in the tm1aMcph1tm1a/ mice are consistent with conductive impairment. We noticed a few tm1aMcph1tm1a/ mice had ABR threshold elevation higher than 40 dB  also suggest a more complex explanation, as these cells expressed residual MCPH1 protein but were derived from a patient with microcephaly Very recently, three different MCPH1 mutations previously. One possible explanation for this is that hearing impairment can easily be missed in the mouse. Also, owing to practical difficulties OM or hearing impairment has not been reported in human patients or mouse models with tm1a/tm1aMcph1 mice. Similar to studies of other Mcph1 mutants, we found that Mcph1-deficient mice have defects in DNA damage repair revealed by the increased prevalence of micronucleated normochromatic erythrocytes. Eye abnormalities revealed by gross morphology and histopathology present to varying degrees in the mutants implicating Mcph1 function in vision, but have not previously been reported in MCPH1 patients or mouse models.Besides OM, hearing impairment and smaller brain and skull sizes, we observed other defects in Mcph1 was proposed as a potential tumour suppressor because decreased levels of Mcph1 were detected in several types of human cancer including breast and ovarian cancers tm1a/tm1aMcph1 mutants suggests genomic instability so is consistent with a role in cancer. However, the four available Mcph1 mutant mouse lines have not been reported to show any excess of tumours, although none have been systematically aged and examined appropriately to detect tumours. Furthermore, there is anecdotal evidence that the incidence of cancer in MCPH1 patients is low MCPH1 expression in human cancer cells and increased micronuclei in the mice reported here on the one hand and the lack of reported tumour development in mouse Mcph1 mutants and MCPH1 patients on the other hand may reflect the small numbers of individuals studied appropriately.tm1a/tm1aMcph1 mice carry can produce reporter knockouts, conditional knockouts, and null alleles following exposure to site-specific recombinases Flp and Cre tm1aMcph1tm1a/ mouse could provide useful tools for further research to unravel the underlying mechanism of OM. The discovery of a role for Mcph1 in predisposition to OM expands our knowledge of genetic factors underlying OM. Rapid advances in sequencing technologies have already proved valuable in finding novel OM genes The knockout-first allele which


Dear Editor,Happiness has several main components including the emotional component the presence of contentment and affection; the social component- the presence of good social relationships and the ability to receive support; and finally the cognitive component that causes the happy person to interpret information in a special way that leads to joy and optimism.2]3]4][3][4]2][[4][3][4][3][4]2]Consequently, peoples’ assessment of themselves and their lives can include cognitive aspects- like judgments about life satisfaction- or emotional aspects like mood and affect.6]7]8][7][8]6][[8][7][8][7][8]6]14] [14] 13][ [14] 13]Our study demonstrated no difference in happiness and life satisfaction between men and women. There were also no differences in the feeling of well-being between men and women across different educational groups. However, the difference between happiness across various faculties was significant. There was a significant difference between men and women in relation to happiness and socioeconomical, cultural, emotional, and mental conditions. Marital divorce and financial status were independent variables and happiness and life satisfaction were predictive variables; parental education was considered as the control variable. Furthermore, we failed to observe any difference among men and women in relation between happiness and needs, expectations and wishes. The mean±SD of happiness score in this study was 47.13±14.3 that is comparable to other studies in Iran. In comparison with studies performed after 1990, Argyle and Leu reported a 35.6 percent.[