Brigitta Saul

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Vivekanand Jha

Fortis Escorts Heart Institute

Prof. Nagwa Mohamed A . Aref

Ain Shams University

Prof. Dr. Moustafa Moharam

Sohag University

Dr.Mahmoud Lotfy

Minufiya University

Michael J. Gonzalez

Universidad de Puerto Rico Rio

Nevcivan Guldas

Dokuz Eylül University

Uwamere O Edeghor

University of Calabar, Calabar, Nigeria.

John Egbere

UNIVERSITY OF JOS, NIGERIA

Pradipta Saha

Burdwan University

James Ebdon

University of Brighton

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Papers by Brigitta Saul

A Direct Capture Method for Purification and Detection of Viral Nucleic Acid Enables Epidemiological Surveillance of SARS-CoV-2

medRxiv (Cold Spring Harbor Laboratory), May 10, 2021

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individual... more Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities.

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NovelGLRA1Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating

The Journal of Neuroscience, 1999

Missense mutations as well as a null allele of the human glycine receptor α1 subunit geneGLRA1res... more Missense mutations as well as a null allele of the human glycine receptor α1 subunit geneGLRA1result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A ofGLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature α1 polypeptide. After recombinant expression, homomeric α1P250Tsubunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase inKivalues. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the ...

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A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors

American journal of human genetics, 1996

Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) giv... more Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1(null) allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1 spd-ot), the loss of the GlyR alpha 1 subunit is effectively compensated in ...

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The frameshift mutation oscillator (Glra1spd-ot) produces a complete loss of glycine receptor α1-polypeptide in mouse central nervous system

Neuroscience, 1997

Mice homozygous for the recessive mutation oscillator (Glra1(spd-ot)) suffer from a complex motor... more Mice homozygous for the recessive mutation oscillator (Glra1(spd-ot)) suffer from a complex motor disorder leading to death within three weeks after birth. Symptoms of this disorder mimic poisoning by strychnine, the antagonist of the inhibitory glycine receptor. The syndrome has previously been correlated to a 7 base pair microdeletion within the Glra1 gene (chromosome 11) encoding the alpha1-subunit of the adult glycine receptor isoform. As shown by [3H]strychnine binding and western blot analysis employing subunit-specific antibodies, spinal cord of homozygous oscillator mice was totally devoid of alpha1-polypeptide, characterizing the Glra1(spd-ot) gene as a functional null allele of Glra1. Moreover, tissue levels of the postsynaptic anchoring protein gephyrin were drastically reduced in the Glra1(spd-ot)/Glra1(spd-ot) genotype. In contrast, immunoanalysis revealed a persisting low-level expression of non-alpha1 glycine receptor polypeptides. Spinal glycine receptor content was also significantly reduced in the +/Glra1(spd-ot) genotype. This reduction coincided with increased acoustic startle responses in heterozygous animals consistent with haplotype insufficiency of glycine receptor function. Lethality of the murine null allele Glra1(spd-ot) contrasts with the situation in the human, where homozygosity for a GLRA1 null allele produces the phenotype of the non-lethal disorder hyperekplexia (startle disease; stiff baby syndrome). This suggests a disparate regulation of glycine receptor subunit genes and/or diverse compensatory pathways in mice and humans.

Point mutation of glycine receptor α1 subunit in the spasmodic mouse affects agonist responses

FEBS Letters, 1994

Homozygotic spasmodic (spd/spd) mice suffer from a motor disorder resembling poisoning by the gly... more Homozygotic spasmodic (spd/spd) mice suffer from a motor disorder resembling poisoning by the glycine receptor antagonist strychnine. Here, a point mutation was identified in the glycine receptor α1 subunit gene of the spasmodic mouse which predicts an alanine‐to‐serine exchange at position 52 of the mature polypeptide. Upon expression in Xenopus laevis oocytes, α1A52S receptor channels displayed reduced responses to glycine, β‐alanine and taurine when compared to recombinant α1 glycine receptors. As glycine receptor content in spinal cord and native molecular weight appeared unaltered, this suggests that the spasmodic phenotype results from an altered neurotransmitter sensitivity of the mutant α1A52S subunit.

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The Inhibitory Glycine Receptor

Annals of the New York Academy of Sciences, 1994

... Q. Rev. Biophys. 17. VANDENBERG, RJ, CA HANDFORD & PR SCHOFIELD. 1992. Distinct agonist-a... more