Biology subjects

Harris, C.

Publications and source records attributed to Harris, C..

30 records · Page 2Linked to original sources

bioRxiv2022

Applications of a Novel Reciprocating Positive Displacement Pump in the Simulation of Pulsatile Arterial Blood Flow

Pulsatile arterial blood flow plays an important role in vascular system mechanobiology, especially in the study of mechanisms of pathology. Limitations in cost, setup time, sample size, and control across current in-vitro and in-vivo methods prevent future exploration of novel treatments. Presented is the verification of a novel reciprocating positive displacement pump aimed at resolving these issues through the simulation of human ocular, human fingertip and skin surface, human cerebral, and rodent spleen organ systems. A range of pulsatile amplitudes, frequencies, and flow rates were simulated using pumps made of 3D printed parts incorporating a tubing system, check valve and proprietary software. Volumetric analysis of 430 total readings across a flow range of 0.025ml/min to 16ml/min determined that the pump had a mean absolute error and mean relative error of 0.041 ml/min and 1.385%, respectively. Linear regression of flow rate ranges yielded R2 between 0.9987 and 0.9998. Waveform analysis indicated that the pump could recreate accurate beat frequency for flow ranges above 0.06ml/min at 70BPM. The verification of accurate pump output opens avenues for the development of novel long-term in-vitro benchtop models capable of looking at fluid flow scenarios previously unfeasible, including low volume-high shear rate pulsatile flow.

bioengineering↗

bioRxiv2022

Validation of a small molecule inhibitor of PDE6D-RAS interaction with potent anti-leukemic effects

RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights on the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival.

cancer biology↗

bioRxiv2021

Testing and Validation of Reciprocating Positive Displacement Pump for Benchtop Pulsating Flow Model of Cerebrospinal Fluid Production and Other Physiologic Systems

BackgroundThe flow of physiologic fluids through organs and organs systems is an integral component of their function. The complex fluid dynamics in many organ systems are still not completely understood, and in-vivo measurements of flow rates and pressure provide a testament to the complexity of each flow system. Variability in in-vivo measurements and the lack of control over flow characteristics leave a lot to be desired for testing and evaluation of current modes of treatments as well as future innovations. In-vitro models are particularly ideal for studying neurological conditions such as hydrocephalus due to their complex pathophysiology and interactions with therapeutic measures. The following aims to present the reciprocating positive displacement pump, capable of inducing pulsating flow of a defined volume at a controlled beat rate and amplitude. While the other fluidic applications of the pump are currently under investigation, this study was focused on simulating the pulsating cerebrospinal fluid production across profiles with varying parameters. MethodsPumps were manufactured using 3D printed and injection molded parts. The pumps were powered by an Arduino-based board and proprietary software that controls the linear motion of the pumps to achieve the specified output rate at the desired pulsation rate and amplitude. A range of [Formula] to [Formula] was tested to evaluate the versatility of the pumps. The accuracy and precision of the pumps output were evaluated by obtaining a total of 150 one-minute weight measurements of degassed deionized water per output rate across 15 pump channels. In addition, nine experiments were performed to evaluate the pumps control over pulsation rate and amplitude. Resultsvolumetric analysis of a total of 1200 readings determined that the pumps achieved the target output volume rate with a mean absolute error of -0.001034283 [Formula] across the specified domain. It was also determined that the pumps can maintain pulsatile flow at a user-specified beat rate and amplitude. ConclusionThe validation of this reciprocating positive displacement pump system allows for the future validation of novel designs to components used to treat hydrocephalus and other physiologic models involving pulsatile flow. Based on the promising results of these experiments at simulating pulsatile CSF flow, a benchtop model of human CSF production and distribution could be achieved through the incorporation of a chamber system and a compliance component.

bioengineering↗

bioRxiv2021

Anxiety attenuates learning advantages conferred by statistical stability and induces loss of volatility-attuning in brain activity

Anxiety can alter an individuals perception of their external sensory environment. Previous studies suggest that anxiety can increase the magnitude of neural responses to unexpected (or surprising) stimuli. Additionally, surprise responses are reported to be boosted during stable compared to volatile environments. Few studies, however, have examined how learning is impacted by both threat and volatility. To investigate these effects, we used threat-of-shock to transiently increase subjective anxiety in healthy adults during an auditory oddball task, in which the regularity could be stable or volatile, while undergoing functional Magnetic Resonance Imaging (fMRI) scanning. We then used Bayesian Model Selection (BMS) mapping to pinpoint the brain areas where different models of anxiety displayed the highest evidence. Behaviourally, we found that threat-of-shock eliminated the accuracy advantage conferred by environmental stability over volatility in the task at hand. Neurally, we found that threat-of-shock led to both attenuation and loss of volatility-attuning of neural activity evoked by surprising sounds across most subcortical and limbic brain regions including the thalamus, basal ganglia, claustrum, insula, anterior cingulate, hippocampal gyrus and also the superior temporal gyrus. Conversely, within two small clusters in the left medial frontal gyrus and extrastriate area, threat-of-shock boosted the neural activity (relative to the safe and volatile condition) to the levels observed during the safe and stable condition, while also inducing a loss of volatility-attuning. Taken together, our findings suggest that threat eliminates the learning advantage conferred by statistical stability compared to volatility. Thus, we propose that anxiety disrupts behavioural adaptation to environmental statistics, and that multiple subcortical and limbic regions are implicated in this process.

neuroscience↗

bioRxiv2021

The Effect of A1/A2 Reactive Astrocyte Expression on Hydrocephalus Shunt Failure

Understanding the composition characteristics of the glial scar contributing to the high failure rate of neuroprosthetic devices implanted in the brain has been limited, to date, with the evaluation of cells, tissue, and biomarkers obstructing the implant. However, there remains a critical knowledge gap in gene expression profiles of the obstructing cells. This first-time study investigates the phenotypic expression specific to astrocyte scarring from those cells on hydrocephalus shunt surfaces at the time of failure, aimed at the development of therapeutic approaches to target reactive astrocytes for improved functional outcome. Recent evidence has indicated that the tissue obstructing shunts is over 80% inflammatory, with a more exaggerated astrocytic response. To understand how to mitigate the astrocyte immune response to shunts, we performed gene expression profiling of the C3 and EMP1 genes to quantify if astrocytes were classically activated and pro-inflammatory (A1) or alternatively activated and anti-inflammatory (A2), respectively. Shunt catheters were removed from patients at the time of failure and categorized by obstructed vs non-obstructed shunts. RNAscope fluorescent in situ hybridization and quantitative PCR analysis of the C3 and EMP1 expressed genes revealed that a heterogeneous mixed population of both the A1 and A2 reactive phenotype exist on the shunt surface. However, the number of A2 reactive astrocytes are significantly higher on obstructed shunts compared to A1 reactive astrocytes. ELISA data also confirmed higher levels of IL-6 for obstructed shunts involved in A2 reactive astrocyte proliferation and glial scar formation on the shunt surface. Since TNF- and IL-1{beta} propel resting astrocytes into an A2 reactive state, by simply blocking the secretion or action of these cytokines, astrocyte activation and attachment on obstructing shunts could be inhibited.

bioengineering↗

bioRxiv2021

Efficient, selectable marker free gene targeting in soybean using novel Ochrobactrum haywardense-mediated delivery

We report robust selectable marker-free gene targeting (GT) system in soybean, one of the most economically important crops. A novel efficient Ochrobactrum haywardense-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants in 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with [~] 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without a need of a selectable marker within the template. High-resolution Southern by Sequencing (SbS) analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require selectable marker within the donor DNA, and generates non-chimeric plants with heritable GT.

plant biology↗

bioRxiv2021

Intrapopulation adaptive variance supports selective breeding in a reef-building coral

The long-term persistence of coral reefs under climate change requires heritable drivers of thermal tolerance which support adaptation. The genomic basis of thermal tolerance has been evaluated across strong spatial and environmental gradients, but this variation also exists within populations due to neutral evolutionary processes. Small scale heterogeneity in coral bleaching is ubiquitous, so we used corals from a single reef to examine genomic signatures of bleaching performance, their biochemical correlates and the downstream consequences of selective breeding. In the absence of directional selection due to environmental differences, adult corals from a single population exhibit strong genomic patterns related to natural bleaching tolerance and symbiosis state, including functional differentiation in signaling pathways, protein and amino acid modification and metabolism. Conversely, growth, development and innate immune responses did not distinguish bleaching tolerance in adult corals. The genomic signatures of these gene ontologies influence biochemical patterns in healthy corals, primarily via cell-signaling pathway impacts on peptides and amino acids. Thermal tolerance in this population is highly heritable, with significantly higher survivorship under temperature stress in larvae and juveniles reared from thermally tolerant parents than those from sensitive parents. Using a select and re-sequence approach, certain gene ontologies were reproducibly impacted, while antioxidant activity and cell signaling ontologies were disproportionately selected in thermally tolerant corals, demonstrating the genomic drivers of successful selective breeding. These data show that intrapopulation variance in the absence of historical selection supports the adaptive capacity of coral reefs under climate change.

ecology↗

bioRxiv2020

Unique features of stimulus-based probabilistic reversal learning

Reversal learning paradigms are widely-used assays of behavioral flexibility with their probabilistic versions being more amenable to studying integration of reward outcomes over time. Prior research suggests differences between initial and reversal learning, including higher learning rates, a greater need for inhibitory control, and more perseveration after reversals. However, it is not well-understood what aspects of stimulus-based reversal learning are unique to reversals, and whether and how observed differences depend on reward probability. Here, we used a visual probabilistic discrimination and reversal learning paradigm where male and female rats selected between a pair of stimuli associated with different reward probabilities. We compared accuracy, rewards collected, omissions, latencies, win-stay/lose-shift strategies, and indices of perseveration across two different reward probability schedules. We found that discrimination and reversal learning are behaviorally more unique than similar: latencies to select the better option, win-stay strategies, and perseveration were of a different pattern in reversal learning compared to discrimination learning. Additionally, fit of choice behavior using reinforcement learning models revealed a lower sensitivity to the difference in subjective reward values (greater exploration) and higher learning rates for the reversal phase. Interestingly, a consistent reward probability group difference emerged with a richer environment associated with longer reward collection latencies than a leaner environment. We also replicated previous reports on sex differences in reversal learning. Future studies should systematically compare the neural correlates of fine-grained behavioral measures to reveal possible dissociations in how the circuitry is recruited in each phase.

animal behavior and cognition↗

bioRxiv2020

A genome-wide association study finds genetic variants associated with neck or shoulder pain in UK Biobank

BackgroundCommon types of musculoskeletal conditions include pain in the neck and shoulder areas. This study seeks to identify the genetic variants associated with neck or shoulder pain based on a genome-wide association approach using 203,309 subjects from the UK Biobank cohort and look for replication evidence from the Generation Scotland: Scottish Family Health Study (GS:SFHS) and TwinsUK. MethodsCases in the UK Biobank were determined by a question which asked the participants if they had experienced pain in the neck or shoulder in the previous month influencing daily activities. Controls were the UK Biobank participants who reported no pain anywhere in the last month. A genome-wide association study was performed adjusting for age, sex, BMI and 9 population principal components. Significant and independent genetic variants were then sent to GS:SFHS and TwinsUK for replication. ResultsWe identified 3 genetic loci that were associated with neck or shoulder pain in the UK Biobank samples. The most significant locus was in an intergenic region in chromosome 17, rs12453010, having P = 1.66 x 10-11. The second most significant locus was located in the FOXP2 gene in chromosome 7 with P = 2.38 x 10-10 for rs34291892. The third locus was located in the LINC01572 gene in chromosome 16 with P = 4.50 x 10-8 for rs62053992. In the replication stage, among 4 significant and independent genetic variants, rs2049604 in the FOXP2 gene and rs62053992 in the LINC01572 gene were weakly replicated in GS:SFHS (P = 0.0240 and P = 0.0202, respectively). None of the single nucleotide polymorphisms (SNPs) were replicated in the TwinsUK cohort (P > 0.05). ConclusionsWe have identified 3 loci associated with neck or shoulder pain in the UK Biobank cohort, two of which were weakly supported in a replication cohort. Further evidence is needed to confirm their roles in neck or shoulder pain. SignificanceThis is the first genome-wide association study on neck or shoulder pain. We have identified 3 genetic loci (an intergenic region in chromosome 17, the FOXP2 gene in chromosome 7, and the LINC01572 gene in chromosome 16) that are associated with neck or shoulder pain using the UK Biobank cohort, among which the FOXP2 gene and the LINC01572 gene were weakly replicated by the Generation Scotland: Scottish Family Health Study (P < 0.05). The SNP heritability was 0.11, indicating neck or shoulder pain is a heritable trait. The tissue expression analysis suggested that neck or shoulder pain was related to multiple brain tissues, indicating the involvement of neuron function. The results will inform further research in the characterisation of the mechanisms of neck or shoulder pain.

genetics↗

bioRxiv2018

Integrated Cell Expression Toolkit for low input transcript profiling

Several methods exist for constructing sequencing libraries from single-cell amounts of RNA. However, different library preparation methods are required to enable full length or strand specific 3 end profiling. The Integrated Cell Expression Toolkit (ICE-T) maximizes functionality by integrating these approaches with novel strategies that provide a range of profiling options.\n\nTranscriptome profiling at high cellular resolution has many applications in biology but is hampered by a need to boost the representation of RNA to guard against losses during processing and enable efficient use of high throughput technologies. RNA amplification techniques developed for profiling limiting amounts of RNA by microarray have recently been adapted to NGS1-5. These various, but distinct, techniques enable both full length profiling3 and strand specific 3 end profiling2, 4 ...

genomics↗

bioRxiv2018

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections

AO_SCPLOWBSTRACTC_SCPLOWAntigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of [~]2,000 alleles. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads - this was observed as early as day 3, the earliest that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process.

genomics↗

bioRxiv2017

Cadmium exposure inhibits branching morphogenesis and causes alterations consistent with HIF-1α inhibition in human primary breast organoids

BackgroundDevelopmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cell development have not been measured.\n\nObjectiveThe objective of this study was to quantify the effects of cadmium exposure on normal breast stem cell proliferation and differentiation.\n\nMethodsWe tested the effects of two physiologically relevant doses of cadmium: 250M and 2.5M on reduction mammoplasty patient-derived breast cells using the mammosphere assay, organoid formation in 3D hydrogels, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a HIF-1 transcription factor activity reporter line and pharmaceutical inhibition of HIF-1 in mammosphere and organoid formation assays.\n\nResults2.5M cadmium reduced primary and secondary mammosphere formation and branching structure organoid formation rates by 33%, 40%, and 83%, respectively. Despite no changes in mammosphere formation, 0.25M cadmium treatment inhibited branching organoid formation in hydrogels by 68%. RNA-seq revealed that cadmium treatment downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium treatment also downregulated HIF-1 target genes including LOXL2, ZEB1, and VIM. Cadmium treatment significantly inhibited HIF-1 activity in a luciferase assay, and the HIF-1 inhibitor acriflavine ablated mammosphere and organoid formation.\n\nDiscussionThese findings show that cadmium, at doses relevant to human exposure, inhibited human mammary gland development, potentially through disruption of HIF-1 activity. These findings do not support cadmium being a breast cancer initiator via induction of stem cell proliferation, but instead implicate cadmium as an inhibitor of mammary gland morphogenesis.

pharmacology and toxicology↗