Biology subjects

Morris, P.

Publications and source records attributed to Morris, P..

7 recordsLinked to original sources

bioRxiv2024

CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109 /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 "HIV-1 RNA copy number quantification" (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene of in vitro transcribed RNA molecules at low ({approx} 103 /L) and high concentration ({approx} 109 /L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808 /L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

molecular biology↗

bioRxiv2022

Breathlessness in a virtual world: An experimental paradigm testing how discrepancy between VR visual gradients and pedal resistance during stationary cycling affects breathlessness perception.

IntroductionThe sensation of breathlessness is often attributed to perturbations in cardio-pulmonary physiology, leading to changes in afferent signals. New evidence suggests that these signals are interpreted in the light of prior "expectations". A misalignment between afferent signals and expectations may underly unexplained breathlessness. Using a novel immersive virtual reality (VR) exercise paradigm, we investigated whether manipulating an individuals expectation of effort (determined by a virtual hill gradient) may alter their perception of breathlessness, independent from actual effort (the physical effort of cycling). MethodsNineteen healthy volunteers completed a single experimental session where they exercised on a cycle ergometer while wearing a VR headset. We created an immersive virtual cycle ride where participants climbed up 100 m hills with virtual gradients of 4%, 6%, 8%, 10% and 12%. Each virtual hill gradient was completed twice: once with a 4% cycling ergometer resistance and once with a 6% resistance, allowing us to dissociate expected effort (virtual hill gradient) from actual effort (physical effort of pedalling). At the end of each hill, participants reported their perceived breathlessness. Linear mixed effects models were used to examine the independent contribution of actual effort and expected effort to ratings of breathlessness (0-10 scale). ResultsExpectation of effort (effect estimate {+/-} std. error, 0.63 {+/-} 0.11, p<0.001) and actual effort (0.81 {+/-} 0.21, p<0.001) independently explained subjective ratings of breathlessness, with comparable contributions of 19% and 18%, respectively. Additionally, we found that effort expectation accounted for 6% of participants physical effort of pedalling and was a significant, independent predictor (0.09 {+/-} 0.03; p=0.001). ConclusionsAn individuals expectation of effort is equally important for forming perceptions of breathlessness as the actual effort required to cycle. A new VR paradigm enables this to be experimentally studied and could be used to re-align breathlessness and enhance training programmes.

neuroscience↗

bioRxiv2020

Estimating the breeding population of an elephant seal colony from a single census

Our aim was to develop a method for estimating the number of animals using a single site in an asynchronous species, meaning that not all animals are present at once so that no one count captures the entire population. This is a common problem in seasonal breeders, and in northern elephant seals, we have a model for quantifying asynchrony at the Ano Nuevo colony. Here we test the model at several additional colonies having many years of observations and demonstrate how it can account for animals not present on any one day. This leads to correction factors that yield total population from any single count throughout a season. At seven colonies in California for which we had many years of counts of northern elephant seals, we found that female arrival date varied < 2 days between years within sites and by < 5 days between sites. As a result, the correction factor for any one day was consistent, and at each colony, multiplying a female count between 26 and 30 Jan by 1.15 yielded an estimate of total population size that minimized error. This provides a method for estimating the female population size at colonies not yet studied. Our method can produce population estimates with minimal expenditure of time and resources and will be applicable to many seasonal species with asynchronous breeding phenology, particularly colonial birds and other pinnipeds. In elephant seals, it will facilitate monitoring the population over its entire range.

ecology↗

bioRxiv2020

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration

The problem in meat adulteration and food fraud emphasised the requirement of developing accurate analytical approaches for the quantitative detection in helping the control of meat adulteration. In this study, the droplet digital Polymerase Chain Reaction (ddPCR) assays to quantify the ratios of pork DNA to the total amount of meat DNA were developed by challenging against DNA extracted from a range of gravimetrically prepared matrices of pork in beef. A single copy nuclear DNA gene, {beta}-actin, was employed as a target gene, accompanied with myostatin gene as a cross species target for mammal and poultry meat background in order to quantifying approach. All the developed assays, singleplex, duplex and triplex did not show significant difference in quantification of pork content in beef background and demonstrated a good and comparable performance to the mass fractions. The singleplex assay provided more biases than the other two assays when performing with a low concentration of target species. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and running time. All proposed methods allowed us to quantify pork addition in beef with a limit of quantification (LOQ) estimated at 0.1% (w/w) and a limit of detection (LOD) down to 0.01% (w/w). The developed triplex assay was also tested with commercial processed foods and showed the ability to determine not only the presence of particular pork or beef but also the quantitative purpose directly without standard curves. Hence, the developed ddPCR assays demonstrated a good trueness and precision of the methods in quantifying pork or beef content for meat adulteration. It is expected that these developed approaches can be applied to help regulators to confidently enforce food labelling obligations.

molecular biology↗

bioRxiv2020

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of commonly used polar organic co-solvents: acetonitrile (MeCN), n- butanol (nBuOH), ethyl acetate (EToAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Aggregation was not found to be the driving mechanism of enzyme inactivation, but was in some cases an additional impact of solvent-induced local or global unfolding. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) MeCN, or 6% (v/v) nBuOH over 3 hours. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.

biophysics↗

bioRxiv2018

Chemogenetic ligands for translational neurotheranostics

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

neuroscience↗

bioRxiv2017

Modelling Theta-Band Connectivity Between Occipital And Frontal Lobes: A Methodological MEG Study

Measuring functional connectivity between cortical regions of the human brain has become an important area of research. Modern theory suggests that brain networks exhibit non-stationarity, constantly forming and reforming depending on task demands. A robust means of determining effective connectivity in the short-lived neural responses that occur in event related paradigms would allow the investigation of event related cortico-cortical dynamics. We present such a mathematical model of wave propagation, motivated by current neuroscience literature, and demonstrate the utility of the method in a clinical sample of schizophrenia patients. MEG data were acquired in 10 patients with schizophrenia and 12 healthy controls during a relevance modulation task. Data were filtered into the theta band (4-8Hz) and source localised using a beamformer. The model was implemented using Fourier analysis methods which uncovered an event related travelling wave moving from the visual to frontal cortices. The model was validated using Monte Carlo phase randomisation and compared with another plausible model of wave propagation in the cortex. Results from the clinical sample revealed that wave speed was modulated by task condition and patients were found to have less difference between conditions (ANOVA revealing a significant interaction between group and condition, p<0.05). In conclusion, our method provides a simple and robust means to investigate event related cortico-cortical brain dynamics in individuals and groups in the task positive state.

neuroscience↗