Biology subjects

Sage, J. M.

Publications and source records attributed to Sage, J. M..

7 recordsLinked to original sources

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗

bioRxiv2019

Platelet proteolytic machinery assessment in Alzheimer’s Diseases

AimPlatelets provide substantial information about the proteolytic system profile in neurodegenerative diseases. Assessment of autophagy and proteasome target proteins in platelets may reflect tissue proteolytic machinery profile in central nervous system in Alzheimers diseases (AD). We aimed to demonstrate the optimum assay conditions and identify target proteins in platelet proteolytic machinery. MethodsPlatelet samples were obtained from clinically verified AD patients and age-matched non-demented control subjects that were recruited by University of Kansas Alzheimers disease Center. Autophagosome participating proteins in platelets were identified by Western blotting analysis. Standard gel electrophoresis and electro transfer apparatus were used for protein transfer onto the membrane. Several antibodies were tested to identify the best working antibodies, and their concentrations were optimized. An ELISA kit was used for platelet proteasome protein determination. Infrared imaging technology was used for visualizing the proteins on the membrane. ResultsAutophagosome participating proteins showed elevated levels in AD patient platelet cytosol. Only LC3-I autophagosome protein levels were significantly elevated. The concentrations of platelet lysate proteasome were assessed. AD patients proteasome levels were elevated but they were statistically not important as compared to controls. ConclusionsPlatelets can be used for assessing whether proteolytic system is functional. Blood-based sampling from human donors is less-invasive and analyzing platelet proteolytic system profile may help to develop pharmaceutical intervention approaches for neurodegenerative diseases in general.

neuroscience↗

bioRxiv2019

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls [≥]50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

genomics↗

bioRxiv2017

Impaired Cu-Zn superoxide dismutase (SOD1) and calcineurin (Cn) interaction in ALS: A presumed consequence for TDP-43 and zinc aggregation in Tg SOD1G93A rodent spinal cord tissue.

Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)-enriched TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A - Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previous published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn metal is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.

neuroscience↗

bioRxiv2017

Automated Structural Variant Verification In Human Genomes Using Single-Molecule Electronic DNA Mapping

The importance of structural variation in human disease and the difficulty of detecting structural variants larger than 50 base pairs has led to the development of several long-read sequencing technologies and optical mapping platforms. Frequently, multiple technologies and ad hoc methods are required to obtain a consensus regarding the location, size and nature of a structural variant, with no approach able to reliably bridge the gap of variant sizes between the domain of short-read approaches and the largest rearrangements observed with optical mapping.\n\nTo address this unmet need, we have developed a new software package, SV-Verify, which utilizes data collected with the Nabsys High Definition Mapping (HD-Mapping) system, to perform hypothesis-based verification of putative deletions. We demonstrate that whole genome maps, constructed from electronic detection of tagged DNA, hundreds of kilobases in length, can be used effectively to facilitate calling of structural variants ranging in size from 300 base pairs to hundreds of kilobase pairs. SV-Verify implements hypothesis-based verification of putative structural variants using a set of support vector machines and is capable of concurrently testing several thousand independent hypotheses. We describe support vector machine training, utilizing a well-characterized human genome, and application of the resulting classifiers to another human genome, demonstrating high sensitivity and specificity for deletions [≥]300 base pairs.

genomics↗

bioRxiv2017

High-Definition Electronic Genome Maps From Single Molecule Data

With the advent of routine short-read genome sequencing has come a growing recognition of the importance of long-range, structural information in applications ranging from sequence assembly to the detection of structural variation. Here we describe the Nabsys solid-state detector capable of detecting tags on single molecules of DNA 100s of kilobases in length as they translocate through the detector at a velocity greater than 1 megabase pair per second. Sequence-specific tags are detected with a high signal-to-noise ratio. The physical distance between tags is determined after accounting for viscous drag-induced intramolecular velocity fluctuations. The accurate measurement of the physical distance between tags on each molecule and the ability of the detector to resolve distances between tags of less than 300 base-pairs enables the construction of high-density genome maps.

genomics↗

bioRxiv2017

Platelet phosphorylated TDP-43: An exploratory study for a peripheral surrogate biomarker development for Alzheimer’s disease

Aim: Alzheimers disease (AD) and other forms of dementia create a non-curable disease population in Worlds societies. To develop a blood-based biomarker is important so that the remedial or disease-altering therapeutic intervention for AD patients would be available at the early stage. Materials & Methods: TDP-43 levels were analyzed in post-mortem brain tissue and platelets of AD and control subjects. Results: We observed an increased TDP-43 (<60%) in post-mortem AD brain regions and similar trends were also observed in patients platelets. Conclusion: Platelet TDP-43 could be used as a surrogate biomarker that is measurable, reproducible, and sensitive for screening the patients with some early clinical signs of AD and can be used to monitor disease prognosis.\n\nLay abstractIn this study, we explore to identify an Alzheimers disease-selective phospho-specific antibody that recognizes the diseased form of TDP-43 protein in patients blood-derived platelets. Our results suggest that selective anti-phosphorylated TDP-43 antibody discriminates Alzheimers disease from non-demented controls and patients with amyotrophic lateral sclerosis. Therefore, platelet screening with a selective antibody could potentially be a useful tool for diagnostic purposes for Alzheimers disease.

neuroscience↗