News Article | May 8, 2017
Scientists express concerns about the effect of energy drinks on individuals, particularly teens, with familial long QT syndrome in a new study published in the International Journal of Cardiology Amsterdam, The Netherlands, May 8, 2017 - Caffeinated energy drinks can trigger serious cardiac events including cardiac arrest in individuals not known to have a specific heart disease of genetic origin. Scientists in Australia have now assessed the risk of cardiac events following consumption of energy drinks in patients diagnosed with congenital long QT syndrome (LQTS), a condition that affects 1 in 2000 and that can cause rapid, irregular heartbeat that can lead to sudden death. In their study, published in the International Journal of Cardiology, they report that even small amounts of energy drinks can cause changes in the heart that can lead to life-threatening arrhythmias and recommend cautioning young patients, some of whom may still be unaware of an existing heart condition, about the danger. Used by millions, there has been an explosion in the consumption of "energy drinks" in the past 15 years, the most popular of which are Red Bull® and Monster®. The hemodynamic effects of energy drinks in healthy young adults have been assessed in prior studies with results including increased blood pressure, but no change in heart rate. This is the first study specifically designed to test the effects of these energy drinks in individuals who carry the gene faults (mutations) causing congenital LQTS. "The potential cardiovascular risk of energy drinks continues to emerge as an important public health issue," explained lead investigator Professor Christopher Semsarian, MBBS, PhD, MPH, of the University of Sydney and Centenary Institute, Australia. "The population most at risk is teenagers and young adults, representing the population these drinks are most heavily marketed towards. Since energy drinks are widely available to all ages and over the counter, it is important that cardiovascular effects of these drinks are investigated." The study was designed to assess the acute cardiovascular responses to energy drink consumption in patients with familial LQTS and to discover whether any identified cardiovascular effects correlate with changes in blood levels of the active ingredients - caffeine and taurine. Investigators recruited 24 patients aged 16 to 50. More than half were symptomatic before diagnosis and receiving beta-blocker therapy. Most had undergone genetic testing, 13 of whom had a documented pathogenic or likely pathogenic mutation. Participants were assigned to energy drink or control drink groups for the first study visit. The energy drink consisted of two Red Bull sugar-free cans totaling 160mg of caffeine and 2000mg of taurine, totaling 500ml. The control drink was a cordial-based 500ml drink with no caffeine or taurine. Electrocardiograms and blood pressure were recorded every 10 minutes, while signal-averaged electrocardiogram (SAECG) testing and repeat bloods were collected every 30 minutes for a total observation time of 90 minutes. The results of the study show that three patients (12.5%) exhibited dangerous QT prolongation following energy drink consumption and two of the three had sharp increases in blood pressure. These patients all had a documented family history of sudden cardiac death and two of them had previously experienced severe clinical manifestations and received an implantable cardioverter-defibrillator for recurrent syncope. "Some individual patients may be at a higher risk," commented Professor Semsarian. "We therefore suggest caution in allowing the consumption of energy drinks in young patients with LQTS." In an accompanying commentary, Professor Peter J. Schwartz, MD, Head of the Center for Cardiac Arrhythmias of Genetic Origin, IRCCS Istituto Auxologico Italiano, Milan, Italy commented, "Data suggest that the majority of LQTS patients destined to become symptomatic have the first event well after having become a teenager, which implies that a significant number of youngsters with LQTS will help themselves to energy drinks without knowing their real condition and thus endangering themselves." "When something, in this case energy drinks, is ingested by millions of individuals all over the world, a percentage such as 12.5% is no longer small, and the findings deserve careful consideration," added commentary co-author Federica Dagradi, MD, of the Center for Cardiac Arrhythmias of Genetic Origin, IRCCS Istituto Auxologico Italiano. "We should avoid spreading unjustified alarms and fears, but at the same time, we should not ignore potential dangers."
News Article | May 17, 2017
The Cdh5(PAC)-CreERT2 transgenic mice (iECre) were a gift from R. H. Adams39. Krit1fl/fl and Ccm2fl/fl animals have been previously described40, 41. Tlr4fl/fl, Cd14−/−, Ai14 (R26-LSL-RFP), and R26-CreERT2 animals42, 43, 44, 45 were obtained from the Jackson Laboratories. The Slco1c1(BAC)-CreERT2 transgenic mice have been previously described18. All experimental animals were maintained on a mixed 129/SvJ, C57BL/6J, DBA/2J genetic background unless specifically described. C57BL/6J and timed pregnant Swiss Webster mice were purchased from Charles River Laboratories. Germ-free Swiss Webster mice were purchased from Taconic. Breeding pairs between two and ten months of age were used to generate the neonatal CCM mouse model pups. Mice were housed in a specific pathogen-free facility where cages were changed on a weekly basis; ventilated cages, bedding, food, and acidified water (pH 2.5–3.0) were autoclaved before use, ambient temperature maintained at 23 °C, and 5% Clidox-S was used as a disinfectant. Experimental breeding cages were randomly housed on three different racks in the vivarium, and all cages were kept on automatic 12-h light/dark cycles. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. A group of the resistant Ccm2ECKO colony was exported to the Centenary Institute, Sydney, Australia, where the mice were permanently maintained as an inbred colony in a quarantine facility. After several generations, this colony uniformly converted to lesion susceptibility. Cages were changed on a weekly basis; ventilated cages, bedding, food and acidified water (pH 2.5–3.0) were autoclaved before use. Ambient temperature was maintained between 22–26 °C, and 80% ethanol and F10SC (1:125 dilution of the concentrate, a quaternary ammonium compound) were used as disinfectants. Experimental breeding cages were randomly distributed throughout the vivarium, and all cages were kept on 12-h light/dark cycles. The Sydney Local Health District Animal Welfare Committee approved all animal ethics and protocols. All experiments were conducted under the guidelines/regulations of Centenary Institute and the University of Sydney. Germ-free Swiss Webster mice were purchased from Taconic and directly transferred into sterile isolators (Class Biologically Clean Ltd) under the care of the Penn Gnotobiotic Mouse Facility. Food, bedding and water (non-acidified) were autoclaved before transfer into the sterile isolators. Ventilated cages were changed weekly, and all cages in the vivarium were kept under 12-h light/dark cycles. Microbiology testing (aerobic and anaerobic culture, 16S qPCR) was performed every ten days and faecal samples were sent to Charles Rivers Laboratories for pathology testing on a quarterly basis. Further details regarding the sterile C-section fostering can be found below. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. For all neonatal CCM mouse model experiments, at one day post-birth (P1), pups were intragastrically injected by 30-gauge needle with 40 μg of 4-hydroxytamoxifen (4OHT, Sigma Aldrich, H7904) dissolved in a 9% ethanol/corn oil (volume/volume) vehicle (50 μl total volume per injection). This solution was freshly prepared from pre-measured, 4OHT powder for every injection. Before injection, the pup skin was sanitized using ethanol wipes. The P1 time point was defined by checking experimental breeding pairs every evening for new litters. The following morning (P1), pups were injected with 4OHT. All experimental pups were subjected to this induction regimen. For the Tlr4 rescue experiment (Fig. 2), and all lineage-tracing experiments, an additional dose of 40 μg 4OHT was intragastrically delivered at P2 (P1+2, two total doses). Pups were then harvested as previously described9 at the specified time points. Tissue samples were fixed in 4% formaldehyde overnight, dehydrated in 100% ethanol, and embedded in paraffin. 5-μm-thick sections were used for haematoxylin and eosin (H&E) and immunohistochemistry staining. The following antibodies were used for immunostaining: rat anti-PECAM (1:20, Histo Bio Tech DIA-310), rabbit anti-pMLC2 (1:200, Cell Signaling 3674S), goat anti-KLF4 (1:100, R&D AF3158), and rabbit anti-RFP (1:50, Rockland 600-401-379). Littermate control and experimental animal sections were placed on the same slide and immunostained at the same time under identical conditions. Images were taken at the same time using the same exposure times and colour channels, and were subsequently overlaid using ImageJ. Intra-abdominal abscesses were dissected and triturated in 500 μl of SOC medium. Drops of the mixture were placed on a microscope slide, briefly exposed to heat, and Gram staining was performed using a kit from Sigma Aldrich (77730) following the manufacturer’s protocol. Eyes from euthanized P17 mice were removed and fixed overnight in cold 4% PFA/PBS solution. The following day, retinas were dissected, cut into petals, and stained with isolectin-B4 conjugated to Alexa488 fluorophore (Thermo Fisher I21411) as previously described46. The retinas were then whole-mounted on microscopy slides in a flat, four-petal shape for fluorescence imaging. B. fragilis was purchased directly from the ATCC (strain 25285) and grown in chopped meat glucose (CMG) broth (Anaerobe Systems AS-813) under anaerobic conditions at 37 °C. Autoclaved, degassed caecal contents (ACC) were generated by collecting caecal contents from the colons of euthanized adult mice between 2–8 months of age. Caecal contents were then autoclaved and pulverized in an equal volume of CMG broth. This slurry was filtered through a 70-μm nylon strainer and degassed overnight in the anaerobic chamber. 1 ml of CMG broth was inoculated with B. fragilis and grown overnight to an optical density of between 0.8 and 1.0. An equal volume of ACC was mixed with the overnight bacterial culture. 100 μl of this B. fragilis–ACC mixture was injected intraperitoneally into five-day-old pups with a 31-gauge needle. Control littermates were simultaneously injected intraperitoneally with 100 μl of ACC alone. Pups were harvested at P17. Spleen weight was measured immediately after dissection, and all tissue was subsequently processed as described above. LPS from E. coli O127:B8 was purchased from Sigma (L3129) and administered to the low-lesion-penetrance, resistant Ccm2ECKO neonatal CCM disease model. At P5, a 3 μg dose of LPS dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, a 5 μg dose of LPS was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Peptidoglycan from Bacillus subtilis (a Gram-positive gut commensal) was purchased from Invivogen (tlrl-pgnb3) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Poly(I:C) was purchased from Invivogen (tlrl-picw) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Mouse IL-1β was purchased from Genscript (Z02988) and administered to the resistant Ccm2ECKO neonatal CCM disease model. At P5, a 5 ng dose of IL-1β dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, an 8-ng dose of IL-1β was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Mouse TNFα was purchased from Genscript (Z02918) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the IL-1β experiments. For all experiments using microCT quantification of CCM lesion volume, brains were harvested and immediately placed in 4% PFA/PBS fixative. Brains remained in fixative until staining with non-destructive, iodine contrast and subsequent microCT imaging performed as previously described47. All tissue processing, imaging and volume quantification were performed in a blinded manner by investigators at the University of Chicago without any knowledge of experimental details. We blinded samples at three distinct points in the analysis. First, neonatal CCM model pups were injected with 4OHT without knowledge of genotypes. Second, hindbrains from genotyped animals were given randomized, de-identified labels to provide for blinded microCT scanning by an independent operator. Third, randomized microCT image stacks were analysed in a blinded manner by individuals not involved in any prior experimental steps. Mice were anaesthetized with Avertin and underwent intra-cardic perfusion with 10 ml of cold PBS. The brain was separated from the brainstem, and the cerebellum was separated from the remaining brain and processed in parallel. The tissue was minced with scissors, placed in digestion buffer (RPMI, 20 mM HEPES, 10% FCS, 1 mM CaCl , 1 mM MgCl , 0.05 mg ml−1 Liberase (Sigma), 0.02 mg ml−1 DNase I (Sigma)), and incubated for 40 min at 37 °C with shaking at 200 r.p.m. The mixture was passed through a 100-μm strainer and washed with FACS buffer (PBS, 1% FBS). Cells were resuspended in 4 ml of 40% Percoll (GE Healthcare) and overlaid on 4 ml of 67% Percoll. Gradients were centrifuged at 400g for 20 min at 4 °C and cells at the interface were collected, washed with 10 ml of FACS buffer, and stained for flow cytometric analysis. Neonatal P10 mice were anaesthetized with Avertin and underwent intracardiac puncture/blood draw using a 27-gauge needle/syringe coated with 0.5 M EDTA, pH 8.0 immediately before use. Cells were pelleted by centrifugation at 300g for 5 min at 4 °C. Serum was removed and red blood cells were lysed using ACK lysis buffer. Spleens were dissected in parallel, hand-homogenized using a mini-pestle and red blood cells were lysed using ACK lysis buffer. Cells from both sets of tissues were passed through a 70-μm cell-strainer, pelleted and resuspended in FACS buffer (PBS, 2% FBS, 0.1% NaN ) for immunostaining and subsequent FACS analysis. Cells were isolated from the indicated tissues. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Multi-laser, flow cytometry analysis procedures were performed at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analysers running FACSDiva software (BD Biosciences). Two-laser, flow cytometry analyses were performed at the University of Pennsylvania iPS Cell Core using BD Accuri C6 instruments. FlowJo software (v.10 TreeStar) was used for data analysis and graphics rendering. All fluorochrome-conjugated antibodies used are listed as follows (Clone, Company, Catalog Number): CD11b (M1/70, Biolegend, 101255); CD11c (N418, Biolegend, 117318); CD16/32 (93, Biolegend, 101319); CD16/32 (93, eBiosciences, 56D0161D80); CD19 (6D5, Biolegend, 115510); CD3ε (145D2C11, Biolegend, 100304); CD4 (GK1.5, Biolegend, 100406); CD45 (30-F11, Biolegend, 103121 or 103151), CD8a (53D6.7, Biolegend, 100725); Foxp3 (FJK-16 s, eBiosciences, 50-5773-82); Ly-6G (1A8, Biolegend, 127624); Live/Dead (N/A, Thermofisher, LD34966); NK1.1 (PK136, Biolegend, 108745); RORγt (B2D, eBiosciences, 12-6981-82); Siglec-F (E50D2440, BD, 562757); TCRγδ (UC7-13D5, Biolegend, 107504) At the specified time points, cerebellar endothelial cells were isolated through enzymatic digestion followed by separation using magnetic-activated cell sorting by anti-CD31-conjugated magnetic beads (MACS MS system, Miltenyl Biotec), as previously described9. Lung endothelial cells were isolated through enzymatic digestion, as previously described, followed by separation using anti-CD31-conjugated magnetic beads and the MACS MS system48. Isolated endothelial cells were pelleted and total RNA was extracted using the RNeasy Micro kit (Qiagen 74004). For qPCR analysis, cDNA was synthesized from 300 ng to 500 ng total RNA using the SuperScript VILO cDNA Synthesis Kit and Master Mix (Thermo Fisher 11755050). Real-time PCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher 4368577) using the primers listed (all mouse): Gapdh forward: 5′-AAATGGTGAAGGTCGGTGTGAACG-3′; Gapdh reverse: 5′-ATCTCCACTTTGCCACTGC-3′; Klf2 forward: 5′-CGCCTCGGGTTCATTTC-3′; Klf2 reverse: 5′-AGCCTATCTTGCCGTCCTTT-3′; Klf4 forward: 5′-GTGCCCCGACTAACCGTTG-3′; Klf4 reverse: 5′-GTCGTTGAACTCCTCGGTCT-3′; Krit1 forward: 5′-CCGACCTTCTCCCCTTGAAC-3′; Krit1 reverse: 5′-TCTTCCACAACGCTGCTCAT-3′; Il1b forward: 5′-GCAACTGTTCCTGAACTCAACT-3′; Il1b reverse: 5′-ATCTTTTGGGGTCCGTCAACT-3′; Sele forward: 5′-ATGCCTCGCGCTTTCTCTC-3′; Sele reverse: 5′-GTAGTCCCGCTGACAGTATGC-3′; Tlr4 forward: 5′-ACTGGGGACAATTCACTAGAGC-3′; Tlr4 reverse: 5′-GAGGCCAATTTTGTCTCCACA-3′. As part of the Brain Vascular Malformation Consortium (BVMC) CCM study (Project 1), a large cohort of familial CCM individuals with identical KRIT1(Q455X) mutations were enrolled between 2009–2014 at the University of New Mexico. All study protocols were approved by the Institutional Review Boards at the University of New Mexico and University of California San Francisco (UCSF) and all procedures were performed in accordance with these protocols. Prior to participation in the study, written informed consent was obtained from every patient and properly documented by UNM investigators. At study enrollment, participants received a neurological examination and 3T MRI imaging using a volume T1 acquisition (MPRAGE, 1-mm slice reconstruction) and axial TSE T2, T2 gradient recall, susceptibility-weighted, and FLAIR sequences. Lesion counting by the neuroradiologist was based on concurrent evaluation of axial susceptibility-weighted imaging with 1.5-mm reconstructed images and axial T2 gradient echo 3-mm images. Participants also provided blood or saliva samples for genetic studies. Genomic DNA was extracted using standard protocols. De-identified samples were normalized, plated on 96-well plates, and genotyped at the UCSF Genomics Core Facility using the Affymetrix Axiom Genome-wide LAT1 Human Array. Affymetrix Genotyping Console (GTC) 4.1 Software package was used to generate quality control metrics and genotype calls. All samples had genotyping call rates of ≥ 97% and were further checked for sample mix-ups (sex check, Mendelian errors and cryptic relatedness), resulting in 188 samples for genetic analysis. 21 candidate genes were further examined in the TLR4 and MEKK3–KLF2/4 signalling pathways (TLR4, CD14, MD-2, LBP, MYD88, TICAM1, TIRAP, TRAF1-6, MAP3K3, MEK5, ERK5, MEF2C, KLF2, KLF4, ADAMTS4, ADAMTS5) including 467 SNPs within 20 kb upstream or downstream of each gene locus using UCSC Genome Browser coordinates (GRCh37/hg19). Because total lesion counts are highly right-skewed, raw counts were log-transformed and analysis was performed on residuals (adjusted for age at enrollment and sex). To identify genotypes associated with log-transformed residual counts, linear regression analysis was implemented using QFAM family-based association tests for quantitative traits (PLINK v1.07 software), with stringent multiple testing correction (Bonferroni correction for the number of SNPs tested within each gene) given that some SNPs on the Affymetrix array were in linkage disequilibrium with each other, that is, statistically correlated with R2 > 0.8. The Fehrmann dataset used for eQTL lookups consisted of peripheral blood samples from the UK and the Netherlands49, 50. Samples were genotyped with Illumina HumanHap300, HumanHap370 or 610 Quad platforms. Genotypes were input by Impute v2 (ref. 51) using the GIANT 1000G p1v3 integrated call set for all ancestries as a reference52. Gene expression levels were measured by Illumina HT12v3 arrays. Gene expression pre-processing involved quantile normalization, log transformation, probe centring and scaling, population stratification correction (first four genetic multi-dimensional scaling components were removed from gene expression data) and correction for unknown confounders (first 20 gene expression principal components not associated with genetic variants were removed from gene expression data). Identification of potential sample mix-ups was conducted by MixupMapper21 and finally 1,227 samples remained. All pre-processing steps were performed with the QTL mapping pipeline v1.2.4D (https://github.com/molgenis/systemsgenetics/tree/master/eqtl-mapping-pipeline - downloading-the-software). These results are corroborated by an independently conducted GTEX Consortium study (http://www.gtexportal.org/home/snp/rs10759930 and http://www.gtexportal.org/home/snp/rs778587). TAK-242 was purchased from EMD Millipore (614316) and administered to the neonatal CCM disease model. Five, seven and nine days after birth, a 60-μg dose of TAK-242 was dissolved in DMSO/sterile intralipid (Sigma, I141) vehicle and administered retro-orbitally in a total volume of 30 μl. Control animals were identically injected with sterile DMSO/intralipid vehicle alone. Pups were euthanized and brains dissected 10 days after birth. LPS-RS ultrapure was purchased from Invivogen (tlrl-prslps) and administered to the neonatal CCM disease model. Starting at P5, a 20 μg dose dissolved in sterile PBS was administered retro-orbitally in a total volume of 30 μl every 24 h. Control animals were identically injected with sterile PBS alone. Pups were euthanized and brains dissected 10 days after birth. Experimental breeding pairs of mice, yielding susceptible neonatal CCM pups, were identified by induction of a neonatal CCM litter and evaluation of lesion burden. These breeding pairs then underwent timed matings and at E14.5, both male and female adult mice received antibiotic-laced drinking water mixed with 40 g l−1 of sucralose and red food colouring. Antibiotic water was replaced daily. The following antibiotics were mixed with 0.22-μm-filtered water: penicillin (500 mg l−1), neomycin (500 mg l−1), streptomycin (500 mg l−1), metronidazole (1 g l–1) and vancomycin (1 g l−1). Antibiotics were purchased from the Hospital of the University of Pennsylvania pharmacy. The neonatal CCM model was induced as described above. At P10, pups were euthanized and antibiotic water switched to normal drinking water. Experimental breeding pairs were then mated to obtain third generation, post-antibiotic pups. Co-housed, susceptible Krit1fl/fl females underwent evening–morning timed matings with a single susceptible Krit1ECKO male. Upon detection of a plug in the morning, the females were subsequently separated into singly-housed cages. At E14.5, female mice were received either vancomycin (1 g l−1)-laced or untreated (vehicle) sterile-filtered drinking water, changed daily. The drinking water was further mixed with 40 g l−1 sucralose and red food colouring. Pups were harvested at P11. The entire neonatal gut was dissected, snap-frozen on dry ice, and stored at −80 °C. The QIAamp DNA Stool Mini Kit (Qiagen 51504 or 51604) was used to extract bacterial DNA from the neonatal gut. Before commencing the standard Qiagen protocol, the frozen gut was mixed in the included stool lysis buffer and homogenized with a 5 mm stainless steel bead in a TissueLyser LT (Qiagen 69980) at 50 Hz for 10 min at 4 °C. Concentration of the extracted DNA was equalized and 16 ng of DNA was used per qPCR reaction with universal bacterial 16S rRNA gene primers53, two different sets of previously characterized Bacteroidetes s24-7 primers54, 55, and Firmicutes primers56. Universal 16S rRNA forward: 5′-ACTGAGAYACGGYCCA-3′; universal 16S rRNA reverse: 5′-TTACCGCGGCTGCTGGC-3′; Bacteroidetes s24-7 rRNA set 1 forward: 5′-GGAGAGTACCCGGAGAAAAAGC-3′; Bacteroidetes s24-7 rRNA set 1 reverse: 5′-TTCCGCATACTTCTCGCCCA-3′; Bacteroidetes s24-7 rRNA set 2 forward: 5′-CCAGCAGCCGCGGTAATA-3′; Bacteroidetes s24-7 rRNA set 2 reverse: 5′-CGCATTCCGCATACTTCTC-3′; Firmicutes rRNA forward: 5′-TGAAACTYAAAGGAATTGACG-3′; Firmicutes rRNA reverse: 5′-ACCATGCACCACCTGTC-3′. Evening–morning timed matings to generate donor susceptible or resistant females yielding Krit1ECKO or Ccm2ECKO pups were performed and timed pregnant Swiss Webster females (Charles River 024) served as foster mothers. To prevent delivery of the pups, at E16.5, donor females were injected subcutaneously with 100 μl of a 15 μg ml−1 solution of medroxyprogesterone (Sigma Aldrich, M1629) dissolved in DMSO. The morning of E19.5, the donor mother was euthanized by cervical dislocation and submerged in a warm sterile solution of 1% VirkonS/PBS (weight/volume) for one minute. The uterus was then dissected in a sterile laminar flow hood, submerged in a warm sterile solution of 1% VirkonS/PBS for one minute and quickly rinsed with warm sterile PBS. Pups were then removed from the uterus and fostered to the Swiss Webster recipient female. The following morning, induction of the neonatal CCM model was performed as described above. Timed matings were performed using germ-free Swiss Webster mice housed in sterile isolators under care of the University of Pennsylvania Gnotobiotic Mouse Facility. Simultaneous evening–morning timed matings were also performed using co-housed, susceptible Krit1fl/fl females and Krit1ECKO males previously characterized to yield CCM-susceptible pups. Medroxyprogesterone was administered to donor females and the sterile C-section was performed at E19.5 as described in the previous section. The intact uterus was passed through a J-tube filled with warm 1% VirkonS/PBS that was hermetically sealed to the sterile isolator. Pups were dissected from the uterus inside the sterile isolator and fostered to the recipient germ-free Swiss Webster mother. Approximately one week later, faecal samples were collected for microbiology testing. Germ-free status was further confirmed by 16S qPCR of bacterial DNA isolated from maternal faeces and neonatal guts. Fresh faecal pellets were collected from experimental females yielding susceptible or resistant pups one day after harvesting the pups to determine phenotypic severity. Collection was performed between 16:00 and 18:00, pellets were immediately snap-frozen on dry ice, and stored at −80 °C. DNA was extracted from stool samples using the Power Soil htp kit (Mo Bio Laboratories) following the manufacturer’s protocol. Library preparation was performed by using previously described barcoded primers targeting the V1/V2 region of the 16S rRNA gene57. PCR reactions were performed in quadruplicate using AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen). Each PCR reaction consisted of 0.4 μM primers, 1× AccuPrime Buffer II, 1 U Taq, and 25 ng DNA. PCRs were run using the following parameters: 95 °C for 5 min; 20 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s; and 72 °C for 8 min. Quadruplicate PCR reactions were pooled and products were purified using AMPureXP beads (Beckman-Coulter). Equimolar amounts from each sample were pooled to produce the final library. Positive and negative controls were carried through the amplification, purification and pooling procedures. Negative controls were used to assess reagent contamination and consisted of extraction blanks and DNA-free water. Positive controls were used to assess amplification and sequencing quality and consisted of gBlock DNA (Integrated DNA Technologies) containing non-bacterial 16S rRNA gene sequences flanked by bacterial V1 and V2 primer binding sites. Paired-end 2 × 250 bp sequence reads were obtained from the MiSeq (Illumina) using the 500 cycle v2 kit (Illumina). Sequence data were processed using QIIME version 1.9.1 (ref. 58). Read pairs were joined to form a complete V1/V2 amplicon sequence. Resulting sequences were quality filtered and demultiplexed. Operational taxonomic units (OTUs) were selected by clustering reads at 97% sequence similarity59. Taxonomy was assigned to each OTU with a 90% sequence similarity threshold using the Greengenes reference database60. A phylogenetic tree was inferred from the OTU data using FastTree61. The phylogenetic tree was then used to calculate weighted and unweighted UniFrac distances between each pair of samples in the study62, 63. Microbiome compositional differences were visualized using principle coordinates analysis (PCoA). Community-level differences between mice genetic background as well as disease susceptibility groups were assessed using a PERMANOVA test64 of weighted and unweighted UniFrac distances. To assess significance in the PERMANOVA test, each cage was randomly re-assigned to groups 9,999 times. Differential abundance was assessed for taxa present in at least 80% of the samples, using generalized linear mixed-effects models. For tests of taxon abundance, the cage was modelled as a random effect, as previous research has established that the faecal microbiota of mice are correlated within cages65. The P values were corrected for multiple testing using Benjamini–Hochberg method. Sample sizes were estimated on the basis of our previous experience with the neonatal CCM model and lesion volume quantification by microCT9. Using 40 historically collected, susceptible Krit1ECKO and Ccm2ECKO P10 brains, we calculated a sample standard deviation of 0.250 mm3. Between Krit1ECKO and Ccm2ECKO genotypes, an F-test to compare variances confirmed no significant difference (P = 0.340). Thus, for a two-group comparison of lesion volumes, each sample group requires seven animals for a desired statistical power of 95% (β = 0.05), and a conventional significance threshold of 5% (α = 0.05) assuming an effect size of 50% (0.5) and equal standard deviations between sample groups. These predictive calculations were corroborated by our recent publication in which larger effect sizes (>90%) were found to be statistically significant with four to five samples per group9. All experimental and control animals were littermates and none were excluded from analysis at the time of harvest. Experimental animals were lost or excluded at two pre-defined points: (i) failure to properly inject 4OHT and observation of significant leakage; (ii) death before P10 because of injection or chaos. Given the early time points, no attempt was made to distinguish or segregate results based on neonatal genders. P values were calculated as indicated in figure legends using an unpaired, two-tailed Student’s t-test; one-way ANOVA with multiple comparison corrections (Holm–Sidak or Bonferroni); PERMANOVA; or linear mixed effects modelling. As indicated in the figure legends, the standard error of the mean (s.e.m.), 95% confidence interval, or boxplot is shown. All relevant data are available from the authors upon reasonable request.
News Article | May 11, 2017
They assessed the risk of cardiac events following consumption of energy drinks in patients diagnosed with congenital long QT syndrome (LQTS), a condition that affects 1 in 2000 and that can cause rapid, irregular heartbeat that can lead to sudden death. In their study , published in the International Journal of Cardiology , they report that even small amounts of energy drinks can cause changes in the heart that can lead to life-threatening arrhythmias and recommend cautioning young patients, some of whom may still be unaware of an existing heart condition, about the danger. The hemodynamic effects of energy drinks in healthy young adults have been assessed in prior studies with results including increased blood pressure, but no change in heart rate. This is the first study specifically designed to test the effects of these energy drinks in individuals who carry the gene faults causing congenital LQTS, say the researchers. "The potential cardiovascular risk of energy drinks continues to emerge as an important public health issue," said lead investigator Professor Christopher Semsarian of the University of Sydney and Centenary Institute, Australia. "The population most at risk is teenagers and young adults, representing the population these drinks are most heavily marketed towards. Since energy drinks are widely available to all ages and over the counter, it is important that cardiovascular effects of these drinks are investigated." The study was designed to assess the acute cardiovascular responses to energy drink consumption in patients with familial LQTS and to discover whether any identified cardiovascular effects correlate with changes in blood levels of the active ingredients - caffeine and taurine. Investigators recruited 24 patients aged 16 to 50. More than half were symptomatic before diagnosis and receiving beta-blocker therapy. Most had undergone genetic testing, 13 of whom had a documented pathogenic or likely pathogenic mutation. Participants were assigned to energy drink or control drink groups for the first study visit. The energy drink consisted of two Red Bull sugar-free cans totalling 160mg of caffeine and 2000mg of taurine, totaling 500ml. The control drink was a cordial-based 500ml drink with no caffeine or taurine. Electrocardiograms and blood pressure were recorded every 10 minutes, while signal-averaged electrocardiogram (SAECG) testing and repeat bloods were collected every 30 minutes for a total observation time of 90 minutes. The results of the study show that three patients (12.5%) exhibited dangerous QT prolongation following energy drink consumption and two of the three had sharp increases in blood pressure. These patients all had a documented family history of sudden cardiac death and two of them had previously experienced severe clinical issues. "Some individual patients may be at a higher risk," added Professor Semsarian. "We therefore suggest caution in allowing the consumption of energy drinks in young patients with LQTS." In an accompanying commentary , Professor Peter J. Schwartz, head of the Center for Cardiac Arrhythmias of Genetic Origin, IRCCS Istituto Auxologico Italiano, said: "Data suggest that the majority of LQTS patients destined to become symptomatic have the first event well after having become a teenager, which implies that a significant number of youngsters with LQTS will help themselves to energy drinks without knowing their real condition and thus endangering themselves." The findings drew an immediate response from trade body the British Soft Drinks Association. Gavin Partington, its director general, said: “Undoubtedly anyone sensitive to caffeine should only consume it in moderation but it’s important to bear that in mind in relation to all sources. One 250ml can of energy drink typically contains about the same amount of caffeine as a cup of coffee. The recent EFSA opinion confirms the safety of energy drinks and their ingredients and therefore does not provide any scientific justification to treat energy drinks any differently to the main contributors to daily caffeine intake including tea and coffee.”
Semsarian C.,Centenary Institute |
Semsarian C.,University of Sydney |
Semsarian C.,Royal Prince Alfred Hospital |
Ingles J.,Centenary Institute |
And 3 more authors.
Journal of the American College of Cardiology | Year: 2015
Hypertrophic cardiomyopathy (HCM) is an important genetic heart muscle disease for which prevalence in the general population has not been completely resolved. For the past 20 years, most data have supported the occurrence of HCM at about 1 in 500. However, the authors have interrogated a number of relevant advances in cardiovascular medicine, including widespread fee-for-service genetic testing, population genetic studies, and contemporary diagnostic imaging, as well as a greater index of suspicion and recognition for both the clinically expressed disease and the gene-positive-phenotype-negative subset (at risk for developing the disease). Accounting for the potential impact of these initiatives on disease occurrence, the authors have revisited the prevalence of HCM in the general population. They suggest that HCM is more common than previously estimated, which may enhance its recognition in the practicing cardiovascular community, allowing more timely diagnosis and the implementation of appropriate treatment options for many patients. © 2015 American College of Cardiology Foundation.
Semsarian C.,Centenary Institute |
Semsarian C.,University of Sydney |
Semsarian C.,Royal Prince Alfred Hospital |
Hamilton R.M.,University of Toronto
Heart Rhythm | Year: 2012
Sudden Cardiac Death (SCD) is a major and tragic complication of a number of cardiovascular diseases. While in the older populations, SCD is most frequently caused by underlying coronary artery disease and heart failure, in those aged under 40 years, the causes of SCD commonly include genetic disorders, such as inherited cardiomyopathies and primary arrhythmogenic diseases. As part of the evaluation of families in which SCD has occurred, the role of genetic testing has evolved as an important feature in both establishing an underlying diagnosis and in screening at-risk family relatives. Specifically, in cases where no definitive cause is identified at postmortem, i.e. Sudden Unexpected Death (SUD), the "molecular autopsy" has emerged as a key process in the investigation of the cause of death. The combination of clinical and genetic evaluation of families in which SUD has occurred provides a platform for early initiation of therapeutic and prevention strategies, with the ultimate goal to reduce sudden death among the young in our communities.
News Article | March 1, 2017
Antibacterial compounds found in soil could spell the beginnings of a new treatment for tuberculosis, new research led by the University of Sydney has found. Believed by many to be a relic of past centuries, tuberculosis (TB) causes more deaths than any other infectious disease including HIV/AIDs. In 2015 there were an estimated 10.4 million new cases of TB and 1.4 million deaths from the disease. The bacterium causing TB (Mycobacterium tuberculosis) is becoming increasingly resistant to current therapies, meaning there is an urgent need to develop new TB drugs. In 2015 an estimated 480,000 cases were unresponsive to the two major drugs used to treat TB. It is estimated more than 250,000 TB deaths were from drug-resistant infections. An international collaboration led by University Professors Richard Payne, from the School of Chemistry, and Warwick Britton, from the Sydney Medical School and the Centenary Institute, has discovered a new compound which could translate into a new drug lead for TB. Its findings were published in Nature Communications today. The group was drawn to soil bacteria compounds known to effectively prevent other bacteria growing around them. Using synthetic chemistry the researchers were able to recreate these compounds with structural variations, turning them into more potent compounds called analogues. When tested in a containment laboratory these analogues proved to be effective killers of Mycobacterium tuberculosis. "These analogues inhibit the action of a key protein needed to build a protective cell wall around the bacterium," said Professor Payne. "Without a cell wall, the bacterium dies. This wall-building protein is not targeted by currently available drugs. "The analogues also effectively killed TB-causing bacteria inside macrophages, the cells in which the bacteria live in human lungs." Professor Payne said the findings are the starting point for a new TB drug. Planning for further testing and safety studies is underway. The research was done in collaboration with Colorado State University in the USA, Simon Fraser University in Canada, Warwick University in the UK, Monash University and the University of Queensland. It was funded by Australia's National Health and Medical Research Centre (NHMRC). Professors Payne and Britton also belong to the University's Marie Bashir Institute for Infectious Diseases and Biosecurity. Professor Payne won the Malcolm McIntosh Prize for Physical Scientist of the Year at the 2016 Prime Minister's Science Prizes.
Levesque J.-P.,Materials Medical Research Institute |
Winkler I.G.,Materials Medical Research Institute |
Rasko J.E.J.,Centenary Institute |
Rasko J.E.J.,University of Sydney
BioEssays | Year: 2013
Stem cells and their malignant counterparts require the support of a specific microenvironment or "niche". While various anti-cancer therapies have been broadly successful, there are growing opportunities to target the environment in which these cells reside to further improve therapeutic efficacy and outcome. This is particularly true when the aim is to target normal or malignant stem cells. The field aiming to target or use the niches that harbor, protect, and support stem cells could be designated as "nichotherapy". In this essay, we provide a few examples of nichotherapies. Some have been employed for decades, such as hematopoietic stem cell mobilization, whereas others are emerging, such as chemosensitization of leukemia stem cells by targeting their niche. © 2013 WILEY Periodicals, Inc.
Liu R.,Centenary Institute |
Liu R.,University of Sydney |
Leslie K.L.,Yale University |
Martin K.A.,Yale University
Biochimica et Biophysica Acta - Gene Regulatory Mechanisms | Year: 2015
Smooth muscle cells (SMC) are the major cell type in blood vessels. Their principal function in the body is to regulate blood flow and pressure through vessel wall contraction and relaxation. Unlike many other mature cell types in the adult body, SMC do not terminally differentiate but retain a remarkable plasticity. They have the unique ability to toggle between a differentiated and quiescent "contractile" state and a highly proliferative and migratory "synthetic" phenotype in response to environmental stresses.While there have been major advances in our understanding of SMC plasticity through the identification of growth factors and signals that can influence the SMC phenotype, how these regulate SMC plasticity remains unknown. To date, several key transcription factors and regulatory cis elements have been identified that play a role in modulating SMC state. The frontier in understanding the molecular mechanisms underlying SMC plasticity has now advanced to the level of epigenetics. This review will summarize the epigenetic regulation of SMC, highlighting the role of histone modification, DNA methylation, and our most recent identification of a DNA demethylation pathway in SMC that is pivotal in the regulation of the SMC phenotypic state.Many disorders are associated with smooth muscle dysfunction, including atherosclerosis, the major underlying cause of stroke and coronary heart disease, as well as transplant vasculopathy, aneurysm, asthma, hypertension, and cancer. An increased understanding of the major regulators of SMC plasticity will lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of these diseases. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity. © 2014 Elsevier B.V.
Bonham K.S.,Harvard University |
Orzalli M.H.,Harvard University |
Hayashi K.,Yale University |
Wolf A.I.,Wistar Institute |
And 6 more authors.
Cell | Year: 2014
The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important for host defense. It remains unclear how this common signaling pathway can be activated from multiple subcellular locations. Here, we report that, in response to natural activators of innate immunity, the sorting adaptor TIRAP regulates TLR signaling from the plasma membrane and endosomes. TLR signaling from both locations triggers the TIRAP-dependent assembly of the myddosome, a protein complex that controls proinflammatory cytokine expression. The actions of TIRAP depend on the promiscuity of its phosphoinositide-binding domain. Different lipid targets of this domain direct TIRAP to different organelles, allowing it to survey multiple compartments for the presence of activated TLRs. These data establish how promiscuity, rather than specificity, can be a beneficial means of diversifying the subcellular sites of innate immune signal transduction. © 2014 Elsevier Inc.
Power C.,Centenary Institute |
Rasko J.E.J.,Centenary Institute
Annals of Internal Medicine | Year: 2011
In recent years, stem cells have generated increasing excitement, with frequent claims that they are revolutionizing medicine. For those not directly involved in stem cell research, however, it can be difficult to separate fact from fiction or realistic expectation from wishful thinking. This article aims to provide internists with a clear and concise introduction to the field. While recounting some scientific and medical milestones, the authors discuss the 3 main varieties of stem cells-adult, embryonic, and induced pluripotent- comparing their advantages and disadvantages for clinical medicine. The authors have sought to avoid the moral and political debates surrounding stem cell research, focusing instead on scientific and medical issues. © 2011 American College of Physicians.