Phenotypic analysis of catastrophic childhood epilepsy genes: The Epilepsy Zebrafish Project

AbstractGenetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. A unifying epilepsy classification scheme was developed based on local field potential recordings and blinded scoring from ~3300 larvae. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

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Phenotypic analysis of catastrophic childhood epilepsy genes

Communications Biology ◽

10.1038/s42003-021-02221-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Aliesha Griffin ◽

Colleen Carpenter ◽

Jing Liu ◽

Rosalia Paterno ◽

Brian Grone ◽

...

Keyword(s):

Neurological Disorders ◽

Seizure Activity ◽

Single Gene ◽

Field Potential ◽

Childhood Epilepsy ◽

Phenotypic Analysis ◽

Electrographic Seizure ◽

Starting Point ◽

Gene Mutant ◽

Neuroscience Community

AbstractGenetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen ∼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

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Fields Connected with Particles in Terms of Complex-Riemannian Geometry

Zeitschrift für Naturforschung A ◽

10.1515/zna-1990-0201 ◽

1990 ◽

Vol 45 (2) ◽

pp. 81-94

Author(s):

Julian Ławrynowicz ◽

Katarzyna Kędzia ◽

Leszek Wojtczak

Keyword(s):

Field Potential ◽

Riemannian Metrics ◽

Interaction Matrix ◽

Explicit Calculation ◽

Maxwell System ◽

Curved Space ◽

Starting Point ◽

Autocorrelation Time ◽

The Fourier Transform ◽

Convolution Equations

AbstractA complex analytical method of solving the generalised Dirac-Maxwell system has recently been proposed by two of us for a certain class of complex Riemannian metrics. The Dirac equation without the field potential in such a metric appeared to be equivalent to the Dirac-Maxwell system including the field potentials produced by the currents of a particle in question. The method proposed is connected with applying the Fourier transform with respect to the electric charge treated as a variable, with the consideration of the mass as an eigenvalue, and with solving suitable convolution equations. In the present research an explicit calculation based on linearization of the spinor connections is given. The conditions for the motion are interpreted as a starting point to seek selection rules for curved space-times corresponding to actually existing particles. Then the same method is applied to solids. Namely, by a suitable transformation of the configuration space in terms of elements of the interaction matrix corresponding to the Coulomb, exchange, and dipole integrals, the interaction term in the hamiltonian becomes zero, thus leading to experimentally verificable formulae for the autocorrelation time

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Endogenous and exogenous electric fields as modifiers of brain activity: rational design of noninvasive brain stimulation with transcranial alternating current stimulation

Dialogues in Clinical Neuroscience ◽

10.31887/dcns.2014.16.1/ffroehlich ◽

2014 ◽

Vol 16 (1) ◽

pp. 93-102 ◽

Cited By ~ 15

Keyword(s):

Electric Fields ◽

Brain Stimulation ◽

Rational Design ◽

Brain Activity ◽

Field Potential ◽

Alternating Current ◽

Noninvasive Brain Stimulation ◽

Starting Point ◽

Recent Developments ◽

Transcranial Alternating Current Stimulation

Synchronized neuronal activity in the cortex generates weak electric fields that are routinely measured in humans and animal models by electroencephalography and local field potential recordings. Traditionally, these endogenous electric fields have been considered to be an epiphenomenon of brain activity. Recent work has demonstrated that active cortical networks are surprisingly susceptible to weak perturbations of the membrane voltage of a large number of neurons by electric fields. Simultaneously, noninvasive brain stimulation with weak, exogenous electric fields (transcranial current stimulation, TCS) has undergone a renaissance due to the broad scope of its possible applications in modulating brain activity for cognitive enhancement and treatment of brain disorders. This review aims to interface the recent developments in the study of both endogenous and exogenous electric fields, with a particular focus on rhythmic stimulation for the modulation of cortical oscillations. The main goal is to provide a starting point for the use of rational design for the development of novel mechanism-based TCS therapeutics based on transcranial alternating current stimulation, for the treatment of psychiatric illnesses.

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Ontogenetic study of the fusion of floral organs in the normal and "solanifolia" mutant of tomato (Lycopersicon esculentum)

Canadian Journal of Botany ◽

10.1139/b87-030 ◽

1987 ◽

Vol 65 (2) ◽

pp. 215-221 ◽

Cited By ~ 5

Author(s):

K. N. Chandra Sekhar ◽

V. K. Sawhney

Keyword(s):

Lycopersicon Esculentum ◽

Single Gene ◽

Corolla Tube ◽

Lateral Growth ◽

Floral Organs ◽

Lack Of Fusion ◽

Organ Type ◽

Gene Mutant ◽

Ontogenetic Study ◽

Single Ovary

A comparative study on the ontogeny of the fusion of floral organs of the normal (cv. Pearson) and a single-gene mutant, "solanifolia" (sf/sf), of tomato (Lycopersicon esculentum Mill.) was conducted. In the normal, floral organs were laterally fused, although the degree and the region of fusion varied in each organ type. In the mutant, the various organs either did not fuse or, if they did, were individually recognizable. The sepals and petals of mutant flowers, unlike those of the normal, did not form a calyx cup and a corolla tube, respectively, and this was related to the limited lateral growth of mutant primordia and the absence of growth in the interprimordial region. Also, petal primordia of the mutant were narrower in width at inception. The stamens of normal flowers were fused by interweaving rows of lateral and adaxial hairs on the anthers. The mutant stamens produced lateral and adaxial hairs, yet they were free. The nonfusion of mutant stamens was related to the smaller primordium widths, greater distance between the primordia, and the larger apex diameter at the time of stamen initiation. The gynoecium of normal flowers consisted of a single ovary, style, and stigma formed by the fusion of carpel primordia. In the mutant, the gynoecium consisted of several carpels, laterally adhered to each other, and each had a recognizable style and stigma. The lack of fusion of mutant carpels was attributed to the larger apex diameter of the mutant during carpel initiation.

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Comprehensive phenotypic analysis of single-gene deletion and overexpression strains of Saccharomyces cerevisiae

Yeast ◽

10.1002/yea.1843 ◽

2011 ◽

Vol 28 (5) ◽

pp. 349-361 ◽

Cited By ~ 74

Author(s):

Katsunori Yoshikawa ◽

Tadamasa Tanaka ◽

Yoshihiro Ida ◽

Chikara Furusawa ◽

Takashi Hirasawa ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Deletion ◽

Single Gene ◽

Phenotypic Analysis ◽

Single Gene Deletion

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A Role for Gαz in regulating seizure susceptibility

10.1101/567628 ◽

2019 ◽

Author(s):

Rainbo Hultman ◽

Okechi Boms ◽

Stephen Mague ◽

Dalton Hughes ◽

Victor Nadler ◽

...

Keyword(s):

Cortical Neurons ◽

Molecular Mechanisms ◽

Seizure Activity ◽

Dorsal Hippocampus ◽

Field Potential ◽

Brain Regions ◽

Seizure Susceptibility ◽

Electrographic Seizure ◽

Pilocarpine Model ◽

Local Field Potential Lfp

AbstractMuch about the molecular mechanisms underlying seizure susceptibility remains unknown. A number of studies have indicated that the neurotrophic factor BDNF plays an important role in mediating seizure susceptibility. Recently, we found that the heterotrimeric G – protein, Gz, which is known to endogenously couple to monoaminergic receptors, such as serotonin, norepinephrine and dopamine receptors, regulates BDNF-induced signaling and development in cortical neurons. Interestingly, several of the receptors that Gz endogenously couples to have also been shown to be associated with seizure phenotypes (5HT1A-serotonin and D2 dopamine). Here we characterized seizure susceptibility in Gz-null mice, behaviorally and electrographically, finding that Gz-null mice have increased seizure susceptibility using a modified version of the pilocarpine model of status epilepticus. Local field potential (LFP) data recorded from six brain regions-amygdala, dorsal hippocampus, ventral hippocampus, motor cortex, somatosensory cortex, and thalamus-showed robust electrographic seizure activity for Gz-null mice compared with low or no seizure activity in wild-type controls.

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CRISPR-Cas9 mutagenesis and single gene reintegration suggests functional diversity within the Candida albicans TLO gene family

Access Microbiology ◽

10.1099/acmi.cc2021.po0016 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Jessica Fletcher ◽

Gary Moran ◽

Derek Sullivan

Keyword(s):

Growth Rate ◽

Candida Albicans ◽

Gene Family ◽

Biofilm Formation ◽

Single Gene ◽

Hyphal Growth ◽

Detectable Effect ◽

Null Mutant ◽

Phenotypic Analysis ◽

Functional Differences

Candida albicans has between 10-15 Telomere-associated ORF family(TLO)genes, whereas its closest relative, Candida dubliniensis, has two. The Tlo proteins are components of the Mediator complex which plays an important role in transcriptional regulation. CRISPR-Cas9 mutagenesis was used to generate a TLOnull mutant of C. albicans. Phenotypic analysis of the mutant showed significantly reduced fitness, with major defects in growth rate, morphogenesis, stress resistance and virulence in a Galleria mellonellamodel. Clade representative TLOα1, TLOβ2 and TLOγ11constructs were reintroduced into the null mutant background to determine if members of the TLO gene family exhibit functional differences. The genes were reintroduced under the control of the TET1 and ENO1promoters. TLOα1and TLOβ2expression restored stress tolerance and growth rate, in some cases to the level of the WT. TLOβ2expression also showed a dramatic effect on morphology resulting in constitutive true hyphal growth. Moderate expression of TLOγ11 had no detectable effect on many of the phenotypes tested, however overexpression increased biofilm formation in Spider medium, and also conferred increased resistance to cell wall stressors. These data suggest that individual TLO genes have distinct functions and that the diversity within the TLO family may contribute to the relative success of C. albicans as a coloniser and pathogen of humans.

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Development of a Single-Gene, Signature-Tag-Based Approach in Combination with Alanine Mutagenesis To Identify Listeriolysin O Residues Critical for theIn VivoSurvival of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.06196-11 ◽

2012 ◽

Vol 80 (6) ◽

pp. 2221-2230 ◽

Cited By ~ 13

Author(s):

Jody A. Melton-Witt ◽

Susannah L. McKay ◽

Daniel A. Portnoy

Keyword(s):

Listeria Monocytogenes ◽

Single Gene ◽

Saturation Mutagenesis ◽

Single Amino Acid ◽

Screening Methods ◽

Listeriolysin O ◽

Phenotypic Analysis ◽

Content Type

ABSTRACTListeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogenListeria monocytogenes. LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis ofL. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termedinvivoanalysis bysaturation mutagenesis andsignature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzedin vivoeach conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using commonin vitrotechniques suggested that the functional contributions of some residues could only have been revealed throughin vivoanalysis.

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The age of single-gene neurological disorders is not dead

Brain ◽

10.1093/brain/awq161 ◽

2010 ◽

Vol 133 (7) ◽

pp. 1865-1868

Author(s):

P. F. Chinnery

Keyword(s):

Neurological Disorders ◽

Single Gene

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Genetic Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae

Phytopathology ◽

10.1094/phyto-97-0106 ◽

2007 ◽

Vol 97 (1) ◽

pp. 106-112 ◽

Cited By ~ 12

Author(s):

S. G. Gordon ◽

S. A. Berry ◽

S. K. St. Martin ◽

A. E. Dorrance

Keyword(s):

Single Gene ◽

Phytophthora Sojae ◽

Stem Rot ◽

Soybean Plant ◽

Phenotypic Analysis ◽

Plant Introductions ◽

Two Generations ◽

Rps Gene ◽

Serious Disease ◽

The Individual

Phytophthora sojae, which causes Phytophthora root and stem rot of soybean, is a serious disease worldwide and is managed primarily by deploying cultivars with resistance. Thirty-two soybean plant introductions (PIs), all but three of which were from South Korea, were proposed as new sources of single-gene resistance to P. sojae. The objective of this study was to characterize the inheritance of resistance to P. sojae in these PIs. Twenty-two soybean populations from crosses of these PIs and the susceptible cv. Williams were inoculated with P. sojae OH17 (vir 1b, 1d, 2, 3a, 3b, 3c, 4, 5, 6, 7), and OH25 (vir 1a, 1b, 1c, 1k, 7). These isolates were selected because they are virulent on soybeans with all known Rps genes and many Rps gene combinations. Thirteen of the twenty-two populations had consistent segregation responses following inoculations between the two generations. In two PIs, resistance was conferred by two genes to OH17 and three genes to OH25. Resistance to both isolates was conferred by a single gene in PI 398440 although the individual families were not resistant to the same isolates. The data suggest that six of the populations have three-Rps gene combinations as previously proposed, while another four may have either a novel Rps gene or a four-Rps gene combination. Based on this phenotypic analysis, novel and uncharacterized Rps genes may be present in this material. More importantly, these PIs may serve as sources of novel Rps genes that can be used to more effectively manage Phytophthora root and stem rot.

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