Localized cardiomyocyte lipid accumulation is associated with slowed epicardial conduction in rats.

Transmural action potential duration differences and transmural conduction gradients aid the synchronization of left ventricular repolarization, reducing vulnerability to transmural reentry and arrhythmias. A high-fat diet and the associated accumulation of pericardial adipose tissue are linked with conduction slowing and greater arrhythmia vulnerability. It is predicted that cardiac adiposity may more readily influence epicardial conduction (versus endocardial) and disrupt normal transmural activation/repolarization gradients. The aim of this investigation was to determine whether transmural conduction gradients are modified in a rat model of pericardial adiposity. Adult Sprague-Dawley rats were fed control/high-fat diets for 15 wk. Left ventricular 300 µm tangential slices were generated from the endocardium to the epicardium, and conduction was mapped using microelectrode arrays. Slices were then histologically processed to assess fibrosis and cardiomyocyte lipid status. Conduction velocity was significantly greater in epicardial versus endocardial slices in control rats, supporting the concept of a transmural conduction gradient. High-fat diet feeding increased pericardial adiposity and abolished the transmural conduction gradient. Slowed epicardial conduction in epicardial slices strongly correlated with an increase in cardiomyocyte lipid content, but not fibrosis. The positive transmural conduction gradient reported here represents a physiological property of the ventricular activation sequence that likely protects against reentry. The absence of this gradient, secondary to conduction slowing and cardiomyocyte lipid accumulation, specifically in the epicardium, indicates a novel mechanism by which pericardial adiposity may exacerbate ventricular arrhythmias.

High resolution optical mapping of cardiac electrophysiology in pre-clinical models.

Optical mapping of animal models is a widely used technique in pre-clinical cardiac research. It has several advantages over other methods, including higher spatial resolution, contactless recording and direct visualisation of action potentials and calcium transients. Optical mapping enables simultaneous study of action potential and calcium transient morphology, conduction dynamics, regional heterogeneity, restitution and arrhythmogenesis. In this dataset, we have optically mapped Langendorff perfused isolated whole hearts (mouse and guinea pig) and superfused isolated atria (mouse). Raw datasets (consisting of over 400 files) can be combined with open-source software for processing and analysis. We have generated a comprehensive post-processed dataset characterising the baseline cardiac electrophysiology in these widely used pre-clinical models. This dataset also provides reference information detailing the effect of heart rate, clinically used anti-arrhythmic drugs, ischaemia-reperfusion and sympathetic nervous stimulation on cardiac electrophysiology. The effects of these interventions can be studied in a global or regional manner, enabling new insights into the prevention and initiation of arrhythmia.

Optimal management strategies for primary headache in the emergency department.

PURPOSE: We sought to evaluate the factors associated with better outcomes for emergency department (ED) patients treated for primary headache. METHODS: This was a health records review of consecutive patients over a 3-month period presenting to two tertiary EDs and discharged with a diagnosis of primary headache. The primary outcome was the need for second round medications, defined as medications received > 1 h after the initial physician-ordered medications were administered. We performed multivariate logistic regression analysis to determine treatment factors associated with need for second round medications. RESULTS: We included 553 patients, mean age was 42.2 years and 72.9% were females. The most common diagnoses were headache not otherwise specified (48.8%) and migraine (43%). Ketorolac IV (62.2%) and metoclopramide IV (70.2%) were the most frequently administered medications. 18% of patients met the primary outcome. Dopamine antagonists (OR 0.3 [95% CI 0.1-0.5]) and non-steroidal anti-inflammatory drugs (NSAIDs) (OR 0.5 [95% CI 0.3-0.8]) ordered with initial medications were associated with reduced need for second round medications. Intravenous fluid boluses ≥ 500 ml (OR 2.8 [95% CI: 1.5-5.2]) and non-dopamine antagonist antiemetics (OR 2.2 [95% CI 1.2-4.2]) were associated with increased need. Opioid use approached statistical significance for receiving second round medication (p = 0.06). CONCLUSION: We determined that use of dopamine antagonists and NSAIDs were associated with a reduced need for second round medications in ED primary headache patients. Conversely, non-dopamine antagonist antiemetic medications and intravenous fluids were associated with a significantly increased need for second round medications. Careful choice of initial therapy may optimize management for these patients.

RéSUMé: OBJECTIF: Nous avons cherché à évaluer les facteurs associés à de meilleurs résultats pour les patients des services d'urgence traités pour des céphalées primaires. MéTHODES: Il s'agissait d'un examen des dossiers médicaux de patients consécutifs sur une période de 3 mois se présentant à deux services d'urgence tertiaires et sortis avec un diagnostic de céphalée primaire. Le résultat primaire était la nécessité d'une deuxième série de médicaments, définis comme des médicaments reçus > 1 heure après l'administration des premiers médicaments prescrits par le médecin. Nous avons effectué une analyse de régression logistique multivariée pour déterminer les facteurs de traitement associés au besoin de médicaments de second tour RéSULTATS: Nous avons inclus 553 patients, l'âge moyen était de 42,2 ans et 72,9 % étaient des femmes. Les diagnostics les plus fréquents étaient les céphalées non spécifiées autrement (48,8 %) et la migraine (43 %). Le kétorolac IV (62,2%) et le métoclopramide IV (70,2 %) étaient les médicaments les plus fréquemment administrés. 18 % des patients ont atteint le résultat primaire. Les antagonistes de la dopamine (OR 0,3 [IC 95 % : 0,1-0,5]) et les anti-inflammatoires non stéroïdiens (AINS) (OR 0,5 [IC 95 % : 0,3-0,8]) commandés avec les médicaments initiaux étaient associés à un besoin réduit de médicaments de deuxième série. Les bolus liquidiens intraveineux ≥ 500 ml (OR 2,8 [IC 95 % : 1,5-5,2]) et les antiémétiques non antagonistes de la dopamine (OR 2,2 [IC 95 % : 1,2-4,2]) étaient associés à un besoin accru. L'utilisation d'opioïdes a approché la signification statistique pour la réception d'un médicament de deuxième série (p = 0,06). CONCLUSION: Nous avons déterminé que l'utilisation d'antagonistes de la dopamine et d'AINS était associée à un besoin réduit de médicaments de second tour chez les patients souffrant de céphalées primaires aux urgences. À l'inverse, les médicaments antiémétiques non antagonistes de la dopamine et les fluides intraveineux étaient associés à un besoin significativement accru de médicaments de second tour. Un choix judicieux du traitement initial peut optimiser la prise en charge de ces patients.

Epicardial Adipose Tissue Accumulation Confers Atrial Conduction Abnormality.

BACKGROUND: Clinical studies have reported that epicardial adipose tissue (EpAT) accumulation associates with the progression of atrial fibrillation (AF) pathology and adversely affects AF management. The role of local cardiac EpAT deposition in disease progression is unclear, and the electrophysiological, cellular, and molecular mechanisms involved remain poorly defined. OBJECTIVES: The purpose of this study was to identify the underlying mechanisms by which EpAT influences the atrial substrate for AF. METHODS: Patients without AF undergoing coronary artery bypass surgery were recruited. Computed tomography and high-density epicardial electrophysiological mapping of the anterior right atrium were utilized to quantify EpAT volumes and to assess association with the electrophysiological substrate in situ. Excised right atrial appendages were analyzed histologically to characterize EpAT infiltration, fibrosis, and gap junction localization. Co-culture experiments were used to evaluate the paracrine effects of EpAT on cardiomyocyte electrophysiology. Proteomic analyses were applied to identify molecular mediators of cellular electrophysiological disturbance. RESULTS: Higher local EpAT volume clinically correlated with slowed conduction, greater electrogram fractionation, increased fibrosis, and lateralization of cardiomyocyte connexin-40. In addition, atrial conduction heterogeneity was increased with more extensive myocardial EpAT infiltration. Cardiomyocyte culture studies using multielectrode arrays showed that cardiac adipose tissue-secreted factors slowed conduction velocity and contained proteins with capacity to disrupt intermyocyte electromechanical integrity. CONCLUSIONS: These findings indicate that atrial pathophysiology is critically dependent on local EpAT accumulation and infiltration. In addition to myocardial architecture disruption, this effect can be attributed to an EpAT-cardiomyocyte paracrine axis. The focal adhesion group proteins are identified as new disease candidates potentially contributing to arrhythmogenic atrial substrate.

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48-120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the ß-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

High-Throughput Analysis of Optical Mapping Data Using ElectroMap.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

ElectroMap: High-throughput open-source software for analysis and mapping of cardiac electrophysiology.

The ability to record and analyse electrical behaviour across the heart using optical and electrode mapping has revolutionised cardiac research. However, wider uptake of these technologies is constrained by the lack of multi-functional and robustly characterised analysis and mapping software. We present ElectroMap, an adaptable, high-throughput, open-source software for processing, analysis and mapping of complex electrophysiology datasets from diverse experimental models and acquisition modalities. Key innovation is development of standalone module for quantification of conduction velocity, employing multiple methodologies, currently not widely available to researchers. ElectroMap has also been designed to support multiple methodologies for accurate calculation of activation, repolarisation, arrhythmia detection, calcium handling and beat-to-beat heterogeneity. ElectroMap implements automated signal segmentation, ensemble averaging and integrates optogenetic approaches. Here we employ ElectroMap for analysis, mapping and detection of pro-arrhythmic phenomena in silico, in cellulo, animal model and in vivo patient datasets. We anticipate that ElectroMap will accelerate innovative cardiac research and enhance the uptake, application and interpretation of mapping technologies leading to novel approaches for arrhythmia prevention.

Clostridium difficile infection in pregnant and postpartum women in 2 hospitals and a review of literature.

BACKGROUND: Healthcare-associated Clostridium difficile infection (CDI) in pregnant/postpartum women is underreported, especially outside of North America. We report a cluster of cases in 2 neighboring secondary care hospitals in South-East England. The objective of this study was to identify the epidemiology and risk factors for infection. METHODS: An investigation into a cluster of cases of confirmed CDI in pregnant/postpartum women was performed over a 12-month period, from June 2016 to June 2017. RESULTS: Eleven cases, in 10 patients, were identified, including 1 patient who had a relapse. Eight of 10 patients developed symptoms after hospital discharge. All patients had received broad-spectrum antibiotics prior to CDI onset. Environmental vectors, such as labor room mattresses, that were found difficult to effectively decontaminate after heavy contamination with blood, feces, and other body fluids may have been possible reservoirs. An infection control care bundle was successful in preventing further cases. CONCLUSIONS: Antibiotic use and exposure to the organism in a contaminated labor room environment are likely risk factors for healthcare-associated CDI in postpartum women. Active surveillance is necessary to prevent these infections, as these cases often present after hospital discharge.

Developmental validation of the ParaDNA® Body Fluid ID System-A rapid multiplex mRNA-profiling system for the forensic identification of body fluids.

Identifying the biological origin of forensic traces can provide crucial evidence to aid criminal investigations. Current forensic practice for the identification of body fluids mostly uses protein-based presumptive tests. Such tests cannot identify all of the forensically relevant fluids and have issues of cross-reactivity. More recently, messenger RNA methods have been developed that have expanded the range of body fluids that can be positively identified. However, these methods are slow and require expert scientists to run the processes and interpret the results. The ParaDNA® Body Fluid ID System has been designed to provide a simple, fast and robust way to identify forensically relevant body fluids in a lab or field-deployable manner. The system can analyse and detect mRNA targets for six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual blood and peripheral blood. Utilising the ParaDNA Sample Collector and instruments, minimal training is required to enable both forensic scientists and non-specialist personnel to analyse biological samples directly, without prior sample processing, in approximately 90 min. The developmental validation studies described here were designed to address requirements of end users, based on the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Body Fluid ID System on a range of mock evidence items. The data collected demonstrate that the Body Fluid ID System can automatically determine the presence of specific body fluid mRNA markers in single-source and mixed samples on multiple substrate types and body fluids could be identified with as little as 0.05ng total RNA and 1µl of the relevant fluid. Results can either be used to support confirmation of source from previously obtained STR DNA profiling results or to improve sample success rates by making better informed evidential submissions.

Development of HyBeacon® probes for specific mRNA detection using body fluids as a model system.

HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed.

Structural and Vibrational Properties of Iodopentafluorobenzene: A Combined Raman and Infrared Spectral and Theoretical Study.

A combined study of the vibrational spectroscopy of iodopentafluorobenzene by new Raman and Fourier transform infrared (FTIR) spectroscopies, over the spectral range 300 to 3200 cm-1 (Raman) and 50 to 3400 cm-1 (FTIR), with a state-of-the-art theoretical investigation is reported. This has enabled reliable identification of numerous fundamental, overtone, and combination band transitions in unprecedented detail. The theoretical analysis, beyond the double-harmonic approximation, is based on generalized second-order vibrational perturbation theory (GVPT2) with a hybrid coupled cluster/density functional theory (CC/DFT) approach. Anharmonic contributions to structural parameters, rotational constants, vibrational frequencies, and spectral intensities are incorporated. The procedures, of general applicability, enable rigorous comparison of theoretical methods with experimental results in vibrational spectroscopy.

Is there a group of patients at greater risk for hematoma following thyroidectomy? A systematic review and meta-analysis.

OBJECTIVE: The purpose of this study was to perform a systematic review and meta-analysis to determine whether a difference exists in hematoma rates following thyroidectomy for any of the following subgroups of patients: Graves disease, toxic nodular goiter (TNG), and malignancy. STUDY DESIGN: Systematic review and meta-analysis. METHODS: A systematic literature search was performed for all relevant English and French language studies (1946-2015) using Ovid MEDLINE, EMBASE, and PubMed. Three authors independently extracted data and analyzed articles for quality using the Newcastle-Ottawa Quality Assessment Scale. Our primary outcome of interest was hematoma requiring re-operation. RESULTS: A total of 301 studies were screened, with 11 studies meeting the inclusion criteria. The results of our analysis demonstrated that Graves disease is the only indication for thyroidectomy that appears to have an increased risk of postoperative hematoma formation, pooled odds ratio = 1.58 (1.09-2.31); P = 0.02. Malignancy and TNG did not demonstrate significantly higher rates of postoperative hematoma formation. CONCLUSION: This study demonstrates that of patients undergoing thyroidectomy, Graves disease is the only indication in which patients are at increased risk of postoperative hematoma formation. This information may help guide future decisions regarding the implementation of outpatient thyroidectomy. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1483-1490, 2017.

Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).

Alzheimer's disease-related peptide PS2V plays ancient, conserved roles in suppression of the unfolded protein response under hypoxia and stimulation of γ-secretase activity.

The PRESENILIN1 and PRESENILIN2 genes encode structurally related proteases essential for γ-secretase activity. Of nearly 200 PRESENILIN mutations causing early onset, familial Alzheimer's disease (FAD) only the K115Efx10 mutation of PSEN2 causes truncation of the open reading frame. If translated, the truncated product would resemble a naturally occurring isoform of PSEN2 named PS2V that is induced by hypoxia and found at elevated levels in late onset Alzheimer's disease (AD) brains. The function of PS2V is largely unexplored. We show that zebrafish possess a PS2V-like isoform, PS1IV, produced from the fish's PSEN1 rather than PSEN2 orthologous gene. The molecular mechanism controlling formation of PS2V/PS1IV was probably present in the ancient common ancestor of the PSEN1 and PSEN2 genes. Human PS2V and zebrafish PS1IV have highly divergent structures but conserved abilities to stimulate γ-secretase activity and to suppress the unfolded protein response (UPR) under hypoxia. The putative protein truncation caused by K115Efx10 resembles PS2V in its ability to increase γ-secretase activity and suppress the UPR. This supports increased Aß levels as a common link between K115Efx10 early onset AD and sporadic, late onset AD. The ability of mutant variants of PS2V to stimulate γ-secretase activity partially correlates with their ability to suppress the UPR. The cytosolic, transmembrane and luminal domains of PS2V are all critical to its γ-secretase and UPR-suppression activities. Our data support a model in which chronic hypoxia in aged brains promotes excessive Notch signalling and accumulation of Aß that contribute to AD pathogenesis.

Developmental Validation of the ParaDNA® Screening System - A presumptive test for the detection of DNA on forensic evidence items.

Current assessment of whether a forensic evidence item should be submitted for STR profiling is largely based on the personal experience of the Crime Scene Investigator (CSI) and the submissions policy of the law enforcement authority involved. While there are chemical tests that can infer the presence of DNA through the detection of biological stains, the process remains mostly subjective and leads to many samples being submitted that give no profile or not being submitted although DNA is present. The ParaDNA(®) Screening System was developed to address this issue. It consists of a sampling device, pre-loaded reaction plates and detection instrument. The test uses direct PCR with fluorescent HyBeacon™ detection of PCR amplicons to identify the presence and relative amount of DNA on an evidence item and also provides a gender identification result in approximately 75 minutes. This simple-to-use design allows objective data to be acquired by both DNA analyst and non-specialist personnel, to enable a more informed submission decision to be made. The developmental validation study described here tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Screening System on a range of mock evidence items. The data collected demonstrates that the ParaDNA Screening System identifies the presence of DNA on a variety of evidence items including blood, saliva and touch DNA items.

Transgenic zebrafish recapitulating tbx16 gene early developmental expression.

We describe the creation of a transgenic zebrafish expressing GFP driven by a 7.5 kb promoter region of the tbx16 gene. This promoter segment is sufficient to recapitulate early embryonic expression of endogenous tbx16 in the presomitic mesoderm, the polster and, subsequently, in the hatching gland. Expression of GFP in the transgenic lines later in development diverges to some extent from endogenous tbx16 expression with the serendipitous result that one line expresses GFP specifically in commissural primary ascending (CoPA) interneurons of the developing spinal cord. Using this line we demonstrate that the gene mafba (valentino) is expressed in CoPA interneurons.

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord.

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine "irregular" patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.

Independent and cooperative action of Psen2 with Psen1 in zebrafish embryos.

Presenilin1 (PSEN1) and presenilin2 (PSEN2) are involved in the processing of type-1 transmembrane proteins including the amyloid precursor protein (APP), Notch and several others. PSEN1 has been shown to be crucial for proteolytic cleavage of Notch in developing animal embryos. Mouse embryos lacking Psen1 function show disturbed neurogenesis and somite formation, resembling Notch pathway mutants. However, loss of Psen2 activity reveals only a minor phenotype. Zebrafish embryos are a valuable tool for analysis of the molecular genetic control of cell differentiation since endogenous gene expression can be modulated in subtle and complex ways to give a phenotypic readout. Using injection of morpholino antisense oligonucleotides to inhibit protein translation in zebrafish embryos, we show that reduced Psen2 activity decreases the number of melanocytes in the trunk but not in the cranial area at 2 days post fertilisation (dpf). Reduced Psen2 activity apparently reduces Notch signalling resulting in perturbed spinal neurogenin1 (neurog1) expression, neurogenesis and trunk and tail neural crest development. Similar effects are seen for reduced Psen1 activity. These results suggest that Psen2 plays a more prominent role in Notch signalling and embryo development in zebrafish than in mammals. Intriguingly, decreased Psen2 activity increases the number of Dorsal Longitudinal Ascending (DoLA) interneurons in the spinal cord while decreased Psen1 activity has no effect. However, the effect on DoLAs of reduced Psen2 can be ameliorated by Psen1 loss. The effects of changes in Psen2 activity on DoLA interneurons and other cells in zebrafish embryos provide bioassays for more detailed dissection of Psen2 function.

Interference with splicing of Presenilin transcripts has potent dominant negative effects on Presenilin activity.

Missense mutations in the PRESENILIN1 (PSEN1) gene frequently underlie familial Alzheimer's disease (FAD). Nonsense and most splicing mutations result in the synthesis of truncated peptides, and it has been assumed that truncated PSEN1 protein is functionless so that heterozygotes for these mutations are unaffected. Some FAD mutations affecting PSEN1 mRNA splicing cause loss of exon 8 or 9 sequences while maintaining the reading frame. We attempted to model these exon-loss mutations in zebrafish embryos by injecting morpholino antisense oligonucleotides (morpholinos) directed against splice acceptor sites in zebrafish psen1 transcripts. However, this produced cryptic changes in splicing potentially forming mRNAs encoding truncated presenilin proteins. Aberrant splicing in the region between exons 6 and 8 produces potent dominant negative effects on Psen1 protein activity, including Notch signalling, and causes a hydrocephalus phenotype. Reductions in Psen1 activity feedback positively to increase psen1 transcription through a mechanism apparently independent of gamma-secretase. We present evidence that the dominant negative effects are mediated through production of truncated Psen1 peptides that interfere with the normal activity of both Psen1 and Psen2. Mutations causing such truncations would be dominant lethal in embryo development. Somatic cellular changes in ageing cells that interfere with PSEN1 splicing, or otherwise cause protein truncation, might contribute to sporadic Alzheimer's disease, cancer and other diseases.