MEIS1, BTBD9 and MAP2K5/SKOR1 are the only known genes located within these loci and their association with RLS was subsequently confirmed in a number of follow up GWAS. Following this finding, our group reported the MEIS1 risk haplotype to be associated with its decreased expression at the mRNA and protein levels. Here we report the effect of the risk variants of the three other genes strongly associated with RLS. While these variants had no effect on the mRNA levels of the genes harboring them, we find that the homeobox transcription factor MEIS1 positively regulates the expression of the transcription co-repressor SKOR1. This regulation appears mediated through the binding of MEIS1 at two specific sites located in the SKOR1 promoter region and is modified by an RLS associated SNP in the promoter region of the gene. Our findings directly link MEIS1 and SKOR1, two significantly associated genes with RLS and also prioritize SKOR1 over MAP2K5 in the RLS associated intergenic region of MAP2K5/SKOR1 found by GWAS.Restless Legs syndrome (RLS) is a common sleep disorder for which the genetic contribution remains poorly explained. In 2007, the first large scale genome wide association study (GWAS) identified three genomic regions associated with RLS.  RLS is a circadian sensorimotor dysregulation disorder that can lead to severe sleep disturbances and impaired quality of life3. Epidemiological studies have established that RLS is a common neurological disorder with a prevalence of up to 15% in Central Europe and North America12.Restless Legs syndrome (RLS), also known as Willis-Ekbom disease (WED) or Wittmaack-Ekbom syndrome, is a common sleep-related sensorimotor disorder characterized by an urge to move the legs to relieve uncomfortable sensations. These sensations occur, or worsen, during rest, such as before falling asleep or during the night13. One of these loci was in an intronic region of the homeobox gene MEIS1. A second locus was in the BTBD9 gene, which encodes a BTB (POZ) domain. The third locus was between the MAP2K5 gene, which encodes a mitogen-activated protein kinase and the SKOR1 gene, which encodes a transcription factor 13. During the same year, an independent GWAS studying cases and controls from Iceland also identified an association between BTBD9 and periodic limb movements in sleep (PLMS), a motor feature strongly associated with RLS14. A third independent GWAS conducted with cases from the United States also replicated the association of MEIS1 and BTBD9 with RLS15. Two years later our group further confirmed the association between MEIS1 and RLS as we established that the intronic risk haplotype (rs12469063/rs2300478: GG/GG) led to decreased expression of the gene at both the mRNA and protein levels in both brain tissues and lymphoblastoid cell lines (LCL) derived from patients16.In 2007, a genome-wide association study (GWAS) conducted using German and French-Canadian RLS cases and control individuals identified variants in three genomic regions located on chromosomes 2p, 6p and 15q17. However, there can be negative side effects to the administration of dopaminergic drugs, like augmentation of overall RLS symptoms18. Iron deficiency anemia, end stage renal disease and multiple pregnancies increase the risk of RLS. The fact that iron supplements relieve symptoms in many of these cases strongly implicated iron in disease pathogenesis19. Studies suggest a dysfunction in the transfer of iron between the serum and the central nervous system, possibly involving the blood brain barrier22.The first line of treatment for RLS are dopamine agonists like levodopa, suggesting that reduced dopaminergic activity is involved in the pathogenesisMEIS1, BTBD9, MAP2K5 and SKOR1). The positive interaction between MEIS1 and SKOR1 was further explored using specific protein-DNA binding studies and promoter reporter assays.To extend on these previous reports and explore the pathogenic molecular mechanisms we looked for interactions between the different RLS-associated genes. First, we investigated the effects of the risk variants on the mRNA expression levels of the genes harboring them and the other RLS associated genes  to test for variations in the expression levels of these genes in RLS patients with these risk variants, similar to our previous report16.Given our previous report highlighting the decreased levels of BTBD9  that is located in intron 5 of BD9 Fig. . When mRMAP2K5 and SKOR1. No effect was observed to be driven by this risk allele  on the mRNA expression of either MAP2K5  located in the intergenic region between 2K5 Fig.  or SKOR1OR1 Fig. ; unfortuSKOR1 expression, which varied according to the MEIS1 risk haplotype. Using LCL we observed a significantly decreased expression (P = 0.0202) of SKOR1 in RLS cases who were homozygous carriers of the MEIS1 risk haplotype (rs12469063/rs2300478: GG/GG) by comparison to individuals who were homozygous for the MEIS1 non-risk haplotype (AA/TT). The same SKOR1 expression measures were made using thalamus samples obtained from individuals carrying the same MEIS1 haplotypes and these confirmed a similar decrease of expression(P = 0.0174). Analysis of the pons samples from the same individuals did not show a significant decrease of expression (P = 0.1519)  were found to be reliable as all were prone to produce various patterns of non-specific signals.After establishing the effect of each risk variant on the expression level of the genes harboring them, we looked for links between each of the risk variants and the other susceptibility genes. No significant effect on expression was observed between these genes except for the case of 19) Fig. . The samMEIS1 risk haplotype (GG/GG) to be associated with decreased levels of both its mRNA and protein16. Hence, we decided to test if silencing MEIS1 would result in a reduction of SKOR1 mRNA. Using an siRNA  specifically targeting MEIS1 we reduced the expression of the gene (~70% mRNA decrease) in HeLa cells and found a coincident 64% decrease of SKOR1 mRNA level (P < 0.0001)  Fig. . This obMEIS1 is a homeobox gene that encodes a DNA binding transcription factor, we hypothesized that it might regulate the expression of SKOR1 by binding to its promoter region. A literature search revealed that MEIS1 and members of another family of homeobox genes, PBXs  form in vivo heterodimeric DNA binding complexes with each other and their DNA binding is more intense when together than when either Meis1 or Pbx1 are alone26. We searched for the presence of MEIS1/PBX1 consensus binding sites27 , a second one at ~1.76 kb upstream (TGACAGagTGAg) and a third one at ~2.6 kb downstream of the ATG, in intron 2 of the canonical isoform of SKOR1 (GGACAGtaTGAT). Our search was focused on the hexameric consensus binding site of MEIS1. Moreover a search for the octameric consensus binding site of MEIS1 (TGATTG/TAT), as reported by Penkov et al., did not reveal any such sites in upstream regulatory region and the fist intron of SKOR1 gene28. It is also noteworthy that an examination of Meis1 ChIP-Seq (Chromatin immunoprecipitation followed by high throughput sequencing) data made in the mouse (GEO database #GSM2188919), shows two Meis1 binding sites upstream Skor1 gene (mm8 genome assembly) which suggest they are likely real elements involved in the activation of Skor1 . The purified proteins were used in electrophoretic mobility shift assay (EMSA) experiments testing the three different human MEIS1/PBX1 binding sequences (~8.7 kb and ~1.76 kb sites upstream to the ATG and the ~2.6 kb site downstream to the ATG)   murine forms of the proteins (Meis1 or Pbx1) we prepared purified proteins using TG) Fig. . The ~2.TG) Fig.  and therTG) Fig. . Overallype Fig. , this in31. The average age at enrollment of RLS patients was 52.5 ± 14.9 years, with 38.5% men. Patients were diagnosed based on the international RLS study group (IRLSSG) criteria32. The average age at enrollment of the control population was 53.0 ± 16.1 years, with 38.2% men. There is no significant difference in sex and age between the cases and controls. The frequency of the risk allele (C) of rs4776976 is 0.848 in RLS cases and 0.776 in controls, respectively; and the genotype distribution fits with the Hardy-Weinberg equilibrium. The p value for the association test of the C risk allele is 0.0001267. The RLS risk allele (C) of rs4776976 is the most common allele in the European population (0.8) while the non-risk T allele of rs4776976 is the less frequent (0.2); as observed on gnom Aggregation Database33.An association study including unrelated and consecutively recruited RLS patients in Montreal, Canada (n = 401) and controls (n = 588) of European ancestry, recruited as previously describedSKOR1 expression and so our mRNA expression data was revisited using the two possible genotypes (C and T)  and so it was divided into two separate fragments (P1 contained the ~8.7 kb binding site and nearby rs4776976 SNP (C or T) and P2 the ~1.76 kb   Fig. .MEIS1 siRNA, a significant decrease (∼40%) of luciferase expression occurred when driven by the P1 fragment with the rs4776976 C risk allele (P < 0.001), by comparison the P1 fragment with the T rs4776976 allele does not affect the luciferase expression. This suggests that the presence of C allele that represents the common RLS risk allele results in binding of MEIS1/PBX1 to the P1 segment of SKOR1 promoter region. When the luciferase expression was driven by the P2 fragment a significant decrease (P < 0.01) also occurred, thus confirming this potential MEIS1/PBX1 binding site was recognized by MEIS1 in our assay. This experiment demonstrates that MEIS1 physically acts as a direct activator of SKOR1 and shows that the rs4776976 SNP plays an important role for the binding of MEIS1 on SKOR1 promoter, where the presence of C allele results in SKOR1 gene being regulated by MEIS1.In the presence of 34. We previously showed that the RLS associated risk haplotype within the MEIS1 non-coding regions is associated with a decreased expression of this gene in the RLS patient LCL and thalamus samples16. Here we show that one of the consequences of this reduced expression is reduced expression of SKOR1. No other risk variants in the other GWAS RLS susceptibility loci show any significant correlation with the expression levels of the genes harboring them. Considering that MEIS1, a homeobox gene, is a transcription factor with precise temporal and spatial gene expression during development35, this link could be a result of transcriptional regulatory function of MEIS1 on SKOR1, which we confirmed using EMSA and a luciferase reporter assay.It is important to understand if genes identified using GWA studies interact with each other, how they exert their biological effects and which are the variants driving the associationSKOR1 ATG start site that is a regulatory SNP (rSNP), as it affects the MEIS1 regulation of SKOR1. We found that when the risk allele is present, reduced MEIS1 expression leads to reduced SKOR1 expression, whereas with the non-risk allele there are no changes in the expression of SKOR1 . These data suggest that a reduced level SKOR1 expression might be relevant to the development of a subset of RLS cases, and therefore it will be important to understand what are the downstream effects of reduced SKOR1 expression. Finally, these data also suggest that the variant driving the association with RLS in the SKOR1 locus is rs4776976.We also report a new SNP in the ∼8.7 kb upstream region of the SKOR1 strongly suggest that for the GWAS locus MAP2K5/SKOR1, the gene associated with RLS is SKOR1 and not MAP2K5. SKOR1 or SKI family transcriptional corepressor 1 is highly expressed in the central nervous system of human and mouse. In human, it appears to become more restricted to Purkinje cells of the cerebellum during adulthood36. The murine Skor1 interacts with a homeodomain transcription factor (Lbx1) to cooperatively repress transcription selectively in dorsal horn interneurons of the developing spinal cord37. It also appears to be a transcriptional corepressor which among others can regulate cell fate determination in murine dorsal spinal cord37. The necessity of Lbx1/Skor1 for generation of GABAergic phenotypes in dorsal horn interneurons of spinal cord potentially implicate them in the modulation of pain and sensory input processing of RLS40.Our findings directly linking MEIS1 and MEIS1 genomic region as well the MEIS1 protein as a transcription factor with the SKOR1 promoter sequences opens an avenue for future studies on the regulatory function of MEIS1 in the RLS pathogenesis mechanism as well as emphasizing the importance of the other candidate gene, SKOR1. Keeping in mind SKOR1’s expression pattern, as well as its proposed function as a developmental co-repressor, we can proceed to the subsequent research into RLS molecular mechanisms with a greater focus on the regulatory function of MEIS1 and SKOR1, two highly significantly associated genes with RLS.In a post GWAS era, one of the initial strategies to progress from the tag SNPs to the mechanisms underlying the disorders is to use expression studies on human patient cells harboring risk variants. In this study, establishing a link between the risk haplotype within the 22.Brain tissue specimens  were obtained from autopsy brain tissues of 31 individuals with an RLS diagnosis from the Harvard Brain Tissue Resource Center. Final diagnosis was made by an RLS expert based on available questionnaires and medical records, but blinded to the genotype information; these samples were previously used in our earlier RLS studies2 in RPMI 1640 medium (Invitrogen) supplemented with 15% (v/v) heat-inactivated fetal calf serum (Sigma-Aldrich), 0.29 mg/ml of l-glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin (Invitrogen). Healthy cells were harvested at an approximate density of 1 × 106 cells/ml.Selected LCL from RLS patients previously established by transformation with EB virus using standard protocols were grown at 37 °C and 5% CO® Lipid Tissue kit (Qiagen) or from 5 million lymphoblastoid cells using the RNeasy® kit (Qiagen). Single-stranded cDNA synthesis was performed from 1 µg of total RNA using a mix of oligo-dT and random primers and the Quantitect® Reverse Transcription kit (Qiagen) or the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). Quantitative RT-PCR was performed using the TaqMan method (Applied Biosystems) with probes and primers designed by Applied Biosystems recognizing BTBD9 (Hs00537653_m1) and MAP2K5 (Hs00177134_m1) and a custom probe designed at the junction of two exons of SKOR1 (AJ1RULU). PCR conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 sec (denaturation) and 60 °C for 1 min . Fluorescent signals were captured using the ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). The level of expression was determined by converting the threshold cycle (Ct) values using the 2−∆∆Ct method. Expression levels were normalized using the human 18 S ribosomal RNA (rRNA) gene with commercial primer-probe mix (Applied Biosystems) or the human polR2A control (Hs00172187_m1). All experiments were run in triplicate. Independent cDNA synthesis was carried out twice. ANOVA test and post-hoc Tukey’s test was used for statistics of all tissues with three genotypes. t-test was used for statistics of BTBD9 expression in thalamus and pons with only two genotypes.Total RNA was extracted from 0.2 g of frozen brain tissue using the RNeasyMEIS1 was realised by transfection of 100pmol of the MEIS1 siRNA  for 48 hours in HeLa cells using the jetPRIME® (Polyplus) transfection reagent. The negative control cells were transfected with low GC duplex control siRNA. Total RNA was then extracted following a classical protocol using TRIzol® (Invitrogen) and SKOR1 expression measured as described above. This experiment was run in triplicate and t-test was used for statistics.The artificial 70% decreased expression of SKOR1 promoter has been tested at the three potential sites with an electrophoretic mobility shift assay. Using vectors (PCS2) designed to express either wild-type or mutated  MEIS1 or Pbx1 (provided by Dr. Featherstone), purified proteins were prepared using in vitro transcription/translation (IVTT) . On the other hand, DNA probes were prepared by annealing of the complementary following oligonucleotides  in SSC buffer, marked with dATP 32 P using the Large (Klenow) Fragment DNA polymerase (Invitrogen) and purified using Microspin™ G50 columns . The resulting proteins and DNA probes were incubated for 20 minutes at room temperature with Poly DiDc 0,1 U/μL (Novagen) and BindBuffer  for DNA-protein interaction and the mix was then loaded on a 30% acrylamide:bisacrylamide gel which has been pre-run at 100 V for 30 minutes. Electrophoresis is performed at 125 Volts for 45 minutes. The gel is then dried under vacuum at 80 °C for 45 minutes and autoradiography is performed with a time of exposure of 48 hours.The binding of MEIS1 and Pbx1 on SKOR1 promoter into PGL3 vectors expressing the firefly luciferase gene. The ~20 kb of SKOR1 promoter region is too long for its cloning as one piece so we cut this region into two segments; each one containing one of the potential MEIS1/Pbx1 binding site. P1 is the most upstream segment from the ATG and contains the ~8.7 kb MEIS1/Pbx1 binding site. This fragment of 5,287 bp was amplified with Phusion DNA polymerase (New England Biolabs) from BAC clone RP11-207J8 and using the following primers: fwd_5′-gag ctc tta cgc gtg cta gcc cgg gct cga gag ggt gcc tgt ggt gtg gga cgg tag g-3′, rev_5′-ctg act aat tga gat gca gat cgc aga tct taa att gtc ttg acc cct tgc tgg ttt tt-3′. This P1 fragment was produced with 2 alternatives  and using QuikChange® Site-Directed Mutagenesis (Agilent). The resultant PCR amplicons were then cloned into the XhoI site of pGL3-promoter vector (Promega) containing a SV40 promoter upstream of the firefly luciferase gene with the sequence and ligation independent cloning (slic) method41. P2 is the closest segment to the ATG start codon and contains the ~1.76 kb MEIS1/Pbx1 binding site. This fragment of 1,535 bp was amplified from same BAC clone using the following primers: fwd_5′-gct ctt acg cgt gct agc ccg ggg tcg acg cca aaa aga ggg aag aac c-3′, rev_5′-cta att gag atg cag atc gca gat ctc gag acc agg tcc cac ttg act tg-3′. The cloning of P2 fragment was identical to P1. Resultant clones were screened for the presence of the insert and the sequence of positive clones was verified using Sanger sequencing (Genome Quebec Innovation Centre).To perform luciferase assays, we inserted genomic fragments from the 2 and Student’s t-tests were used to determine differences in sex and age, respectively. The association test between the marker rs4776976 and RLS was performed using the PLINK program42.Genotyping of rs4776976 (C/T) was done using TaqMan assays C__11771023_10, following the manufacturer’s instructions. The genotypes were called using the Applied Biosystems 7900 Fast Real-Time Polymerase Chain Reaction (PCR) System and Safety Data Sheet (SDS) software (v. 2.2.2). The goodness-of-fit test with one degree of freedom (df) was applied to check for the genotype distribution deviation from the Hardy–Weinberg equilibrium. The χMEIS1 or negative control) using the attractene transfection reagent (Qiagen). After 48 hours, cells were rinsed with PBS and lysed with 100 ul of passive lysis buffer from the Dual-Luciferase® reporter assay (Promega). Firefly and Renilla luciferase activity was then read using the same kit and a Synergy 4 microplate reader (Biotek). ANOVA test was used for statistics. The luciferase expression was measured as a ratio between firefly and Renilla expression.At day 1, 100,000 HeLa cells per well were plated in a 24-well plate. At day 2, transfection was performed with 100 ng or 200 ng of one of the following vector expressing the firefly luciferase , 100 ng of the vector containing the Renilla luciferase and 25 pmol of siRNA .All statistical analyses of expression studies were performed using GraphPad Prism 5.0 software (GraphPad Software Inc.) by one-way ANOVA followed by post-hoc Tukey’s test for significant results. Student’s t test was only used for Fig. Lymphoblastoid cell lines (LCL) were prepared from individuals who signed an informed consent form before entering the study; the biobanking of LCL has been reviewed and approved by the institutional review board of McGill University . RLS brain tissues were provided by the Harvard Brain Tissue Resource Center, which is supported in part by a Public Health Service Grant (R24MH068855), with permission from the RLS Brain Bank Tissue Review Committee through the RLS Foundation. Control brain tissues were provided by the Douglas-Bell Canada Brain Bank . In the case control association study of rs4776976 with RLS, all participants signed informed consent at enrollment, and the study protocols were approved by the institutional ethics review boards. This study was approved by Comité d’éthique de la recherche du Centre hospitalier de l′Université de Montréal and McGill University ethics, all methods were performed in accordance with the relevant guidelines and regulations of McGill University (REB NEU-14-051).Supplemental Data


Carassius auratus in the Dongting water system  can perform normal meiosis. In artificial autotriploid Carassius auratus , female individuals undergo normal meiosis and produce mature gametes, while male individuals cannot. To better understand the effects of triploidization on meiosis in fish, we study the structure, methylation level, and expression level of meiosis-related genes  in diploid Carassius auratus , Carassius auratus red var., 3nCC and 3nRR. The results show that, compared with their diploid ancestors (2nCC and RCC), Dmc1 and Ph1 genes are hypomethylated in all 3nCC and female 3nRR, while are hypermethylated in male 3nRR. Correspondingly, Dmc1 and Ph1 genes are highly expressed in all 3nCC and female 3nRR, while are lowly expressed in male 3nRR. These results indicate that high expression of meiosis-related genes can contribute to restoration of bivalent pairing during meiosis in autotriploid Carassius auratus. This study provides new insights into the effect of DNA methylation on the fertility in triploid fish.Triploid is usually considered to be unable to perform normal meiosis due to the abnormal behavior of the three sets of chromosomes. But autotriploid  Polyploidization, the addition of a complete set of chromosomes to the genome, represents one of the most dramatic mutations known to occur Mallet . InterspDmc1 (DNA meiotic recombinase 1) and Ph1 (polyhomeotic-like protein 1) as important meiosis-related genes control the behavior of chromosomes during meiosis. The expression product of Dmc1 is a recombinant enzyme used in meiosis and plays a vital role during synapsis  (♀) × Megalobrama amblycephala  (♂)  was found in the Carassius auratus  were randomly selected from the Dongting Lake water system. 3nCC came from the self-crossing offspring of the male and female triploid Carassius auratus that were randomly selected from the Dongting Lake water system. Red crucian carp  and 3nRR were provided by the Engineering Center of Polyploid Fish Breeding of the National Education Ministry located at Hunan Normal University. All materials were obtained during breeding season (April) (2017–2018) and were cultured in open pools (0.067 ha) with suitable pH (7.0–8.5), water temperature (22–24°), dissolved oxygen content (5.0–8.0 mg/L), and adequate forage. Fish treatments were carried out according to the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in China and approved by the Animal Care Committee of Hunan Normal University (Permit number: 4237). All samples were anesthetized with 100 mg/L MS-222  before blood collection. Peripheral blood cells were extracted surgically.Diploid Chromosome preparations were performed using the peripheral blood cell cultures of all samples. The chromosomes were prepared in accordance with Qin et al. . SpeciesTo detect the DNA content of 3nCC blood and sperm, a flow cytometer (Partec GmbH) was employed. Detailed steps were performed according to Xiao et al. .Ten RCC, 10 2nCC, and 10 3nCC at age 9 months were randomly selected for histological observation of gonad structure, while 10 3nRR were randomly selected for observation of 21 months. Detailed steps were performed according to Qin et al. .Extract RNA according to the instruction of Total RNA Kit I (Omega). The first-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit with gDNA Eraser . The obtained cDNA was stored at − 20 °C.Ph1 and Dmc1 were designed based on the nucleotide sequences found in zebrafish and other Cyprinidae fish  was used to study the expression of hmittgen . Detailehmittgen .Ph1 and Dmc1 genes were found using the Ensembl (http://asia.ensembl.org/index.html) website. Methylation primers were designed by predicting the promoter region CpG islands by the MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) website . The Dmc1 gene coding regions of RCC, 3nRR, 2nCC, and 3nCC are all 1029 bp in length, encoding 342 amino acids. The sequences had been deposited in GenBank . Sequence analysis using Clustal W (2.1) and DNAMAN revealed the existence of high homologies between 3nRR and 3nCC at the protein level, with amino acid identities of 92.3% for Ph1, 100% for Dmc1  compared with 2nCC and RCC, but were lowly expressed in 3nRR male individuals (p < 0.05).Comparison of expression levels of rol Fig. . The resPh1 and Dmc1 gene promoter regions of RCC, 3nRR, 2nCC, and 3nCC was shown in Table Ph1 and Dmc1 in male individuals of 3nRR were 0.983 and 0.883 (Table Ph1 and Dmc1 genes (Fig. The methylation status of the 83 Table , respectnes Fig. .Table 4MTriploids are traditionally considered sterile (Ramsey and Schemske Ph1 and Dmc1 are all important meiosis-related genes. The expression products of the former are necessary for meiotic synapse, and the latter is inducing specific binding of centromere. DMC1 deficiency leads to the abnormal formation of synaptic complex and the disordered separation of homologous chromosomes, and the deletion or mutation of Dmc1 in human and mice can lead to spermatogenesis disorder and premature ovarian failure (Kagawa and Kurumizaka Ph1 gene can promote homologous chromosome pairing, and it is also necessary for homologous chromosome pairing (Riley and Chapman Ph1 and Dmc1 gene coding regions of 3nCC and 3nRR were 99% and 100% respectively compared with their diploid ancestors. It is concluded that the Dmc1 and Ph1 genes of 3nCC and 3nRR have no significant difference in nucleic acid sequence and can encode amino acids normally. These results suggest that autotriploid has no significant effect on the structure of Ph1 and Dmc1 genes.Ph1 and Dmc1 genes are hypomethylated in all 3nCC individuals and female 3nRR individuals, while hypermethylated in male 3nRR individuals. The results of quantitative real-time PCR showed that Ph1 and Dmc1 genes were highly expressed in all 3nCC individuals and female 3nRR individuals, but a low expression in male 3nRR individuals. During the breeding season, 3nCC can extrude normal gametes and 3nRR can extrude eggs of different sizes without extruding semen. In meiosis, polyploids adopt a special mechanism of diploid meiosis to avoid the problem of gametes’ imbalance caused by complex chromosome combinations in metaphase I (Maestra et al. Ph1 and Dmc1 genes, homologous chromosome pairing is promoted, resulting in the production of eggs of different sizes. Due to the high expression of the Ph1 and Dmc1 genes, the 3nCC has diploidized to some extent. The level of gene expression is regulated by the degree of methylation of the gene promoter region. In the data presented here, we cannot explain why the Ph1 and Dmc1 genes are hypermethylated in male 3nRR.Genome merging and doubling brings about plenty of gene redundancy (Soltis et al.


Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kD heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of hsp70 using RNAi did not affect longevity.It has been known for over 20 years that  Drosophila 70 kD inducible heat shock protein (HSP70) is synthesized in salivary glands, Malpighian tubules, brain, wing imaginal discs and many tissues after a heat shock  have been previously obtained , and standardized to a single fly equivalent.The temporal profiles of GAL4 expression were obtained by quantification of β-galactosidase activity with a CPRG assay  staining  supplemented with protease inhibitors . Extracts were mixed with 50µl 2× SDS loading buffer , incubated for 5 min at 100° and centrifuged for 5 min at 10,000 g. 20 µg samples were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose membrane. HSP70-myc was detected with the mouse 9E10 monoclonal anti-myc antibody  at 1:5000 or the mouse 5A5 monoclonal anti-human HSP70 antibody  at 1:1000. α-Tubulin was used as loading control and detected with the mouse DM1A monoclonal anti-chicken α-Tubulin  at 1:5000. Primary antibodies were incubated at 4° overnight. After washes, the nitrocellulose membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  at 1:5000 at 4° overnight. The ECL advance Western blotting detection kit  was used for signal detection. Results were visualized and imaged with ChemiGenius bio-imaging system . ImageJ  was used for the measurement of optic density of specific bands. HSP70-myc level was normalized to α-Tubulin for quantification. Two-way ANOVA with Bonferroni post-test was performed with GraphPad Prism 5 to compare HSP70-myc expression levels.Control animals were obtained by crossing w males with homozygous UAS-hsp70 females (UAS control) and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS-hsp70 females with homozygous GAL4 driver males. For each cross, 20-30 males and 50 virgin females were mated for two days. At least 200 0-4 h eggs were collected from each cross and transferred with forceps onto petri plates filled with media . The plates were then incubated at 25°. Hatching rates were assessed 26-30h after egg transfer by scoring the number of empty eggshells. Pupation and adult emergence rates were scored 5-6 days and 10-12 days after egg transfer respectively.Control animals were obtained by crossing w males with homozygous UAS-hsp70 females (UAS control) and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS-hsp70 females with homozygous GAL4 driver males. 0-2 days old males and females were collected from control and experimental crosses and aged at 25° in separate vials with media  until tested. Paraquat oxidative stress tests were performed as previously described  and homozygous GAL4 driver males with w females (GAL4 control). Experimental animals were generated by crossing homozygous UAS females with homozygous GAL4 driver males. 0-2 days old males and females were sorted and collected from control and experimental crosses under nitrogen anesthesia for less than three minutes. The longevity of each genotype is obtained from four vials of males and four vials of females  kept at 25°. Animals were transferred to vials with fresh media and the mortality recorded at least twice a week. Four to eight replicates were measured. Each replicate used independent cultures and parents. Kaplan-Meier (Log-rank) analysis with GraphPad Prism 5 was used to determine the significance of experimental animals that displayed a mean longevity higher or lower than both kinds of control animals.hsp70 by the UAS-hsp70-RNAi transgenes. Supplemental Table S1 contains the data and statistical analysis of the survival during development. Supplemental Table S2 contains the data and statistical analysis of the stress resistance experiments. Supplemental Table S3 contains the data and statistical analysis of the longevity experiments with the UAS-hsp70 transgene. Supplemental Table S4 contains the data and statistical analysis of the longevity experiments with the UAS-hsp70-RNAi transgene. The reagent table provides the information and source of transgenic strains, DNA plasmids and antibodies used in this study. Supplemental material available at figshare: https://doi.org/10.25387/g3.9916223.Strains and plasmids are available upon request. All data necessary for confirming the conclusions of the article are present within the article, figures and supplemental files. Supplemental Figure S1 contains western-blot data obtained with an HSP70 antibody. Supplemental Figure S2 shows the outcome of the survival data to thermal, oxidative and metabolic stresses of 3 days and 10 days old females overexpressing HSP70. Supplemental Figure S3 shows the outcome of the longevity data of females overexpressing HSP70. Supplemental Figure S4 reports the cloning steps performed to obtain the pCX9 plasmid. Supplemental Figure S5 contains western-blot data that show the down-regulation of E. coli β-galactosidase  (P < 0.0001) but modest effect on the expression level.Transgenic UAS-hsp70 fly strains that carry the Hsp70Ab cDNA under the control of a UAS promoter have already been obtained and described (tibodies  and S1.  of GAL4 . Therefo of GAL4 . Three a of GAL4 . At ten  0.0001) . AlthougP < 0.0001) but not with the D42 driver (P = 0.5751). With both drivers, the location of the UAS transgene has a significant effect on the expression level and the range of expression obtained is wider than with the da-GAL4 driver. Between the genotypes with the lowest and highest expression level, there is a ∼40% (10 days) to ∼130% (3 days) difference with da-GAL4, a ∼135% (3 days) to ∼165% (10 days) with DJ694 and a ∼410% (3 days) to ∼560% with D42 (10 days).In agreement with the GAL4 expression profile, HSP70-myc levels increased significantly between three and ten days with the DJ694 driver . High temperature stress was tested by exposure at 36°. Oxidative stress was assessed by feeding the free radical generator paraquat. Desiccation stress was also examined with a dry starvation assay. At least three and up to 10 replicates were processed per stress, sex, age, UAS-hsp70 insertion and GAL4 driver. The results from each replicate are compiled in the The longevity of flies with both UAS-hsp70 and GAL transgenes was measured and compared to control flies lacking the GAL4 or UAS transgene (Table S3). Consistent results are only seen across all UAS-hsp70 insertions in males with the ubiquitous driver . All insLife span extensions were consistently obtained across replicates with only two (#3.2 and #10.1) out of the seven insertions tested. Both insertions driven by DJ694 or D42 extend the longevity of males in all replicates except for #3.2 that revealed an extension in 5 out of 6 replicates with D42. The extensions associated with #3.2 appear to be also consistent in the females but with a lower magnitude. The extensions associated with #10.1 is seen in females with DJ694 but not with D42. The increased longevity with these insertions is not correlated with the level of hsp70 expression. With D42, #3.2 and #10.1 express the highest and lowest levels respectively. With DJ694, #3.2 and #10.1 express high levels that are similar to the #9.1 insertion that does not display any change in all replicates. Since the extension of longevity is not observed with all the insertions tested and is not correlated with HSP70 levels, the longevity phenotypes observed with the #3.2 and #10.1 are an artifact of the transgene insertion site.hsp70 cDNA section was excised from the pCX1 plasmid . Seven out of the nine RNAi insertions completely prevented the detection of the transgenic HSP70-myc whereas it remains detectable with R5.2 and unaffected with R5.12. Next the effectiveness of the R3.12 insertion to reduce endogenous hsp70 expression was examined with the three GAL4 drivers . As one would expect from the anatomical location of GAL4 expression, almost complete or strong reduction of heat-induced HSP70 is observed with daGAL4 and DJ694 respectively whereas D42 expression is too localized to affect whole animal extracts. Finally, the effects of the DJ694-driven R3.12 and R5.14 insertions on endogenous HSP70 expression were compared (The compared . Both inThe longevity with both UAS-hsp70-RNAi and GAL transgenes was measured and compared to control flies lacking the GAL4 or UAS transgene (Table S4). Two out of the three RNAi insertions tested with all drivers did not show any effects in both sexes . These rHsp70 mRNA (Hsp70 are present (Hsp70 mutants have demonstrated that HSP70 is required for basal and induced thermotolerance of larvae and adults (Hsp70 with any of the three GAL4 drivers did not alter the response of adult flies to thermal, oxidative or metabolic stresses. Although it has long been known that increasing Hsp70 expression is sufficient to increase the thermotolerance of cultured cells (in vivo in the whole organism (Hsp70 expression is sufficient to affect thermotolerance of embryos (Hsp70 overexpression on the resistance to oxidative and wet starvation stresses has been investigated in the presence of additional Hsp70 copies and did not have any consequences with or without heat shock (Hsp70 expression alone is insufficient to affect the resistance of the organism to thermal, oxidative or metabolic stress.The UAS/GAL4 system allowed extensive and precise control of Hsp70 expression within a constant environment. The manipulation of HSP70 level during the pre-adult stages did not cause any visible morphological defect, lethality or developmental delays. Under normal conditions, p70 mRNA . Develop present  but the at shock . The UASHsp70 expression with RNAi transgenes did not affect longevity. Although the western-blot analysis confirmed the effectiveness of the transgenes, it remains possible that there is residual expression. The null Hsp70 mutant does completely prevent expression and has been recently reported to exhibit a modest reduction of the life span (Hsp70 is required for longevity and that only minimal amount is needed accounting for the lack of correlation between longevity and Hsp70 RNA levels in wild-type strains (Hsp70 levels above wild-type levels without any environmental modifications that affect the expression of additional genes. Ubiquitous GAL4-mediated Hsp70 overexpression reduced male longevity while ubiquitous overexpression by heat treatment of males carrying extra Hsp70 copies had the opposite effect (Hsp70 has detrimental effects on longevity that are mitigated or abolished by the simultaneous expression of one or several gene products induced by the heat treatment. After extended periods of recovery from heat shock HSP70 intracellular granules are formed to control HSP70 activity by sequestration (Hsp70 with the da-GAL4 driver during the pupal stage may not be detrimental enough to cause lethality and instead caused the emergence of sick animals. It is unlikely that the detrimental effect is due to overexpression in the nervous system or muscles since it is not observed with the D42 or DJ694 drivers. It will be worth investigating additional GAL4 drivers with different tissue-specificity in order to identify the anatomical location responsible for the detrimental effect in males.The reduction of Hsp70 in the nervous system, muscles in both sex or ubiquitously in females had neither beneficial nor detrimental effects on life span indicating that HSP70 is insufficient to extend longevity. It is important to note that identical results were obtained in muscles with the MHC-GAL4 driver and independent UAS-hsp70 strains (Hsp70 copies would suggest that HSP70 extends longevity in combination with one or several gene products induced by the heat treatment. The identification of HSP70 partners could easily be investigated by measuring the longevity of animals carrying a UAS-hsp70 transgene as well as a UAS transgene for a gene to be tested.The overexpression of  strains . However


Cdk6, which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6, other Pax6-regulated cell cycle genes are likely to be primary mediators of Pax6’s actions on cortical progenitor cell cycles. Following acute cortex-specific deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6’s actions. We then used flow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase.Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene  Cerebral corticogenesis requires precise spatio-temporal control of the rates of progenitor cell division. Previous research on the developing mouse embryo established that the transcription factor Pax6 is a key regulator of this process . Pax6 isCdks drive progression through the eukaryotic cell cycle by forming complexes with cyclins . In mostHere we addressed several questions. Are the effects of Pax6 on the cell cycle likely to be mediated primarily through actions on Cdk6, or does Pax6 control multiple cell cycle regulators in parallel? Do the effects of Pax6 vary between cell cycle phases? Are levels of Cdk6 normally constant throughout the cell cycle of cortical progenitors? Does Pax6 modulate Cdk6 levels equally or differentially across the phases?Pax6-/- mutants  to test for changes in the expression of a set of cell cycle regulators following acute cortical deletion of Pax6. The set we tested contained all cell cycle regulators that were shown in previous work to have altered cortical expression levels in constitutive  mutants . Here wePax6Sey allele . Dissociated cell suspensions diluted to 1.8 × 106 ml-1 were fixed and permeabilised in 100% ethanol and stored at -20°C.All mice were bred in-house. The University of Edinburgh Animal Welfare and Ethics Board and a license from the Home Office UK issued under the UK Animals (Scientific Procedures) Act 1986 regulated all procedures. For constitutive inactivation of Pax6, we used the 6- here; . For conP allele  and a grP allele  with BAC1CreERT2 . Dams wecDNA was synthesized from RNA with a Superscript reverse transcriptase reaction  and qRT-PCR was done with a Quantitect SYBR Green PCR kit  and a DNA Engine Opticon Continuous Fluorescence Detector . Primer pairs are listed in Table Aliquots of permeabilised and fixed cells suspended in fluorescence activated cell sorting (FACS) buffer were reacted with Hoechst 33342 . Primary antibodies used were: anti-Pax6 ; anti-Tuj1 ; anti-PH3 ; anti-Ki67 ; anti-Cdk6 . We used secondary-only and fluorescence-minus-one controls to set thresholds for primary antibody-specific staining. Cells were analyzed using an LSRII flow cytometer and FlowJo V10 software . In all of our experiments, we used the same gate for forward scatter for both wild-type and mutant progenitor cells. Since forward scatter values in flow cytometry are directly linked to cell size, this indicates that the wild-type and mutant cells were in the same size range.-1 IdU and then 1.5 h later with the same dose of BrdU. They were sacrificed after 30 min and histological sections were cut through the cortex. Primary antibodies used were anti-BrdU/IddU (which recognizes both BrdU and IddU)  and anti-BrdU . Nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes). Analysis was as described in  and littermate control Pax6fl/+;Emx1CreER embryos at E9.5. All embryos contained a floxed-stop GFP reporter allele  in gene expression at E12.5 and this was sustained at E13.5  of progenitors in Pax6+/+ and Pax6-/- E12.5 and E14.5 cortex using double labeling with iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) . At E14.5, Tc was ∼15% shorter in Pax6-/- cortex than in Pax6+/+ cortex   . At E12.Pax6+/+ and Pax6-/- embryos . This was only the case in G1 at E14.5 (p < 0.01). These findings on rostral versus caudal differences are in excellent agreement with previous work showing a rostral [high] to caudal [low] gradient of Pax6 expression at E12.5 and E13.5 that flattens by E14.5  directly affect the lengths of these phases.Our new results support a central role for Cdk6 in Pax6’s regulation of cortical progenitor cell cycles. Figure  Figures . OscillaThis proposal has similarity to the mechanism proposed to regulate an engineered minimal control network in fission yeast . This covice versa. An additional complexity would be explaining the dynamics of this scenario. Changes in Pax6 protein levels would need a certain time to influence transcription of the Cdk6 gene and the mRNA would then need to be translated to alter Cdk6 protein levels. Although we do not exclude the possibility that fluctuations in Pax6 levels might contribute to the temporal pattern of change of Cdk6 levels, the simplest suggestion is that Pax6 reduces Cdk6 levels overall and that the oscillations in Cdk6 levels during the cell cycle are driven by other mechanisms. Our previous work showed that Pax6 overexpression significantly reduced Cdk6 levels . G1 length is increased in neurogenic progenitors compared with proliferative progenitors . This isDM, MM, and Y-TH performed the experiments. DP and JM helped to design the experiments, obtained the funding, and supervised the work. DP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Density functional theory was applied to simulate the structure of crystalline KAlF4 while a quantum chemistry ab initio simulation was performed to explore the structure of molten KAlF4. Two crystal polymorphs demonstrated to be present in solid KAlF4. At the temperature below 673 K, it belongs to the tetragonal crystal system within the P4/mbm space group, while the high temperature phase is attributed to the monoclinic crystal system within the P21/m space group. Both polymorph KAlF4 phases are characterized by a layered structure consisting of K+ and [AlF6]3− octahedra, each of the [AlF6]3− octahedra equivalently shares four corners with other four [AlF6]3− octahedra along the layer. The layered structure became unstable at higher temperatures and crashed when the temperature exceeded the melting point. It demonstrated that the molten KAlF4 consisted of predominant [AlF4]− and a small amount of [AlF6]3−. The Raman spectrum of molten KAlF4 simulated by using a quantum chemistry ab initio method agreed well with the experimental Raman spectrum.In situ high temperature X-ray diffraction and Raman spectroscopy were used to investigate the temperature dependent micro-structure of KAlF Akdeniz concluded that the Raman peaks observed at 621 cm−1 for NaAlF4 are related to the Al-F bond of isolated [AlF4]− clusters with a bond length of 1.69 Å 18]) vers4, a simulation based on a quantum chemistry ab initio method was also carried out. A series of aluminum fluoride model clusters with typical characteristic structures were proposed and optimized for geometric configuration before the simulation. The restricted Hartree-Fock (RHF) 3− octahedra, each [AlF6]3− octahedron equivalently shares four corners with other four [AlF6]3− octahedra within the layer. The [AlF6]3− octahedron consists of a six-coordinated aluminum atom which is linked to six fluorine ions including four bridging fluorine ions and two non-bridging fluorine ions. 4 crystal, drawn by the VESTA software 3− octahedra, where each [AlF6]3− octahedron shares four corners with other [AlF6]3− octahedra within the layer. The potassium ions between the [AlF6]3− octahedron layers were used to keep the charge balanced. Comparing with the room temperature phase, the structure of the high temperature phase is more distorted due to the temperature impact, resulting in a more complicated XRD spectrum, as evidenced in 4 was calculated  from room temperature to 823 K and at a heating rate of 5 K/min afterwards. The high temperature spectra were recorded 5 min after the sample reached the target temperature. 6]3−n clusters (by ab initio method) which refers to our previous work 3− octahedra with the high temperature phase. As clearly shown in 4 structure as it evolves from the room to high temperature structure. The room temperature and high temperature phase are drawn in a simple diagram. In 4 turns from the tetragonal to monoclinic structure approximately at 723 K, the space group changes from P4/mbm to P21/m. The cell length becomes much longer, and one of the cell angles increases from 90.000 to 108.478°. As a consequence, the distribution of the bond length and bond angle of KAlF4 becomes more complicated.Two new Raman bands at 319 and 481 cmAs the sample temperature increased further, the sample was fully melted at 893 K and is characterized by the molten structure as evidenced in 4]− is the main species in molten KAlF4. 4]−n model clusters presented in molten KAlF4. After being optimized for geometry configuration, these model clusters were used for a quantum chemistry ab initio method to calculate the Raman spectra. The characteristic vibrational wavenumber of the Al-F symmetric stretching vibrational modes of the [AlF4]−n clusters were then estimated accordingly.According to the literatures ,30, [AlF4]− tetrahedra in the neighbor of an isolated [AlF4]− tetrahedron in the melt according to the crystal KAlF4 structure in KAlF4. In 4]−n cluster. As the number of molecules in the clusters increased, the characteristic wavenumber of the Al-F symmetric stretching vibrational mode of [AlF4]−n appeared to increase. The symmetric stretching vibrational wavenumber of the model cluster with seven molecules of [AlF4]− was therefore estimated to be 625.6 cm−1, which was in good agreement with the values observed in the experiment and reported in the literature − tetrahedron, appeared at 863 K. Simultaneously, the intensity of the Raman band of 628 cm−1 increased while the intensity of the Raman band of 536 cm−1 decreased. The six-coordinated Al atom began to turn into four-coordinated. At 893 K, KAlF4 had been completely melted, revealing the symmetric stretching vibrations of AlIV-Fnb of the [AlF4]− tetrahedron and little symmetric stretching vibrations of AlVI-Fnb of the [AlF6]3− octahedron were in the melt.As shown in 4 was investigated by in situ high temperature X-ray diffraction and Raman spectroscopy. The major vibrational modes of temperature dependent Raman spectra of KAlF4 were assigned based on the calculated spectra by both density functional theory and quantum chemistry ab initio methods.The structure evolution of KAlF4 was found to present in two different polymorph phases at room and high temperature. Both polymorph KAlF4 phases are characterized by a layered structure consisting of K+ and [AlF6]3− octahedra, where each [AlF6]3− octahedron equivalently shares four corners with other four [AlF6]3− octahedra along the layer. While the room temperature phase is in the tetragonal cell (space group P4/mbm), the high temperature phase undergoes a monoclinic distortion (space group P21/m) together with an ordering of the K atoms.Below the melting point, solid KAlF4]− tetrahedra when the temperature exceeds the melting point. It was hence indicated that in molten KAlF4, there is a principal amount of [AlF4]− and a small amount of [AlF6]3−. This was further confirmed by quantum chemistry ab initio simulations.The layer structure decomposed with increasing temperature and finally turned to [AlF


ELF5) and ETS‐homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis‐regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site‐specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC‐seq in these lines revealed novel peaks of open chromatin at the 5’ end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C‐seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP‐seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA‐mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.E74‐like factor 5 ( Of note, mutations in CFTR do not correlate with lung disease severity, the major cause of morbidity in CF.Many monogenic disorders have significant phenotypic variability that may be attributed to genetic elements outside the causative locus. These sites (modifier loci) are often identified by genome‐wide‐association studies (GWAS).CFTR. The high frequency of the disorder facilitated a replicated GWAS, which identified single nucleotide polymorphisms (SNPs) in an intergenic region of chromosome 11p13 that significantly associated with CF lung disease severity.EHF) and E74‐like factor 5 (ELF5) on the 5’ side and Apaf‐1‐interacting protein (APIP) on the 3’ side.CF impacts about 70,000 people worldwide EHF is abundantly expressed in differentiated epithelial tissues, particularly in the prostate, pancreas, salivary gland and airway.CFTR regulation, as it binds to an airway selective enhancer element of CFTR and represses its expression.EHF is a strong candidate CF modifier gene and also a potential CF therapeutic target since elevated CFTR substrate may enhance the efficacy of correctors and potentiators.EHF expression, since ChIP‐seq with an antibody specific for ELF5 showed its occupancy at the EHF promoter and first intron in the breast cancer cell line T47D.In contrast, the role of ELF5 has been studied in breast cancer, where it drives lung metastasis by recruiting myeloid‐derived suppressor cells.cis‐regulatory elements (CREs) at chr11p13 that may control the expression of these two epithelial selective ETS transcription factors. Using CRISPR/Cas9 technology, three key CREs were deleted from the region in airway epithelial cells, and subsequent changes in gene expression and chromatin architecture were examined. We also took an unbiased screening approach to identify additional CREs at chr11p13 by activating multiple DNase I hypersensitive sites (DHS) across the region using CRISPRa,EHF by investigating open chromatin and 3D architecture in cell types with different abundance of ELF5. These genomic context studies revealed a critical role for ELF5 in coordinating the complex regulatory environment at chr11p13. Our results are relevant to the involvement of both ELF5 and EHF in lung disease, since both genes are expressed in human bronchial epithelium.Although to date, the highest p‐value SNPs identified in the replicated GWAS do not correlate with the transcription of genes at 11p13, this may reflect a paucity of data on gene expression at the single‐cell level. However, revealing the regulatory interactions of the EHF and ELF5 TFs is critical to understanding their role in lung epithelial biology. Here we investigate the 22.1A549,2.2Luciferase promoter:enhancer constructs were generated as described previously.2.3EHF intron 6, 11.2521 and 11.2516 were identified using Benchling Two pairs of gRNAs flanking 2.4RNA was extracted from cells using TRIzol (In Vitrogen) according to manufacturer's instructions. Reverse transcription used TaqMan Reverse Transcription Reagents (Life Technologies) by the standard protocol. qPCR assays were performed using SYBR Green reagents (Life Technologies) and primers listed in Table 2.5Chromatin immunoprecipitation (ChIP)‐seq was performed as previously described 2.64C‐seq was performed as previously described 2.7ELF5 and EHF promoters were designed within 1 kb 5’ to the transcriptional start sites (TSS). Two to four sgRNAs were designed to target DHS cores at chr11p13 using Benchling Single guide RNAs (sgRNAs) targeting 2.8Omni‐ATAC‐seq was performed on 50,000 Calu3, LNCaP and T47D cells as described previously2.9ELF5 depletion in T47D cells was performed using Silencer select siRNA  as previously described.2.10t tests. All statistical analysis was performed in Prism software (GraphPad).Error bars denote standard deviation (SD) or standard error of the mean (SEM) as noted in figure legends, which also define the statistical analysis used. This was either two‐way ANOVA plus multiple comparisons tests or Student's unpaired 33.1EHF locus coincides with an extended track of the H3K27ac active histone mark, indicative of a stretch enhancer in airway epithelial cells.EHF intron 6 lies at the 3’ end of this feature and coincides with a region of high occupancy by multiple transcription factors (ENCODE data EHF intron 6 (right) are shown in Figure ELF5, EHF and APIP genes was assayed by RT‐qPCR  expression.The R Figure B. RemovaEHF are recruited to the intron 6 element or that loss of this site alters chromatin architecture at the locus, thus facilitating occupancy of activating factors at the EHF promoter or its enhancers. To investigate the latter hypothesis, we first assayed CTCF occupancy at specific sites across the chr11p13 region by ChIP‐qPCR. CCCTC‐binding factor (CTCF) is an architectural protein involved in higher order chromatin organization. It is recruited to invariant sites in the genome  boundaries) in addition to variant structural elements that may be cell‐type specific.EHF intron 6 element binds CTCF with high affinity in several airway cell types,EHF intron 6 element was associated with a substantial (~3‐fold) decrease in CTCF occupancy at the adjacent site DHS11.2516 , most notably close to 11.2516 and within the first intron of EHF. With a viewpoint located 20kb 5’ to the EHF promoter (11.2516), interactions with the ELF5 and EHF promoters were notably reduced and those with HB11.1485 element were slightly diminished (black arrows). These results are consistent with our earlier data from WT cells showing that DHS11.2516 and the EHF intron 6 element interact directly.EHF promoter viewpoint shows a modest loss of interactions across the whole region, most prominently between HB11.1485 and the ELF5 promoter (black dotted line). In summary, these data suggest that looping of the CRE at DHS11.2516 to the EHF and ELF5 promoters is somewhat dependent on the EHF intron 6 element and moreover that this element contributes to the maintenance of locus architecture.To examine the impact of loss of the s Figure D. A mini3.2ELF5 and EHF promoters (though not APIP), by luciferase assays in 16HBE14o‐ bronchial epithelial cells.ELF5, EHF and APIP expressions were measured using RT‐qPCR .The 11.2521 CRE was shown previously to be a strong enhancer of both the ELF5 and EHF promoters in transient assays,ELF5 upon deletion of this CRE suggests that features of the genomic architecture of the region may have a dominant role in regulating gene expression. To address this hypothesis, we measured CTCF occupancy at known binding sites across the region using ChIP‐qPCR. The 11.2521 CRE itself binds CTCF with low affinity in 16HBE14o‐ cells though not in other airway cell types.Since the 11.2521 CRE is an enhancer of the ELF5 promoter, loss of the 11.2521 CRE increased its interactions with DHS11.2512/11.2513 and an uncharacterized element immediately 3’ to EHF (black arrows). However, the deletion had no impact on interactions with the HB11.1485 viewpoint, while associations with the EHF promoter were modestly reduced (black dotted line). These data suggest that the 11.2521 CRE may inhibit ELF5 promoter interactions with previously uncharacterized enhancer elements, and that its functions depend upon specific chromatin architecture at chr11p13.Next, to investigate the impact of removal of the 11.2521 CRE on chromatin architecture across chr11p13, we performed 4C‐seq on the deletion clones and compared them to WT A549 c9 cells , by luciferase assays in 16HBE14o‐ cells.ELF5, EHF and APIP expressions were measured using RT‐qPCR .The 11.2516 CRE was also shown previously to be a strong enhancer of both the R Figure B. RemovaEHF intron 6 (~1.4‐fold increase), though these alterations did not reach statistical significance . These data are consistent with our previous results showing direct interaction of 11.2516 with these sites.ELF5 promoter, EHF promoter and EHF intron 6 (black arrows). Loss of the 11.2516 CRE also coincided with a modest reduction of interactions between the EHF promoter and the rest of the region, with the exception of an unidentified element located between HB11.1485 and 11.2516 (black arrow), which showed an enhanced association. These data suggest that 11.2516 has an important role in the maintenance of chromatin architecture at the chr11p13 region.Next, to examine the impact of removal of the 11.2516 CRE on chromatin architecture, we performed 4C‐seq on the deletion clones and compared them to WT A549 c9 cells Figure D. Again,3.4ELF5 transcript abundance is very low ELF5 to EHF transcripts is highest in T47D cells, and the reverse is seen in the LNCaP line ‐seq ELF5).None of the airway cell lines used in this work and our earlier analysis of the chr11p13 region w Figure . HoweverELF5 and EHF loci in the different cell lines  between the ELF5 promoter viewpoint, the novel peak of open chromatin at 11.2513 and a site in EHF intron 1, also associated with an ATAC‐seq peak. Interactions with the 3’ TAD boundary and an element 3’ of 11.2527 were also seen.Since expression of EHF promoter viewpoint, the major specific difference seen in Calu3, LNCaP and T47D cells was a great reduction in interactions with the HB11.1485 5’ sub‐TAD boundary when the ELF5 gene is highly expressed (T47D) (black arrows). A minor reduction of interactions with the EHF intron 6 CRE was also observed in LNCaP and T47D cells (black arrows). In addition, in Calu3 cells only, interactions were evident with the airway enhancers (11.2516 and 11.2521), EHF intron 6 and the 11.2525/11.2526 sites closest to the highest p‐value SNP (rs10742326) from the GWASELF5.Using the 3.5ELF5 this locus may have an important role in the functional genomics of the chr11p13 region. Our earlier in vitro studies suggested that the 11.2516 and 11.2521 CREs enhanced activity of both the ELF5 and EHF promoters p65 subunit [p65], and Replication and transcriptional activator [Rta] (SP‐dCas9‐VPR) to target sites. A minimum of two guide RNAs were designed to target the ELF5 and EHF promoters and multiple airway DHS across chr11p13 in 16HBE14o‐ and A549 cells . Expression of ELF5 in 16HBE14o‐ cells was also increased significantly by CRISPRa at the EHF intron 6 (~3.5‐fold), 11.2521 (~7.5‐fold), 11.2522 (~3‐fold), 11.2523 (~5‐fold), 11.2529 (~4.5‐fold) and 11.2530 (~4‐fold) DHS . The modest response to CRISPRa at chr11p13 suggests that either additional sequences, which are not associated with regions of open chromatin in airway cells, are key in transcriptional activation of ELF5 and EHF, or that a more complex regulatory mechanism exists for the genes in this region. The latter explanation would be consistent with our 4C‐seq observations suggesting close interactions between the ELF5 and EHF genes and their CREs.The data shown in the previous sections suggest that in cells that express s Figure . Targetis Figure . APIP ex3.6ELF5 and EHF loci and their CREs might underlie the complex regulatory environment at chr11p13. We first performed siRNA‐mediated depletion of ELF5 in T47D cells, which express ELF5 and EHF, and assayed expression of both genes by RT‐qPCR. Primers located in exons 5 and 6 for ELF5 and exons 3 and 4 for EHF  analysis of two replica experiments is shown in Figure EHF locus, most notably in introns 1 and 6 and close to its 3’ end (black arrows). Each of the peaks of ELF5 binding also coincides with a peak of open chromatin in T47D cells, mapped by ATAC‐seq , suggesting that ELF5 may have a major impact across 11p13, both through controlling other genes and by autoregulation. These results are highly relevant to primary bronchial and tracheal epithelial cells, which also express both ELF5 and EHF, albeit with much lower ELF5 abundance than in T47D cells.A previous study4ELF5 and EHF within the region and defined important aspects of the chromatin architecture relevant to the regulation of genes at chr11p13.EHF locus.Intense interest in the chr11p13 region arose through its association with lung disease severity in CF; however, the genetic mechanisms underlying the functions of this region are not well understood. Our data reveal a complex regulatory network at chr11p13 that coordinates the expression of the ETS transcription factors ELF5 and EHF. We previously identified several putative airway epithelial cell‐selective enhancer elements of EHF intron 6 and the two airway epithelial cell‐selective enhancers at 11.2516 and 11.2521 using CRISPR/Cas9 protocols had unexpected effects on gene expression, and were associated with modest changes in CTCF occupancy and local higher order chromatin structure. Firstly, removal of the two enhancers that were shown to activate the ELF5 and EHF gene promoters in transient luciferase assaysELF5 or EHF expression. Removal of either element enhanced ELF5 transcript abundance and had no impact on EHF levels. Though these data do not support a simple enhancer role for the 11.2521 and 11.2516 CREs, these elements are both marked with active histones (H3K27Ac) and furthermore lack repressive marks in several airway cell types, supporting their role as enhancers in the genomic context.ELF5 and EHF gene expression levels in their endogenous location, perhaps providing functional redundancy. These factors may include features of the higher order chromatin structure and organization. This interpretation would be consistent with the luciferase assays showing enhancer activity, since within the reporter gene constructs potential CREs are sited next to a gene promoter without consideration for architectural barriers such as CTCF sites. It is also possible that eRNA transcription arising at these CREs may repress nearby gene expression.EHF intron 6  and an altered chromatin architecture associated with expression of ELF5. Two sites are of particular interest: 11.2513, is a peak of open chromatin in primary HTE cellsEHF locus, which is not evident in primary HTE cells, but is clearly seen in both ELF5‐expressing cell types studied here. Upon removal of the 11.2521 enhancer from airway cells, the ELF5 promoter gains interactions with this element , which are essential for cell motilitySPDEF),MUC5AC) gene,EHF locus and represses its expression, it is likely that this ETS factor also plays an important role in lung biology. Modulation of ELF5 expression would be predicted to impact many of the EHF‐mediated processes discussed above. Although EHF remains the primary candidate for influencing lung phenotype differences observed in CF patients, ELF5 likely also plays an important role through its regulation of EHF expression. These observations are not only relevant to CF but also to other airway diseases associated with inflammation, defective wound response and lung tissue remodelling, including asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis.Returning to the role of chr11p13 as a modifier of lung disease severity, we do not address here the mechanisms underlying the association of high p‐value SNP close to the chr11.2525 site. However, our observations on cell‐specific recruitment of CREs across the modifier region and most importantly on the direct regulation of The authors confirm that there are no conflicts of interest.HS, JSE, KML and AH conceived and designed the project. HS, JSE, KML, SY, JLK, MN, CC, EMM and S‐HL designed and performed experiments. HS and AH wrote the manuscript. Click here for additional data file. Click here for additional data file.


All proliferating cells need to match metabolism, growth and cell cycle progression with nutrient availability to guarantee cell viability in spite of a changing environment. In yeast, a signaling pathway centered on the effector kinase Snf1 is required to adapt to nutrient limitation and to utilize alternative carbon sources, such as sucrose and ethanol. Snf1 shares evolutionary conserved functions with the AMP-activated Kinase (AMPK) in higher eukaryotes which, activated by energy depletion, stimulates catabolic processes and, at the same time, inhibits anabolism. Although the yeast Snf1 is best known for its role in responding to a number of stress factors, in addition to glucose limitation, new unconventional roles of Snf1 have recently emerged, even in glucose repressing and unstressed conditions. Here, we review and integrate available data on conventional and non-conventional functions of Snf1 to better understand the complexity of cellular physiology which controls energy homeostasis. Cell growth and proliferation require a high amount of energy for biosynthetic pathways. Cells take energy from nutrient intake and both unicellular and multicellular eukaryotes have evolved systems that allow dynamic sensing of energy sources, mainly sugars. The class of Snf1/AMPK (Sucrose non-fermenting/AMP-activated protein kinase) plays a key role as a guardian of cellular energy Saccharomyces cerevisiae and its conventional roles in the regulation of metabolism, stress response and aging. In addition, we also focus on recent advances showing emerging functions of Snf1 on the modulation of key processes such as endocytosis and cellular trafficking as well as cell cycle, proliferation and metabolism.Here we discuss the mechanisms of action of Snf1, a member of the Snf1/AMPK family in Protein kinase Snf1 in yeast is a heterotrimeric complex made by the catalytic α subunit Snf1, a regulatory β subunit  and the γ subunit Snf4 SNF1 gene) was identified in a screening of mutants unable to grow in presence of sucrose as carbon source 5The catalytic α subunit  are present. They share partially redundant functions, since only the triple mutant sip1Δsip2Δgal83Δ strain showsgrowth defects when glycerol or ethanol are added as carbon sources 711In SNF1, the gene encoding the γ subunit, SNF4, was identified by isolation of a sucrose non fermenting mutant 10SNF4 deletion causes a decreased kinase activity of Snf1, whereas deletion of the AIS domain of Snf1 fully complement the phenotype of a snf4Δ strain 19snf4Δ strain Similarly to sak1Δtos3Δelm1Δ strain shows the same phenotype of a snf1Δ strain, such as growth defects in presence of limiting glucose or alternative carbon sources like glycerol or ethanol Snf1 complex is activated through phosphorylation of the Thr210 of the α subunit by one of the three constitutively active upstream kinases Sak1, Tos3 and Elm1 22Although Snf1 phosphorylation is a key step for its activation, a non-phosphorylatable Snf1 mutant (Snf1-T210A) retains a low catalytic activity, originating intermediate phenotypes 24262728On the other side, in response to high glucose concentrations Snf1 is inactivated through dephosphorylation of Thr210 by the Gcl7 protein phosphatase , which is targeted to Snf1 by the adaptor subunit Reg1 303233Active Snf1 phosphorylates serine and threonine residues contained in the consensus pattern Φ-x-R-x-x-S/T-x-x-x-Φ, where Φ is a hydrophobic residue Differently from its mammalian homolog AMPK, yeast protein kinase Snf1 is not allosterically activated by AMP 37The structure of the kinase domain of Snf1 showed that it is a dimer, which represents an inactive form of the kinase, since Thr210 is inaccessible for phosphorylation by the activating kinases (i) phosphorylation of Ser214, inside the activation loop, downregulates Snf1 function (ii) SUMOylation of the catalytic α subunit Snf1 inhibits its activity, possibly by attenuating its levels in the cell and/or favoring the inactive conformation of the kinase (iii) the SAGA acetyl transferase complex deubiquitylates Snf1 affecting the stability of the complex and its kinase activity  (iv) the ubiquitin-associated motif (UBA) of the α subunit Snf1 indirectly regulates SNF1 gene expression and Snf1 interaction with the γ subunit Snf4 Interestingly, some recent evidence indicates additional mechanisms that regulate Snf1 activity: http://www.yeastgenome.org, 92 of which are also Snf1 substrates (identified by high throughput or low throughput assays). In Figure 2 we clustered them on the base of their function. Apart from the most known interactors involved in Snf1 complex regulation, transcription, histone modification, signaling and metabolism, there are many proteins which regulate translation, ribosome function, intracellular transport/trafficking and cell cycle. In addition, some of them are also proteins of the ubiquitin/proteasome machinery, chaperones and Fe/S cluster proteins . Remarkably, only a few of them have been extensively investigated for the physiological relevance of Snf1-dependent phosphorylations, suggesting that many functions of Snf1 are still to be discovered.A total of 216 proteins physically interacting with Snf1 are annotated in SGD , maltose (the MAL regulon) and galactose (GAL4) HXT2, HXT4) TPS1, essential for the metabolism of trehalose Mig1 is the most important glucose-regulated transcriptional repressor 4852CAT8 gene is activated by Snf1 through inhibition of Mig1 Besides Mig1, Snf1 regulates the activity of other transcription factors. Cat8 and Sip4, which bind Carbon Source Responsive Elements (CSRE), regulate the expression of gluconeogenic genes 4961ADY2 gene. In fact, Snf1 stimulates the binding of Gcn5 and the acetylation of histone H3 at ADY2 promoter, promoting the transcription of this gene Snf1 has been reported to phosphorylate Ser10 of histone H3 and to promote the acetylation on Lys14 of histone H3 by Gcn5, a component of the SAGA complex INO1 expression 65Besides its role in regulating the transcription of several genes involved in metabolism, Snf1 directly regulates, through phosphorylation, important metabolic enzymes. In fact, together with the class of transcription factors and regulators, proteins linked to metabolism are the most abundant among Snf1 interactors . Probably the most impactful function exerted by Snf1 as a direct regulator of metabolism is the regulation of the acetyl-CoA carboxylase Acc1 Grr1-dependent degradation of Pfk27 Snf1 was also shown to phosphorylate Pfk27, the second isoform of 6-phosphofructo-2-kinase Moreover, Snf1 phosphorylates Gpd2, the glycerol-3-phosphate dehydrogenase required for anaerobic growth, thus inhibiting glycerol synthesis during the diauxic shift. In fact, it was reported that Snf1 phosphorylates Gpd2 on Ser72 priming Gpd2 for subsequent phosphorylation on Ser75, probably by Yck1 In yeast, the main pathway activated by glucose is the PKA pathway, involved in metabolism, growth and proliferation 71727176Notably, recent data nicely complement observations of a cross-talk between Snf1 and PKA. Indeed, the adenylate cyclase Cyr1 and Snf1 interact in a nutrient-independent manner The Target Of Rapamycin Complex I (TORC1) is a highly conserved nutrient-responsive regulator of cell growth and metabolism in all eukaryotes 80818381858780Kog1 is phosphorylated by Snf1 under glucose deprivation, as AMPK does on the ortholog Raptor in vitro kinase assay and epistasis analysis, Sch9 has been shown to be also a target of Snf1, indeed total Sch9 phosphorylation is reduced in snf1Δ mutant In the presence of nutrients, TORC1 phosphorylates and activates Sch9, the ortholog of S6K in yeast which, together with other substrates, drives ribosome biosynthesis 9192Interestingly, Orlova and coworkers showed that rapamycin treatment results in a significant increase of Thr210 phosphorylation on Snf1 snf1Δ cells, the translocation of GFP-Atg8 to the vacuole is reduced by 50% compared to the wild type TORC1 activity is involved in the regulation of autophagy, a cellular recycling system that degrades proteins and organelles by delivery to the vacuole in response to nutrient deprivation 98ENA1, responsible for Na+ ions detoxification Besides nutritional deprivation, Snf1 is also involved in the response to other cellular stresses. Snf1 activity protects against toxicity caused by cadmium 106SNF1 gene and Snf1 activation cause increased and decreased resistance to ER stress, respectively snf1Δ cells are more sensitive to tunicamycin, a known inducer of ER stress In addition, protein kinase Snf1 regulates the Unfolded Protein Response (UPR), the evolutionary conserved pathway activated when improperly folded proteins accumulate and induce endoplasmic reticulum (ER) stress 109111Thus, even though these data indicate an interesting role for Snf1 in the regulation of ER stress response, more work is needed to understand the underlying molecular mechanism.Interesting results from Simpson-Lavy and collaborators recently show cross-talk between Snf1 and protein kinases involved in DNA damage (Mec1/ATR and Tel1/ATM) Given the role of AMPK kinase in the regulation of energy homeostasis, it is not surprising the existence of a strong relationship between aging and the AMPK pathway 115116117118The first evidence of the involvement of Snf1/AMPK in the aging process in yeast occurred several years ago when Asharafhi and co-workers discovered that Snf1 activity increased in replicative aging even in the presence of abundant glucose in the environment SNF1 gene have a very short CLS 125On the contrary, in a yeast model of Chronological Life Span , Snf1 activity is critical for the extension of CLS in caloric restriction condition and cells deleted for Taken together these data indicate that Snf1 activity promotes longevity in CLS while accelerates RLS, showing distinct and opposite mechanisms in the regulation of aging, as also reported for other proteins in yeast 129Recent data suggest that Snf1 function is not limited to conditions of carbon limitation. One of such functions is the regulation of proteins involved in endocytosis and cellular trafficking, indeed several interactors and substrates of Snf1 are in this cluster . Snf1 interacts with and phosphorylates the α-arrestin Rod1 27131Snf1 also regulates Arf3 135snf1Δ and snf1as mutant, whose activity can be chemically inhibited) show a slow growth phenotype and an increased fraction of cells in G1 phase, in synthetic medium supplemented with 2% glucose 2525Several reports indicate that Snf1 could be active in the presence of high glucose 54133137The relevant role of the catalytic activity of Snf1 in regulating proliferation in glucose-repressed condition is further supported by the fact that Snf1 loss reduces the expression of G1-specific genes 25SNF1-T210A mutant shows a slow growth phenotype and a delayed G1/S transition. 25Snf1-T210 is weakly phosphorylated in 2% glucose, confirming that it is partially active in that growth condition 21139Snf1 exerts its function in mitosis too and active Snf1 is localized at the division site from the time of bud emergence to cytokinesis snf1Δ cells, further supporting the emerging roles of Snf1 in non-limiting nutrient condition too It is amazing that cells lacking Snf1 and growing in synthetic media containing 2% glucose, show an extensive transcriptional reprogramming, being the most upregulated genes mainly involved in transmembrane transport and metabolic processes such as aminoacid biosynthesis, iron homeostasis and redox metabolism Many studies have reported that AMPK activity is altered in several diseases, such as inflammation, diabetes and cancer 145147145Saccharomyces cerevisiae is a powerful model for studying fundamental aspects of eukaryotic cell biology and to validate the increasing downstream targets of the class of Snf1/AMPK protein kinases which control the complexity of cellular physiology.Therefore, the expansion of the repertoire of AMPK substrates, as well as more in-depth studies of the molecular mechanisms by which AMPK is activated, will help to better understand the roles of this kinase in the regulation of human pathologies. In this context, the yeast unicellular organism


It is a clear liquid at room temperature, allowing it to be mistaken for water if unlabelled. Ingestion of H2O2 can have serious sequelae even in low volumes and concentrations.We present a case study of H2O2 poisoning and discuss the pertinent radiographical findings.Hydrogen peroxide (H It is a clear liquid at room temperature, allowing it to be mistaken for water if unlabelled. Ingestion of H2O2 can have serious sequelae even in low volumes and concentrations. We present a case study of H2O2 poisoning and discuss the pertinent radiographical findings.Hydrogen peroxide  and mildly hypertensive (155/100 mmHg) but not hypoxic (SpO2 97% on room air). A clinical examination revealed dysphonia with mild erythema and oedema of the oropharynx and uvula; the abdominal and respiratory examinations were otherwise unremarkable. Blood and biochemical investigations were also normal.A healthy 21-year-old male unintentionally ingested approximately one mouthful of 3% HA CT scan was performed to exclude gastrointestinal perforation, which demonstrated pneumatosis and mucosal thickening throughout the stomach and proximal duodenum, as well as extensive portal venous gas –3.−1 was commenced prior to transfer to a tertiary intensive care unit.The patient was intubated owing to concerns for developing airway involvement and gastrointestinal perforation, and conservative treatment with intravenous piperacillin/tazobactam 4.5 g four times daily and intravenous pantoprazole infusion at 8 mg hourAn upper endoscopy performed 3 days after the ingestion was normal, with no evidence of mucosal injury. A repeat CT scan at this time revealed interval partial resolution of the bowel wall thickening and complete resolution of the pneumatosis and portal venous gas . The pat2O2 is an oxidizing agent that is available in concentrations ranging from 3% to 90%.1 It is found in numerous household products, including disinfectants, hair dyes, bleaches and stain removers. It is a clear, colourless liquid at room temperature,2 allowing it to be mistaken for water if unlabelled, as in this case. Recently, H2O2 has also been sold for consumption in small volumes following promotion of its purported natural health benefits,3 despite multiple documented fatalities from its ingestion2 and no evidence to demonstrate health benefits of any kind. Upon contact with the enzyme catalase in the gastric mucosa, H2O2 undergoes rapid decomposition into oxygen and water (2H2O2 → 2H2O + O2 + heat).4 If the amount of oxygen liberated exceeds the maximum solubility of blood, bubbles migrate through the epithelial interstices and gas embolism may occur, manifesting as pneumatosis3 or gas within the portal venous system,5 brain6 and coronary arteries.7 Toxicity is also caused by direct caustic injury to the gastric mucosa, resulting in gastritis and potential rupture, as well as cytotoxicity from lipid peroxidation.1 This particular case is unusual because gas embolism usually only occurs with ingestion of the stronger 35% H2O2 solution, with only a few other published case reports of portal venous gas following ingestion of the 3% solution.H2O2, a common household item, can have serious sequelae even in low volumes and concentrations.Ingestion of HRadiographical findings may include gastritis, pneumatosis, perforation and portal venous gas.


Myo10 in pigmentation, with a phenotype unlike those of Myo5a or Myo7a. Adult mice homozygous for a disrupted Myo10 allele on a C57BL/6N background displayed a high degree of penetrance for white patches on their abdomen and dorsal surface. Forepaw syndactyly and hind paw syndactyly were also observed in these mice. Tail epidermal wholemounts showed a complete lack of melanocytes in the hair follicles and interfollicular epidermis. Myo10 has previously been implicated in human pigmentation. Our current study reveals involvement of Myo10 in murine skin pigmentation.Myosins are molecular motors that are well known for their role in cell movement and contractile functions. Although extensively studied in muscle physiology, little is known about the function of myosins in mammalian skin. As part of the Sanger Institute Mouse Genetics Project, we have identified a role for  ATPadenosine triphosphateHair‐GELHair Gene Expression LibraryMyo10Myosin 10Myo5aMyosin 5aMyo7aMyosin 7aTrp1tyrosinase‐related protein 1UVultravioletWTwild type1Skin pigmentation is a highly variable and conserved trait among living organisms. It plays a critical role in social communication, camouflage, mimicry and protection against the harmful effect of UV radiation.Myo5a), Myosin 7a (Myo7a) and Myosin 10 (Myo10) are unconventional myosins and are known to have functions in melanocytes and neurons.Myo5a and Myo7a are known in skin pigmentation, whereas the in vivo involvement of Myo10, while previously studied in primary human cell culturesMyosins are among the motor proteins involved in melanosome cargo transportation. Functionally, myosins are a large superfamily of ATP‐dependent motor proteins that translocate actin filaments. Other functions include cell adhesion, motility, endocytosis, exocytosis and cytokinesis.2Does Myosin 10 play a role in murine skin pigmentation?3Myo10tm2/tm2 C57BL/6N mice from the Sanger Institute Mouse Genetics Project,Myo10 , Myo10tm2/tm2 mice typically displayed abnormal dorsoventral coat patterning with white belly spots and extensive depigmented areas in the tail. Depigmentation was limited to the tail tip in control mice . Unlike Xenopus Myo10 mutants, Myo10tm2/tm2 mice did not show any cranial or skeletal abnormalities.To assess the efficiency of knockout of the s Figure A. SimilaMyo10tm2/tm2 mice prompted us to investigate whether the skin had any melanocyte abnormalities. Confocal imaging of tail epidermal wholemounts revealed a complete lack of pigmented melanocytes in the hair follicles and interfollicular epidermis  showed two distinct clusters of interactions for Myo5a and Myo10  database.Myo10 is predominantly expressed in melanocytes in embryonic and neonatal mice, it is also expressed in other skin compartments such as epidermis and dermal fibroblasts  Myo10 expression in WT and Myo10tm2/tm2 tail. Myo10 probe spans exons 28‐29. RQ value is relative to endogenous control gene B2m. (B) Haematoxylin and eosin staining of the tail and dorsal skin of Myo10tm2/tm2 and WT mice. (C) Myo10tm2/tm2 tail skin sections immunolabelled with the antibody to the melanocyte stem cell marker c‐Kit show the absence of melanocytes in hair follicles when compared to WT (arrows). Asterisks indicate nonspecific staining. (D) STRING interaction network of Myo10 and Myo5a shows two distinct classes of interacting proteins. (E) Gene expression results obtained from Hair‐GEL database show high expression of Myo10 in melanocytes at E14.5 and widespread expression in all skin subpopulations at P5. FPKM, Fragments Per Kilobase of transcript per Million mapped reads; Scale bars 100 μm.Click here for additional data file.Data S1 Materials and MethodsClick here for additional data file.