News Article | May 16, 2017
INVE has signed an agreement that will result in a range of its tilapia products being distributed by one of Thailand’s largest quality tilapia fry and feed providers, Manit Farm. The collaboration is built on the synergy between Manit Farm and INVE Aquaculture – part of the Benchmark Group since 2015 and a supplier of products that improve biosecurity and microbial management – both in the farms’ water supplies and in the gastro-intestinal tracts of the fish themselves. “The deal will allow Manit to distribute a range of INVE products including a biocide, Sanocare CID; a water treatment product, Secure POND; and a feed probiotic, Secure YIELD,” says Mario Hoffmann, Key Account Manager at INVE. Global production of tilapia is expected to reach 6 million tonnes in 2018 and, according to the Thai Department of Fisheries, 197,00 tonnes of this will be produced in Thailand. Thanks to the comparatively low cost and the relative ease of producing tilapia, the sector is seen as an important source of protein to feed a growing human population. Both INVE and Manit Farm are committed to promoting aquaculture in Thailand. “We believe in market synergies among complementary competences and we care for the sustainable growth of the tilapia market in Thailand,” explains Stelios Leontios, INVE’s Commercial Director. Sustainable growth will be achieved if tilapia production is professionalized and modernized, integrating the recent developments in genetics, biosecurity, nutrition and management tools – goals that Manit Farm, which has been in the tilapia hatchery business for over 25 years, is keen to achieve. “In 2007, we established our Tilapia Genetics Center, in collaboration with Benchmark’s Akvaforsk Genetics, focusing on the selection of different strains of tilapia. As a result, Manit Farm now provides Superblack Nile tilapia fry and Superred red tilapia fry, as well as a range of quality feed under both the Manit Farm and OHO brands,” says Khum Amorn Luengnaruemitchai, Managing Director of Manit Farm. INVE, one of the pioneering firms in world aquaculture, which is also part of the Benchmark Group, has more than 32 years’ experience in developing products and solutions for hatcheries and farms worldwide. Through its market and technical knowledge, the company has developed products for nutrition, health and the environment for a range of species. “The combination of fry genetics, biosecurity measures, and specific management protocols – through product and strong technical support – will allow farmers to cost-efficiently produce quality fish,” says Olivier Decamp, INVE’s Farm & Feedmill Product Manager. The agreement will increase the market penetration for INVE’s tilapia products in the Thai market, by capitalizing on Manit Farm’s existing distribution and services network, which already counts more than 1,000 tilapia farmers across the country as customers. It will also help the company to promote one of its main goals. “This partnership will allow INVE Aquaculture to reinforce one of its main ambitions: to care for the growth of aquaculture by being the preferred knowledge partner for aquaculture businesses worldwide,” adds Stelios Leontios.
Frequency of Positive XmnI Gγ polymorphism and coinheritance of common alpha thalassemia mutations do not show statistically significant difference between thalassemia major and intermedia cases with homozygous IVSII-1 mutation
Neishabury M.,University of Social Welfare and Rehabilitation Sciences |
Najmabadi H.,University of Social Welfare and Rehabilitation Sciences |
Najmabadi H.,Genetics Center
Blood Cells, Molecules, and Diseases | Year: 2010
From 362 thalassemia cases referred to adult thalassemia clinic of the Iranian blood transfusion organization (IBTO) for genotyping, 103 cases (28.5%) had a common primary disease factor, IVSII-1 mutation in homozygous state. 61 (59.2%) of these individuals represented thalassemia major and 42 (40.8%) thalassemia intermedia clinical phenotype. To re-evaluate our current diagnostic criteria, XmnIGγ polymorphism and coexistence of alpha thalassemia mutations, frequently recalled as important factors modifying the clinical phenotype of homozygous beta zero thalassemia cases in our country, were examined in both groups. No statistically significant difference in the frequency of positive XmnIGγ polymorphism was observed between thalassemia intermedia and thalassemia major patients. Double gene deletion --Med was observed in only one thalassemia major case, while - a3.7 in heterozygous state (- a3.7/aa) was identified in 6 (9.8%) of thalassemia major and 8 (19%) of thalassemia intermedia patients. - a4.2 was observed in only one thalassemia major case. No statistically significant difference in the frequency of coinheritance of alpha thalassemia was observed between the two groups. These results imply that other interacting mechanisms which modify the phenotype of thalassemia patients is still in the dark in our current diagnostic criteria of thalassemia. © 2009 Elsevier Inc. All rights reserved.
Vaz F.,King's College London |
Hanenberg H.,Heinrich Heine University Düsseldorf |
Hanenberg H.,Riley Hospital |
Schuster B.,University of Würzburg |
And 17 more authors.
Nature Genetics | Year: 2010
Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome. © 2010 Nature America, Inc. All rights reserved.
Scriven P.N.,Guys and St Thomas Hospital NHS Foundation Trust |
Scriven P.N.,King's College London |
Flinter F.A.,Guys and St Thomas Hospital NHS Foundation Trust |
Flinter F.A.,Genetics Center |
And 5 more authors.
European Journal of Human Genetics | Year: 2013
Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation probes was carried out for 59 couples carrying reciprocal translocations. Before treatment, 85% of pregnancies had resulted in spontaneous miscarriage and five couples had achieved a healthy live-birth delivery. Following treatment, 33% of pregnancies failed and 21of 59 couples had a healthy live-born child. The accuracy of diagnosis was 92% (8% false abnormal and 0% false normal results). The overall incidence of 2:2 alternate segregation products was 44%; however, products consistent with 2:2 adjacent segregation were ∼twice as likely from male heterozygotes, and those with 3:1 disjunction were three times more likely from female heterozygotes. Our results indicate that up to three stimulation cycles per couple would give an ∼50% chance of a successful live birth, with the risk of miscarriage reduced to the level found in the general population. In our study, 87% of all normal/balanced embryos available were identified as being suitable for transfer. We conclude that PGD provides benefit for couples with high-risk translocations by reducing the risk of miscarriage and avoiding a pregnancy with an unbalanced form of the translocation; however, for fertile carriers of translocations with a low risk of conceiving a chromosomally unbalanced offspring, natural conception may be a more viable option. © 2013 Macmillan Publishers Limited.
News Article | November 16, 2016
Male C57BL/6J mice (CLEA Japan) were treated with ethylnitrosourea (85 mg kg−1, Sigma-Aldrich) by intraperitoneal injection twice at weekly intervals at the age of 8 weeks. At the age of 25–30 weeks, the sperm of the mice was used for in vitro fertilization with eggs of C57BL/6N mice to obtain F offspring. Mice were provided food and water ad libitum, and were maintained on a 12-h light:12-h dark cycle and housed under controlled temperature and humidity conditions. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba and the RIKEN BioResource Center, University of Texas Southwestern Medical Center at Dallas. EEG/EMG electrode implantation was performed as described previously35, with isoflurane (3% for induction, 1% for maintenance) used for anaesthesia. Seven days after surgery, the mice were tethered to a counterbalanced arm (Instech Laboratories) that allowed free movement and exerted minimal weight. At the age of 12 weeks, male mice were implanted with EEG/EMG electrodes and then screened for sleep/wakefulness behaviour. Examined parameters were total time spent in wake, NREMS and REMS states, episode duration of wake, NREMS and REMS states, appearance of muscle atonia during REMS, and rebound sleep after 4-h sleep deprivation by shaking the cages. For quantitative parameters, we selected mice whose phenotypes deviated from the average by at least 3 standard deviations. After confirming the reproducibility of the sleep phenotype, the mice were selected for offspring production by natural mating or IVF with wild-type females to examine the heritability of the sleep phenotypes. If at least 30% of the male littermates showed sleep phenotypes similar to their father, we considered the sleep abnormalities to be heritable. Single nucleotide polymorphisms of N mice were determined using a custom TaqMan Genotyping assay (Thermo Fisher). The custom probes were designed based on the polymorphism data between C57BL/6J and C57BL/6N (ref. 20). QTL analysis was performed using J/qtl software (Jackson Laboratory). Whole exomes were captured with SureSelectXT2 Mouse All Exon (Agilent) and processed to a paired end 2 × 100-bp run on the Illumina HiSeq2000 platform at the UTSW McDermott Center Next Generation Sequencing Core. Reads were mapped to the University of California Santa Cruz mm9 genome reference sequence for C57BL/6J using Burrows–Wheeler aligner and quality filtered using SAMtools. Cleaned BAM files were used to realign data and call variants using the Genome Analysis ToolKit to detect heterozygous mutations. The recording room was kept under 12-h light:12-h dark cycles and a constant temperature (24–25 °C). To examine sleep–wake behaviour under baseline conditions, EEG/EMG signals were recorded for two consecutive days from the onset of the light phase. EEG/EMG data were visualized and analysed using a MatLab (MathWorks)-based, custom semi-automated staging program followed by visual inspection. EEG signals were subjected to fast Fourier transform analysis from 1 to 30 Hz with 1-Hz bin using MatLab-based custom software. Epochs containing movement artefacts were included in the state totals but excluded from subsequent spectral analysis. Sleep/wakefulness was staged into wakefulness, NREMS and REMS. Wakefulness was scored based on the presence of low amplitude, fast EEG, and high amplitude, variable EMG. NREMS was characterized by high amplitude, delta (1–4 Hz) frequency EEG and low EMG tonus, whereas REMS was staged based on theta (6–9 Hz)-dominant EEG and EMG atonia. Hourly delta density during NREMS indicates hourly averages of delta density which is the ratio of delta power to total EEG power at each 20-s epoch. For the power spectrum of sleep/wakefulness, the EEG power of each frequency bins was expressed as a percentage of the total EEG power over all frequency bins (1–30 Hz) and sleep/wakefulness states35, 36. For sleep deprivation, mice were sleep deprived for 2, 4 and 6 h from the onset of the light phase by gently touching the cages when they started to recline and lower their heads. Food and water were available. To evaluate the effect of sleep deprivation, the NREMS delta power during the first hour after sleep deprivation was expressed relative to the same zeitgeber time (ZT) of the basal recording or relative to the mean of the basal recording. For caffeine and modafinil injection experiments, mice were fully acclimatized for intraperitoneal injection before sleep recording. After 24-h baseline recording, mice received caffeine (Sigma), modafinil (Sigma) or vehicle (0.5% methyl cellulose (Wako)) intraperitoneally at ZT0, followed by 12-h recording. Injections were delivered once per week, with each injection followed by a 6–8-day washout period, during which mice remained in the recording chamber. To examine the sleep/wakefulness behaviour under constant darkness, after 48-h recording under a 12-h light:12-h dark cycle, EEG/EMG recording continued in constant darkness for 3 days. Mice were housed individually in a cage (width 23 cm, length 33 cm, height 14 cm) containing a wireless running wheel (Med Associate ENV-044). Cages were placed in a light-tight chamber equipped with green LED light (100 lx at the bottom of the cage). The rotation numbers of wheels were obtained with 1-min bin using Wheel manager software (Med Associate). Mice were entrained to 12-h light:12-h dark cycle for 7 days, and then released into constant darkness for 3 weeks. The free running period was calculated with linear regression analysis of activity onset using MatLab-based custom software. Circadian activity amplitude was calculated by fast Fourier transform of activity data, which were processed with Bartlett window using MatLab-based custom software. Relative amplitude was normalized to the mean amplitude of the wild- type group. A rabbit polyclonal antibody against the C-terminal 171 amino acids of mouse SIK3 was generated using custom antibody production service (Pacific Immunology). Tissues were homogenized using a rotor-stator homogenizer (Polytron) in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM Na P O , 1.5% Triton X-100,15 mM NaF, 1× PhosSTOP (Roche), 5 mM EDTA, 1× protease inhibitor (Roche)), and then centrifuged at 13,000g at 4 °C. The supernatants were separated by SDS–PAGE and transferred on PVDF membrane. Western blotting was performed according to standard protocols. In situ hybridization was performed as described previously37. In brief, a 0.7–0.8-kb fragment of Nalcn cDNA was inserted into pGEM-T easy (Promega) and used for DIG-labelled probe synthesis. Mice were deeply anaesthetized with sodium pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). Forty-micrometre-thick brain sections were treated with 0.3% Triton X-100, digested with 1 μg ml−1 proteinase K, treated with 0.75% glycine, and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine. After overnight incubation with a digoxigenin (DIG)-labelled probe at 60 °C, the sections were washed and then incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche, 11175041910). The reactions were visualized with a 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium (BCIP/NBT) substrate solution (Roche). HEK293 cells (RCB1637) and HEK293T cells (RCB2202) were obtained from the RIKEN BRC Cell Bank. Cells were cultured in DMEM (Wako) supplemented with 10% FBS, 1% GlutaMAX (Thermo Fisher Scientific), and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO . Cell lines were regularly tested for mycoplasma contamination using MycoAlert (Lonza). Cell lines were regularly renewed by obtaining cell stocks from the Cell Bank for authentication. We used HEK293 and HEK293T cells because of their reliable growth, high efficiency in transfection and morphology suitable for electrophysiological experiments. For generating Sik3Slp knock-in mice, a genomic fragment containing exon 13 of the Sik3 gene was isolated from C57BL/6 mouse genomic BAC clone from a RP23 mouse genomic BAC library (Advanced GenoTEchs Co). A 1.7-kb fragment of FRT-PGK-gb2-neo-FRT-loxP cassette (Gene Bridges) flanked by two flippase recognition target (FRT) sites was inserted before exon 12. The targeting vector also contains a G-to-A substitution at the fifth nucleotide from the beginning of intron 13. The targeting vector was linearized and electroporated into the C57BL/6N ES cell line RENKA. Correctly targeted clones were injected into eight-cell stage ICR mouse embryos, which were cultured to produce blastocysts and then transferred to pseudopregnant ICR females. Resulting male chimaeric mice were crossed with female C57BL/6N mice to establish the Sik3Slp-neo/+ line. To remove the neomycin resistance gene with the FLP-FRT system, Sik3Slp-neo/+ mice were crossed with Actb-FLP knock-in mice. The custom-designed ZFN mRNAs targeting the exon 13–intron 13 boundary region of the Sik3 gene were obtained from Sigma-Aldrich’s Composers Custom ZFN service. Before the final assembly of the ZFN products, Sigma-Aldrich validated the designed ZFN binding sequences in silico using their bioinformatics tools and in vitro using Nero2A cell lines, ensuring high cutting efficiency and specificity using mismatch-specific endonuclease CelI according to the manufacturer’s instructions. The ZFN mRNAs were injected into single-cell stage C57BL/6J mouse zygotes at the University of Texas Southwestern Transgenic Core facility. The injected eggs were then transferred to pseudopregnant females to generate F founders. In total, 45 out of 96 F mice were found to be modified at the exon 13–intron 13 boundary region of the Sik3 gene. We crossed one F male mouse that had a 2-bp deletion from the last nucleotide of exon 13 with female C57BL/6N mice to obtain F mice of Sik3Slp/+ ZFN. The F mice were used to confirm the skipping of exon13 in Sik3 mRNA, which was purified from the brains and livers. The F male mice were used for sleep/wakefulness behaviour analysis. To produce a Cas9/single-guide RNA (sgRNA) expression vector, oligonucleotide DNAs (5′-CACCGCGAGCGGCCATCGACCCGC-3′ and 5′-AAACGCGGGTCGATGGCCGCTCGC-3′) were annealed and then inserted into pX330 vector (Addgene). The cleavage activity of the pX330-Sik3Ex1 vector was evaluated by the EGxxFP system38. Genomic DNA containing exon 1 of the Sik3 gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-Sik3Ex1. The pX330-Sik3Ex1 and pCAG-EGxxFP-Sik3Ex1 were transfected into HEK293 cells. As a donor oligonucleotide, a single-stranded 200-nucleotide DNA was synthesized (Integrated DNA Technologies), which contained a Flag-haemagglutinin-coding sequence in the centre and 70-nucleotide arms at the 5′ and 3′ ends. Female C57BL/6J mice or Sik3Slp knock-in mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin at a 48-h interval, and mated with male C57BL/6J mice. The fertilized one-cell embryos were collected from the oviducts. Then, 5 ng μl−1 of pX330-Sik3Ex1 vector and 10 ng μl−1 of the donor oligonucleotide were injected into the pronuclei of these one-cell-stage embryos. The injected one-cell embryos were then transferred into pseudopregnant ICR mice. F mice were genotyped for the presence of Flag-coding sequence in exon1 of the Sik3 gene and for the presence of the Sik3Slp mutation. F mice containing Flag–SIK3 were further examined for the presence of the Cas9 transgene and off-target effects. Candidate off-target sites were identified based on a complete match of 16 bp at the 3′ end, including the PAM sequence. F mice were mated with C57BL/6N mice to obtain F offspring. NalcnDrl mice were produced as described above. To produce the sgRNA expression vector, pX330-NalcnEx9, oligonucleotide DNAs (5′-CACCAGCAATAAACACATTCTGAA-3′ and 5′-AAACTTCAGAATGTGTTTATTGCT-3′) were used. Genomic DNA containing exon 9 of the Nalcn gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-NalcnEx9. As a donor oligonucleotide, a single-stranded 199-nucleotide DNA containing a T-to-A substitution at the centre was synthesized (Integrated DNA Technologies). Nalcn mutant mice of N –N generation were used for sleep/wakefulness analysis. To evaluate Flag-tagged SIK3 protein in brains, we performed peptide mapping of the purified Flag–SIK3 protein. The brains of Flag-Sik3 knock-in mice and Flag-Sik3Slp knock-in mice were quickly dissected after cervical dislocation. Brains were homogenized in detergent-free buffer and then centrifuged (100,000g, 30 min, 4 °C). The supernatant was immunoprecipitated with anti-DDDDK antibody beads (MBL 3325). The eluate was run on a polyacrylamide gel and stained with SilverQuest Silver staining kit (Life technologies). Flag–SIK3 band (150 kDa) was dissected with a fresh blade. The proteins in the bands were reduced with 10 mM dithiothreitol and alkylated with 40 mM iodoacetamide. Each sample was digested with trypsin (4 μg ml−1; Trypsin Gold, Promega) at 37 °C overnight. The extracted peptides were then separated via nano flow LC (Advance LC, Michrom Bioresources) using a C18 column. The LC eluate was coupled to a nano-ionspray source attached to a Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). All MS/MS spectra were searched using Proteome Discoverer 1.3 software (Thermo Fisher Scientific). Peptides were mapped through mouse SIK3 (NP_081774) with 56% coverage. To examine the effect of sleep deprivation on the phosphorylation status of SIK3 protein, five Flag-Sik3 knock-in mice or five Flag-Sik3Slp knock-in mice were ad libitum slept (S) or sleep-deprived (SD) for 4 h by gentle handling immediately after light onset (ZT0–ZT4). Five wild-type mice were used as a negative control. At ZT4, mouse brains were quickly dissected after cervical dislocation, rinsed with cold PBS, and snap frozen in liquid nitrogen. Each half of the brains was lysed in 2 ml of ice-cold lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl , 15 mM NaF, 10 mM Na P O ) freshly supplemented with protease/phosphatase inhibitor cocktail tablets (Roche), and homogenized in a glass tissue homogenizer. After brain homogenate was incubated for 30 min and centrifuged at 13,000g for 20 min at 4 °C, the supernatant was pre-cleared by IgG and Protein G beads for 30 min before immunoprecipitation. Each pre-cleared lysate was added to 50 μl of anti-Flag antibody-conjugated Sepharose beads (Sigma, A2220) and rotated overnight at 4 °C. After washing the beads five times with cold wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl , 15 mM NaF, 10 mM Na P O ), 50 μl of elution buffer (2% SDS, 60 mM Tris-HCl, pH 6.8, 50 mM DTT, 10% glycerol) was added and rotated for 10 min at 4 °C. Elution was repeated twice and combined into one eluate and analysed by western blotting. For each group of Flag knock-in mice, the five eluates of were mixed and equally split into two or three samples for mass spectrometric analysis. Thus, a total of six (Flag-Sik3) or nine (Flag-Sik3 and Flag-Sik3Slp) samples were reduced, alkylated, and trypsin digested overnight. After desalting, each sample was labelled with a different Tandem Mass Tag (TMT) reagent (Thermo Fisher Scientific), then all samples were combined into one mixture for HPLC fractionation using a C18 column. A total of 12 fractions were collected, and analysed separately on the Orbitrap-Fusion mass spectrometry platform (Thermo Fisher Scientific) using a reverse-phase liquid chromatography tandem mass spectrometry (LC–MS/MS) method. We performed data analysis to identify peptides and quantified reporter ion relative abundance using Proteome Discoverer 2.1 (Thermo Fisher Scientific). The relative abundance of quantified SIK3 phosphorylation sites was normalized with wild-type negative control and total SIK3 protein abundance. To express wild-type NALCN, we used pTracer-CMV2-ratNALCN-EF1α-EGFP (a gift from D. Ren)39. A single nucleotide substitution was induced to make pTracer-CMV2-ratNALCN(DRL)-EF1α-EGFP using a KOD plus Mutagenesis kit (Toyobo). HEK293T cells were grown to ~50% confluency in 12-well plates. Using Lipofectamine LTX (2 μl) and PLUS (1 μl) reagents (Thermo Fisher Scientific), the cells were cotransfected with 0.3 μg of each plasmid DNA encoding rat NALCN-EGFP (wild type or DRL), mouse UNC-80, and mouse SRC (Y529F) (constitutively active Src) in 12-well plates. UNC-80 and SRC kinase activate NALCN27, 28. In some experiments, the cells were incubated with 10 μM Gd3+ to inhibit NALCN. The cells were dissociated and plated on 18-mm coverslips coated with poly-l-lysine in fresh culture medium before patch-clamp recordings. All patch-clamp recordings from HEK293T cells were performed >72 h after transfection. Recording patch pipettes were pulled from glass capillaries (1B150F-4, World Precision Instruments) using a micropipette puller (P-97, Sutter Instrument) to give a resistance of ~9 MΩ. The series resistance of whole-cell recordings was ~40 MΩ, which was not compensated. Patch pipettes were filled with solution containing 150 mM CsOH, 120 mM methanesulfonic acid, 10 mM NaCl, 10 mM EGTA, 2 mM Mg ATP and 10 mM HEPES (pH 7.4 adjusted with methanesulfonic acid; osmolarity, 290−299 mOsm l−1 adjusted with CsCl). The cells on coverslips were transferred to a recording chamber under a fluorescence upright microscope (Axio Examiner D1, Zeiss) and continuously perfused with the bath solutions containing 150 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 20 mM glucose, 5 mM NaOH, 2 mM MgCl and 1.2 mM CaCl (pH 7.4 adjusted; osmolarity, 300−310 mOsm l−1). The transfected cells were identified by enhance green fluorescent protein (eGFP) fluorescence. Patch-clamp recordings were performed at room temperature (24 °C) using a computer-controlled amplifier (MultiClamp 700B, Molecular Devices). The signals were digitized with A/D converter (Digidata 1440A, Molecular Devices), and acquired with Clampex (Molecular Devices) at a sampling rate of 50 kHz, and low-pass filtered at 5 kHz. At the end of recording, Gd3+ (10 μM) was used to confirm that the whole-cell currents were mediated through NALCN39. Data were analysed using Clampfit (Molecular Devices). The equilibrium potentials were calculated from I–V curves. Mean membrane conductance was estimated from the regression lines fitted to I–V curves from individual cells. Current, membrane conductance and charge transfer were normalized to membrane capacitance. Patch pipettes and recording system were the same as those used in recordings from HEK293 cells. Acute brain slices containing the DpMe were prepared from post-natal day 12–23 Nalcn+/+ or NalcnDrl/+ mice. After the induction of deep anaesthesia with isoflurane, mice were decapitated and the brains were rapidly removed into an ice-cold cutting solution containing 2.5 mM KCl, 1.25 mM NaH PO , 26 mM NaHCO , 25 mM glucose, 185 mM sucrose, 0.5 mM CaCl and 10 mM MgCl (pH 7.4, when bubbled with 95% O and 5% CO ). The brains were cut coronally into 200–250 μm-thick slices with a vibratome (VT-1200S, Leica). The slices were incubated at 37 °C for 1 h in artificial cerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH PO , 26 mM NaHCO , 10 mM glucose, 2 mM CaCl and 1 mM MgCl (pH 7.4, when bubbled with 95% O and 5% CO ) before recordings. Slices were transferred to a recording chamber perfused with aCSF under an upright microscope (Axio Examiner D1, Zeiss). Patch pipettes were filled with solution containing 125 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.5 mM EGTA, 8 mM phosphocreatine-Na , 4 mM ATP-Mg and 0.3 mM GTP-Na (pH 7.3 adjusted with KOH; osmolality, 290 mOsm l−1). The DpMe was identified with axon bundles. Recordings were made from cells located in the medial part of the DpMe. Cells showing no action potentials following current injection (>1 nA, > 5 ms) were discarded from analysis. Membrane potentials were recorded for 1–10 min. Sik3 hypomorph and UAS-Sik3, UAS-Sik3(S563A) transgenic flies were gifts from M. Montminy and J. B. Thomas40. elav-GS (GeneSwitch) stocks were from the Bloomington stock centre. Flies were reared at 25 °C under 12-h light:12-h dark cycle in 50–60% relative humidity on a standard fly food consisting of corn meal, yeast, glucose, wheat germ and agar. Sleep analysis was performed as described previously41. In brief, male flies (2–5 days old) were individually housed in glass tubes (length, 65 mm; inside diameter, 3 mm) containing standard fly food at one end and a cotton plug on the other end. Sucrose-agar (1% agar supplemented with 5% sucrose) food was used for the GeneSwitch system assay, instead of standard food. The glass tubes were placed in the Drosophila activity monitor (DAM) (Trikinetics) and the locomotor activity of each fly was recorded as the number of infrared beam crossings in 1-min bin. Sleep was defined as periods of inactivity lasting 5 min or longer. Sleep assay were performed for 3 d under 12-h light:12-h dark cycle conditions and then constant darkness conditions. For 12-h light:12-h dark cyles, zeitgeber time (ZT) was used, and for constant darkness, circadian time (CT), with CT0 as 12 h after lights-off of the last 12-h light:12-h dark conditions, was used to indicate the daily time. For conditional expression analysis, we used the GeneSwitch system42 where expression is induced by a steroid hormone antagonist RU486. Flies are monitored for 3 days in tubes without drug in constant darkness and then transferred to new tubes either with vehicle (0.5% DMSO) alone or with 0.5 mM RU486 and then further monitored under constant darkness conditions. The expression of endogenous or transgenic Sik3 genes was confirmed by RT–PCR using RNA from fly heads. The wild-type strain N and the mutant strain PY1479 kin-29(oy38) X were obtained from the Caenorhabditis Genetics Center (CGC)43. All worms were maintained at 20 °C on nematode growth medium (NGM) agar plates seeded with E. coli HB101. For construction of P ::kin-29, kin-29 cDNA was amplified by RT–PCR and inserted into the plasmid pPD-DEST (a gift from Y. Iino) to generate pDEST-KIN-29. Next, we carried out the LR-recombinase reaction (Gateway System, Life Technologies) between pENTR-P (a gift from Y. Iino) and pDEST-KIN-29 to generate P ::kin-29. P ::kin-29 was injected at 30 ng μl−1 together with the injection marker P ::mcherry (10 ng μl−1) and the empty vector pPD49_26 (60 ng μl−1) into the kin-29(oy38) mutant worms. Quiescence during the L4 to adult lethargus was measured using the microfluidic-chamber based assay44. In brief, polydimethylsiloxane-made microfluidic chambers containing liquid NGM and the E. coli HB101 were loaded with early L4 larvae and sealed with a cover glass plus 2% agarose, and set under the microscope. Images were taken every 2 s for 12 to 20 h at 20 ± 0.5 °C using the microscope M205FA (Leica) equipped with the camera MC120HD (Leica) (pixel size: 1,024 μm × 768 μm) controlled by Leica Application Suite V4.3 or the microscope SZX16 (Olympus) equipped with the camera GR500BCM2 (Shodensha) (pixel size: 1,024 μm × 768 μm) controlled by μManager (UCSF). Subtraction between serial images was carried out using Image J, and worms were regarded as quiescent at a specific time point if the difference from the preceding time point was less than 1% of the total body size. The fraction of quiescence was defined as the number of quiescent time points divided by the total number of time points during a period of 10 min. The onset of lethargus quiescence was defined as the time point after which the fraction of quiescence was higher than 0.05 for at least 20 min, whereas the end point was defined as the time point after which the fraction of quiescence was lower than 0.05 for at least 20 min. Occasionally, brief episodes of quiescence were observed outside of lethargus both in wild-type and mutant worms; these episodes were excluded by setting a threshold of 60 min for the minimum duration of lethargus quiescence. Sample sizes were determined using R software based on averages and standard deviations that were obtained from small scale experiments. No method of randomization was used in any of the experiments. The experimenters were blinded to genotypes and treatment assignment. Statistical analysis was performed using SPSS Statistics 22 (IBM) and R software. All data were tested for Gaussian distribution and variance. Homogeneity of variances was tested with Levene’s test. We used Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, one-way repeated measure ANOVA for multiple comparisons with multiple data points, and two-way ANOVA for multiple comparisons involving two independent variables. ANOVA analyses were subjected to Tukey’s post-hoc test. When deviation from normality and lack of homogeneity of variances occurred (P < 0.05), Mann–Whitney U test was used for group comparison. P < 0.05 was considered statistically significant. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
Cortes F.,University of Chile |
Cortes F.,Genetics Center |
Mellado C.,University of Chile |
Pardo R.A.,University of Chile |
And 2 more authors.
American Journal of Medical Genetics, Part A | Year: 2012
In January 2000, Chilean Ministry of Health mandated the addition of folic acid (FA) to wheat flour in order to reduce the risk of neural tube defects (NTDs). This policy resulted in significant increases in serum and red cell folate in women of fertile age 1 year after fortification. To evaluate the effect of wheat flour fortification on the prevalence of NTDs in Chile we designed a prospective hospital-based surveillance program to monitor the frequency of NTDs in all births (live and stillbirths) with birth weight ≥500g at the nine public maternity hospitals of Santiago, Chile from 1999 to 2009. During the pre-fortification period (1999-2000) the NTD rate was 17.1/10,000 births in a total of 120,566 newborns. During the post-fortification period (2001-2009) the NTD rate decreased to 8.6/10,000 births in a total of 489,915 newborns, which translates into a rate reduction of 50% (RR: 0.5; 95% CI: 0.42-0.59) for all NTDs. The rate reduction by type of NTD studied was: 50% in anencephaly (RR: 0.5; 95% CI: 0.38-0.67), 42% in cephalocele (RR: 0.58; 95% CI: 0.37-0.89), and 52% in spina bifida (RR: 0.48; 95% CI: 0.38-0.6). Rates showed significant reduction both in stillbirths and live births: 510.3 to 183.6/10,000 (RR=0.36; 95% CI: 0.25-0.53) and 13.3 to 7.5/10,000 (RR=0.56; 95% CI: 0.47-0.68), respectively. In Chile, fortification of wheat flour with FA has proven to be an effective strategy for the primary prevention of NTDs. © 2012 Wiley Periodicals, Inc.
Cavanagh L.,Genetics Center |
Compton C.J.,31 Thornford Road |
Tluczek A.,University of Wisconsin - Madison |
Brown R.L.,University of Wisconsin - Madison |
M.Farrell P.,University of Wisconsin - Madison
Journal of Genetic Counseling | Year: 2010
This cross-sectional mixed method study was a long-term follow-up evaluation of families who participated in an earlier survey of their understanding of cystic fibrosis (CF) genetics and their infants' false-positive CF newborn screening (NBS) results. Thirty-seven of the original 138 parents participated in the follow-up telephone survey. Results showed parentswho received genetic counseling at the time of their infants' diagnostic sweat tests had significantly higher long-term retention of genetic knowledge than those without genetic counseling. However, both groups still had misconceptions and lacked accurate information about the actual risk associated with being a CF carrier. Most parents either had already informed (65%) or planned to inform (19%) their children about the child's carrier status. Mean child age at the time of disclosure was 9.2 years. Situational prompts were the most common reasons for informing their children. Neither parental knowledge, medical literacy, nor parental education predicted whether parents informed their children about their carrier status. False-positive NBS results for CF were not associated with parental perceptions of child vulnerability 11- 14 years after the testing. Although the sample from this study was small, these findings underscore the benefits of genetic counseling at the time of the diagnostic sweat test and offer information that can assist parents in talking with their children about the implications of having one CFTR mutation. © National Society of Genetic Counselors, Inc. 2009.
Ioannou D.,University of Kent |
Fonseka K.G.L.,University of Kent |
Meershoek E.J.,Kreatech Diagnostics |
Thornhill A.R.,Genetics Center |
And 3 more authors.
Chromosome Research | Year: 2012
Fluorescence in situ hybridisation (FISH) was first applied on in vitro fertilisation (IVF) embryos for the preimplantation genetic diagnosis of sex, then chromosome translocations and later for chromosome copy number (PGS). Because of the controversy surrounding PGS diagnostically, it has been replaced by array-based approaches; however, FISH remains a powerful tool for investigating mechanisms of both postzygotic segregation error and nuclear organisation, especially if most or all of the chromosomes in the karyotype can be analysed. The purpose of this study was to develop and apply a 24 chromosome FISH assay to investigate chromosome-specific rates of gain and loss, nuclear organisation patterns and the veracity of the original PGS result in days 5-6 human embryos. Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per cell for each embryo and the nuclear address of the (mostly centromeric) loci probed. As all embryos were surplus to IVF requirements for both transfer and freezing (and many had an abnormal PGS indication) expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal complement; all were mosaic. Certain patterns emerged, however, namely that chromosome loss was more common than gain and apparent mitotic nondisjunction. Moreover, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, in part, by 24 colour FISH in most but not all cases. © Springer Science+Business Media B.V. 2012.
Ahn J.W.,Genetics Center |
Ogilvie C.,Genetics Center
Advances in Clinical Chemistry | Year: 2016
There has been a huge acceleration in our technical ability to detect variation in the human genome in recent years, and there has been a corresponding effort in clinical diagnostic laboratories to take advantage of this progress for the benefit of patients. There has, however, not been an equivalent increase in our understanding of human genetics and disease, not for lack of effort but due to the far greater complexity of understanding variation than the difficulties of detecting it. This chapter describes how software tools can be used to target clinical genetic diagnostic testing in order to exploit technical and scientific advances both efficiently and cost-effectively, while maximizing clinical utility. © 2016 Elsevier Inc.
Tluczek A.,University of Wisconsin - Madison |
Orland K.M.,University of Wisconsin - Madison |
Cavanagh L.,Genetics Center
Qualitative Health Research | Year: 2011
This study was designed to develop a framework for understanding parents' perspectives about the psychosocial consequences of false-positive newborn screening (NBS) results for cystic fibrosis (CF). Through content analysis of interviews with 87 parents of 44 infants, we found that receipt of genetic information through NBS affected parents on intrapersonal and interpersonal levels within a relational family system. Repercussions included wondering about test accuracy, the child's health, and the future; gaining new perspectives and strengthening relationships; questioning paternity; wondering if other relatives had CF/were carriers; searching for the genetic source; sharing genetic information; supporting NBS; and feeling empathy for parents of affected children. We concluded that abnormal NBS results that involve genetic testing can have psychosocial consequences that affect entire families. These findings merit additional investigation of long-term psychosocial sequelae for false-positive results, interventions to reduce adverse iatrogenic outcomes, and the relevance of the relational family system framework to other genetic testing. © The Author(s) 2011.