Search

Abstract Cellular senescence is considered an important tumour suppression mechanism in response to damage and oncogenic stress in early lesions. However, when senescent cells are not immune-cleared and persist in the tumour microenvironment, they can drive a variety of tumour-promoting activities, including cancer initiation, progression, and metastasis. Additionally, there is compelling evidence demonstrating a direct connection between chemo(radio)therapy-induced senescence and the development of drug resistance and cancer recurrence. Therefore, detection of senescent cells in tissues holds great promise for predicting cancer occurrence earlier, assessing tumour progression, aiding patient stratification and prognosis, and informing about the efficacy of potential senotherapies. However, effective detection of senescent cells is limited by lack of biomarkers and readout strategies suitable for in vivo clinical imaging. To this end, a nanoprobe composed of biocompatible polydopamine (PDA) nanoparticle doped with FDA-approved indocyanine green (ICG) dye, namely PDA-ICG, was designed as a contrast agent for senescence detection using photoacoustic imaging (PAI). In an in vitro model of chemotherapy-induced senescence, PDA-ICG nanoprobe showed an elevated uptake in senescent cells relative to cancer cells. In addition to its improved photostability, 2.5-fold enhancement in photoacoustic signal relative to ICG was observed. Collectively, the results indicate that the PDA-ICG nanoprobe has the potential to be used as a contrast agent for senescence detection of chemotherapy-induced senescence using PAI.

Abstract The circadian clock is a cell-autonomous process that regulates daily internal rhythms by interacting with environmental signals. Reports across species show that infection can alter the expression of circadian genes; however, in teleosts, these effects are influenced by light exposure. Currently, no reports analyze the direct effects of bacterial exposure on the zebrafish clock. Using zebrafish Z3 cells, we demonstrate that exposure to heat-killed Streptococcus pneumoniae (HK-Spn) augments the expression of core repressive factors in a light- and time-dependent manner. In constant darkness, HK-Spn highly upregulated cry1a, per3, and per1b expression. In the presence of light, HK-Spn exposure rapidly and strongly upregulated per2 and cry1a, and this was proportionally increased with light intensity. The combinatorial effect of light and HK-Spn on per2 and cry1a was not duplicated with H2O2, a known byproduct of light exposure. However, the ROS inhibitor N-acetyl cysteine was sufficient to block HK-Spn augmentation of per2, cry1a, and per3. These findings demonstrate that exposure to an inactive bacteria influences the expression of zebrafish clock genes under different light conditions.

Abstract Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the ‘gold standard’ small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.

Abstract Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the ‘fittest hit’ to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Abstract Infrastructure is often a limiting factor in microplastics research impacting the production of scientific outputs and monitoring data. International projects are therefore required to promote collaboration and development of national and regional scientific hubs. The Commonwealth Litter Programme and the Ocean Country Partnership Programme were developed to support Global South countries to take actions on plastics entering the oceans. An international laboratory network was developed to provide the infrastructure and in country capacity to conduct the collection and processing of microplastics in environmental samples. The laboratory network was also extended to include a network developed by the University of East Anglia, UK. All the laboratories were provided with similar equipment for the collection, processing and analysis of microplastics in environmental samples. Harmonised protocols and training were also provided in country during laboratory setup to ensure comparability of quality-controlled outputs between laboratories. Such large networks are needed to produce comparable baseline and monitoring assessments.

Abstract It is hypothesized that disparate brain regions interact via synchronous activity to control behavior. The nature of these interconnected ensembles remains an area of active investigation, and particularly the role of high frequency synchronous activity in simplistic behavior is not well known. Using intracranial electroencephalography, we explored the spectral dynamics and network connectivity of sensorimotor cortical activity during a simple motor task in seven epilepsy patients. Confirming prior work, we see a “spectral tilt” (increased high-frequency (HF, 70–100 Hz) and decreased low-frequency (LF, 3–33 Hz) broadband oscillatory activity) in motor regions during movement compared to rest, as well as an increase in LF synchrony between these regions using time-resolved phase-locking. We then explored this phenomenon in high frequency and found a robust but opposite effect, where time-resolved HF broadband phase-locking significantly decreased during movement. This “connectivity tilt” (increased LF synchrony and decreased HF synchrony) displayed a graded anatomical dependency, with the most robust pattern occurring in primary sensorimotor cortical interactions and less robust pattern occurring in associative cortical interactions. Connectivity in theta (3–7 Hz) and high beta (23–27 Hz) range had the most prominent low frequency contribution during movement, with theta synchrony building gradually while high beta having the most prominent effect immediately following the cue. There was a relatively sharp, opposite transition point in both the spectral and connectivity tilt at approximately 35 Hz. These findings support the hypothesis that task-relevant high-frequency spectral activity is stochastic and that the decrease in high-frequency synchrony may facilitate enhanced low frequency phase coupling and interregional communication. Thus, the “connectivity tilt” may characterize behaviorally meaningful cortical interactions.

Abstract The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

Abstract X-ray Absorption Spectroscopy (XAS) is a widely used X-ray diagnostic method for studying electronic and structural properties of matter. At first glance, the relatively narrow bandwidth and the highly fluctuating spectral structure of X-ray Free Electron Lasers (XFEL) sources seem to require accumulation over many shots to achieve high data quality. To date the best approach to implementing XAS at XFEL facilities has been using monochromators to scan the photon energy across the desired spectral range. While this is possible for easily reproducible samples such as liquids, it is incompatible with many important systems. Here, we demonstrate collection of single-shot XAS spectra over 10s of eV using an XFEL source, with error bars of only a few percent. We additionally show how to extend this technique over wider spectral ranges towards Extended X-ray Absorption Fine Structure measurements, by concatenating a few tens of single-shot measurements. Our results pave the way for future XAS studies at XFELs, in particular those in the femtosecond regime. This advance is envisioned to be especially important for many transient processes that can only be initiated at lower repetition rates, for difficult to reproduce excitation conditions, or for rare samples, such as those encountered in high-energy density physics.

Abstract Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory ‘switch helix’ that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.

Abstract Aquaculture of New Zealand’s endemic green-lipped mussel (Perna canaliculus) is an industry valued at NZ$ 336 M per annum and is ~ 80% reliant on the natural supply of wild mussel spat harvested at a single location—Te Oneroa-a-Tōhē—Ninety Mile Beach (NMB)—in northern New Zealand. Despite the economic and ecological importance of this spat supply, little is known about the population connectivity of green-lipped mussels in this region or the location of the source population(s). In this study, we used a biophysical model to simulate the two-stage dispersal process of P. canaliculus. A combination of backward and forward tracking experiments was used to identify primary settlement areas and putative source populations. The model was then used to estimate the local connectivity, revealing two geographic regions of connectivity in northern New Zealand, with limited larval exchange between them. Although secondary dispersal can double the dispersal distance, our simulations show that spat collected at NMB originate from neighbouring mussel beds, with large contributions from beds located at Ahipara (southern end of NMB). These results provide information that may be used to help monitor and protect these important source populations to ensure the ongoing success of the New Zealand mussel aquaculture industry.

Abstract The microstructure of minerals and rocks can significantly alter reaction rates. This study focuses on identifying transport paths in low porosity rocks based on the hypothesis that grain boundary widening accelerates reactions in which one mineral is replaced by another (replacement reaction). We conducted a time series of replacement experiments of three limestones (CaCO3) of different microstructures and solid impurity contents using FeCl2. Reacted solids were analyzed using chemical imaging, small angle X-ray and neutron scattering and Raman spectroscopy. In high porosity limestones replacement is reaction controlled and complete replacement was observed within 2 days. In low porosity limestones that contain 1–2% dolomite impurities and are dominated by grain boundaries, a reaction rim was observed whose width did not change with reaction time. Siderite (FeCO3) nucleation was observed in all parts of the rock cores indicating the percolation of the solution throughout the complete core. Dolomite impurities were identified to act as nucleation sites leading to growth of crystals that exert force on the CaCO3 grains. Widening of grain boundaries beyond what is expected based on dissolution and thermal grain expansion was observed in the low porosity marble containing dolomite impurities. This leads to a self-perpetuating cycle of grain boundary widening and reaction acceleration instead of reaction front propagation.

Abstract The emergence of high resolution population genetic techniques, such as genotyping-by-sequencing (GBS), in combination with recent advances in particle modelling of larval dispersal in marine organisms, can deliver powerful new insights to support fisheries conservation and management. In this study, we used this combination to investigate the population connectivity of a commercial deep sea lobster species, the New Zealand scampi, Metanephrops challengeri, which ranges across a vast area of seafloor around New Zealand. This species has limited dispersal capabilities, including larvae with weak swimming abilities and short pelagic duration, while the reptant juvenile/adult stages of the lifecycle are obligate burrow dwellers with limited home ranges. Ninety-one individuals, collected from five scampi fishery management areas around New Zealand, were genotyped using GBS. Using 983 haplotypic genomic loci, three genetically distinct groups were identified: eastern, southern and western. These groups showed significant genetic differentiation with clear source-sink dynamics. The direction of gene flow inferred from the genomic data largely reflected the hydrodynamic particle modelling of ocean current flow around New Zealand. The modelled dispersal during pelagic larval phase highlights the strong connectivity among eastern sampling locations and explains the low genetic differentiation detected among these sampled areas. Our results highlight the value of using a transdisciplinary approach in the inference of connectivity among populations for informing conservation and fishery management.

Abstract Human milk oligosaccharides (HMOs) form the third most abundant component of human milk and are known to convey several benefits to the neonate, including protection from viral and bacterial pathogens, training of the immune system, and influencing the gut microbiome. As HMO production during lactation is driven by enzymes that are common to other glycosylation processes, we adapted a model of mucin-type GalNAc-linked glycosylation enzymes to act on free lactose. We identified a subset of 11 enzyme activities that can account for 206 of 226 distinct HMOs isolated from human milk and constructed a biosynthetic reaction network that identifies 5 new core HMO structures. A comparison of monosaccharide compositions demonstrated that the model was able to discriminate between two possible groups of intermediates between major subnetworks, and to assign possible structures to several previously uncharacterised HMOs. The effect of enzyme knockouts is presented, identifying β-1,4-galactosyltransferase and β-1,3-N-acetylglucosaminyltransferase as key enzyme activities involved in the generation of the observed HMO glycosylation patterns. The model also provides a synthesis chassis for the most common HMOs found in lactating mothers.

Abstract Following the first detection in the United Kingdom of Usutu virus (USUV) in wild birds in 2020, we undertook a multidisciplinary investigation that combined screening host and vector populations with interrogation of national citizen science monitoring datasets to assess the potential for population impacts on avian hosts. Pathological findings from six USUV-positive wild passerines were non-specific, highlighting the need for molecular and immunohistochemical examinations to confirm infection. Mosquito surveillance at the index site identified USUV RNA in Culex pipiens s.l. following the outbreak. Although the Eurasian blackbird (Turdus merula) is most frequently impacted by USUV in Europe, national syndromic surveillance failed to detect any increase in occurrence of clinical signs consistent with USUV infection in this species. Furthermore, there was no increase in recoveries of dead blackbirds marked by the national ringing scheme. However, there was regional clustering of blackbird disease incident reports centred near the index site in 2020 and a contemporaneous marked reduction in the frequency with which blackbirds were recorded in gardens in this area, consistent with a hypothesis of disease-mediated population decline. Combining results from multidisciplinary schemes, as we have done, in real-time offers a model for the detection and impact assessment of future disease emergence events.

Abstract Orthopedic and neurological impairments (e.g., muscle contractures, spasticity) are often treated in children and young adults with cerebral palsy (CP). Due to challenges arising from combinatorics, research funding priorities, and medical practicalities, and despite extensive study, the evidence base is weak. Our goal was to estimate the short-term effectiveness of 13 common orthopedic and neurological treatments at four different levels of outcome in children and young adults diagnosed with CP. The outcome levels considered were body structures, specific gait kinematic deviations, overall gait kinematic deviations, and functional mobility. We used three well-establish causal inference approaches (direct matching, virtual twins, and Bayesian causal forests) and a large clinical gait analysis database to estimate the average treatment effect on the treated (ATT). We then examined the effectiveness across treatments, methods, and outcome levels. The dataset consisted of 2851 limbs from 933 individuals (some individuals underwent multiple treatment episodes). Current treatments have medium effects on body structures, but modest to minimal effects on gait and functional mobility. The median ATT of 13 common treatments in children and young adults with CP, measured as Cohen’s D, bordered on medium at the body structures level (median [IQR] = 0.42 [0.05, 0.60]) and became smaller as we moved along the causal chain through specific kinematic deviations (0.21 [0.01, 0.33]), overall kinematic deviations (0.09 [0.03, 0.19]), and functional mobility (-0.01 [-0.06, 0.13]). Further work is needed to understand the source of heterogeneous treatment effects, which are large in this patient population. Replication or refutation of these findings by other centers will be valuable to establish the generalizability of these results and for benchmarking of best practices.

Abstract The molecular determinants of Plasmodium falciparum artemisinin resistance are the single nucleotide polymorphisms in the parasite’s kelch propeller domain, pfk13. Validated and candidate markers are under surveillance in malaria endemic countries using artemisinin-based combination therapy. However, pfk13 mutations which may confer parasite artemisinin resistance in Africa remains elusive. It has therefore become imperative to report all observed pfk13 gene polymorphisms in malaria therapeutic efficacy studies for functional characterization. We herein report all novel pfk13 mutations observed only in the Ghanaian parasite population. In all, 977 archived samples from children aged 12 years and below with uncomplicated malaria from 2007 to 2017 were used. PCR/Sanger sequencing analysis revealed 78% (763/977) of the samples analyzed were wild type (WT) for pfk13 gene. Of the 214 (22%) mutants, 78 were novel mutations observed only in Ghana. The novel SNPs include R404G, P413H, N458D/H/I, C473W/S, R529I, M579T/Y, C580R/V, D584L, N585H/I, Q661G/L. Some of the mutations were sites and ecological zones specific. There was low nucleotide diversity and purifying selection at the pfk13 locus in Ghanaian parasite population. With increasing drug pressure and its consequent parasite resistance, documenting these mutations as baseline data is crucial for future molecular surveillance of P. falciparum resistance to artemisinin in Ghana.

Abstract Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose–response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose–response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

Abstract In the heart, electrophysiological dysregulation arises from defects at many biological levels (from point mutations in ion channel proteins to gross structural abnormalities). These defects disrupt the normal pattern of electrical activation, producing ectopic activity and reentrant arrhythmia. To interrogate mechanisms that link these primary biological defects to macroscopic electrophysiologic dysregulation most prior computational studies have utilized either (i) detailed models of myocyte ion channel dynamics at limited spatial scales, or (ii) homogenized models of action potential conduction that reproduce arrhythmic activity at tissue and organ levels. Here we apply our recent model (EMI), which integrates electrical activation and propagation across these scales, to study human atrial arrhythmias originating in the pulmonary vein (PV) sleeves. These small structures initiate most supraventricular arrhythmias and include pronounced myocyte-to-myocyte heterogeneities in ion channel expression and intercellular coupling. To test EMI’s cell-based architecture in this physiological context we asked whether ion channel mutations known to underlie atrial fibrillation are capable of initiating arrhythmogenic behavior via increased excitability or reentry in a schematic PV sleeve geometry. Our results illustrate that EMI’s improved spatial resolution can directly interrogate how electrophysiological changes at the individual myocyte level manifest in tissue and as arrhythmia in the PV sleeve.

Abstract Studies of the impact of host genetics on gut microbiome composition have mainly focused on the impact of individual single nucleotide polymorphisms (SNPs) on gut microbiome composition, without considering their collective impact or the specific functions of the microbiome. To assess the aggregate role of human genetics on the gut microbiome composition and function, we apply sparse canonical correlation analysis (sCCA), a flexible, multivariate data integration method. A critical attribute of metagenome data is its sparsity, and here we propose application of a Tweedie distribution to accommodate this. We use the TwinsUK cohort to analyze the gut microbiomes and human variants of 250 individuals. Sparse CCA, or sCCA, identified SNPs in microbiome-associated metabolic traits (BMI, blood pressure) and microbiome-associated disorders (type 2 diabetes, some neurological disorders) and certain cancers. Both common and rare microbial functions such as secretion system proteins or antibiotic resistance were found to be associated with host genetics. sCCA applied to microbial species abundances found known associations such as Bifidobacteria species, as well as novel associations. Despite our small sample size, our method can identify not only previously known associations, but novel ones as well. Overall, we present a new and flexible framework for examining host-microbiome genetic interactions, and we provide a new dimension to the current debate around the role of human genetics on the gut microbiome.

Abstract Heart failure (HF) admission is a dominant contributor to morbidity and healthcare costs in dilated cardiomyopathy (DCM). Mid-wall striae (MWS) fibrosis by late gadolinium enhancement (LGE) imaging has been associated with elevated arrhythmia risk. However, its capacity to predict HF-specific outcomes is poorly defined. We investigated its role to predict HF admission and relevant secondary outcomes in a large cohort of DCM patients. 719 patients referred for LGE MRI assessment of DCM were enrolled and followed for clinical events. Standardized image analyses and interpretations were conducted inclusive of coding the presence and patterns of fibrosis observed by LGE imaging. The primary clinical outcome was hospital admission for decompensated HF. Secondary heart failure and arrhythmic composite endpoints were also studied. Median age was 57 (IQR 47–65) years and median LVEF 40% (IQR 29–47%). Any fibrosis was observed in 228 patients (32%) with MWS fibrosis pattern present in 178 (25%). At a median follow up of 1044 days, 104 (15%) patients experienced the primary outcome, and 127 (18%) the secondary outcome. MWS was associated with a 2.14-fold risk of the primary outcome, 2.15-fold risk of the secondary HF outcome, and 2.23-fold risk of the secondary arrhythmic outcome. Multivariable analysis adjusting for all relevant covariates, inclusive of LVEF, showed patients with MWS fibrosis to experience a 1.65-fold increased risk (95% CI 1.11–2.47) of HF admission and 1-year event rate of 12% versus 7% without this phenotypic marker. Similar findings were observed for the secondary outcomes. Patients with LVEF > 35% plus MWS fibrosis experienced similar event rates to those with LVEF ≤ 35%. MWS fibrosis is a powerful and independent predictor of clinical outcomes in patients with DCM, identifying patients with LVEF > 35% who experience similar event rates to those with LVEF below this conventionally employed high-risk phenotype threshold.

Abstract The gut microbiome impacts host health and fitness, in part through the diversification of gut metabolic function and pathogen protection. Elevations in glucocorticoids (GCs) appear to reduce gut microbiome diversity in experimental studies, suggesting that a loss of microbial diversity may be a negative consequence of increased GCs. However, given that ecological factors like food availability and population density may independently influence both GCs and microbial diversity, understanding how these factors structure the GC-microbiome relationship is crucial to interpreting its significance in wild populations. Here, we used an ecological framework to investigate the relationship between GCs and gut microbiome diversity in wild North American red squirrels (Tamiasciurus hudsonicus). As expected, higher GCs predicted lower gut microbiome diversity and an increase in metabolic taxa. Surprisingly, but in line with prior empirical studies on wild animals, gastrointestinal pathogens decreased as GCs increased. Both dietary heterogeneity and an upcoming food pulse exhibited direct effects on gut microbiome diversity, whereas conspecific density and reproductive activity impacted diversity indirectly via changes in host GCs. Our results provide evidence of a gut–brain axis in wild red squirrels and highlight the importance of situating the GC-gut microbiome relationship within an ecological framework.

Abstract The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (

Abstract The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Abstract The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; one in five of these cases could lead to new or refined diagnoses.

Abstract Transmission of SARS-CoV-2 by aerosols has played a significant role in the rapid spread of COVID-19 across the globe. Indoor environments with inadequate ventilation pose a serious infection risk. Whilst vaccines suppress transmission, they are not 100% effective and the risk from variants and new viruses always remains. Consequently, many efforts have focused on ways to disinfect air. One such method involves use of minimally hazardous 222 nm far-UVC light. Whilst a small number of controlled experimental studies have been conducted, determining the efficacy of this approach is difficult because chamber or room geometry, and the air flow within them, influences both far-UVC illumination and aerosol dwell times. Fortunately, computational multiphysics modelling allows the inadequacy of dose-averaged assessment of viral inactivation to be overcome in these complex situations. This article presents the first validation of the WYVERN radiation-CFD code for far-UVC air-disinfection against survival fraction measurements, and the first measurement-informed modelling approach to estimating far-UVC susceptibility of viruses in air. As well as demonstrating the reliability of the code, at circa 70% higher, our findings indicate that aerosolized human coronaviruses are significantly more susceptible to far-UVC than previously thought.

Abstract Pleural mesothelioma is an aggressive malignancy with limited effective therapies. In order to identify therapeutic targets, we integrated SNP genotyping, sequencing and transcriptomics from tumours and low-passage patient-derived cells. Previously unrecognised deletions of SUFU locus (10q24.32), observed in 21% of 118 tumours, resulted in disordered expression of transcripts from Hedgehog pathways and the T-cell synapse including VISTA. Co-deletion of Interferon Type I genes and CDKN2A was present in half of tumours and was a predictor of poor survival. We also found previously unrecognised deletions in RB1 in 26% of cases and show sub-micromolar responses to downstream PLK1, CHEK1 and Aurora Kinase inhibitors in primary mesothelioma cells. Defects in Hippo pathways that included RASSF7 amplification and NF2 or LATS1/2 mutations were present in 50% of tumours and were accompanied by micromolar responses to the YAP1 inhibitor Verteporfin. Our results suggest new therapeutic avenues in mesothelioma and indicate targets and biomarkers for immunotherapy.

Abstract The austral spring climate of 2020 was characterised by the occurrence of La Niña, which is the most predictable climate driver of Australian springtime rainfall. Consistent with this La Niña, the Bureau of Meteorology’s dynamical sub-seasonal to seasonal forecast system, ACCESS-S1, made highly confident predictions of wetter-than-normal conditions over central and eastern Australia for spring when initialised in July 2020 and thereafter. However, many areas of Australia received near average to severely below average rainfall, particularly during November. Possible causes of the deviation of rainfall from its historical response to La Niña and causes of the forecast error are explored with observational and reanalysis data for the period 1979–2020 and real-time forecasts of ACCESS-S1 initialised in July to November 2020. Several compounding factors were identified as key contributors to the drier-than-anticipated spring conditions. Although the ocean surface to the north of Australia was warmer than normal, which would have acted to promote rainfall over northern Australia, it was not as warm as expected from its historical relationship with La Niña and its long-term warming trend. Moreover, a negative phase of the Indian Ocean Dipole mode, which typically acts to increase spring rainfall in southern Australia, decayed earlier than normal in October. Finally, the Madden–Julian Oscillation activity over the equatorial Indian Ocean acted to suppress rainfall across northern and eastern Australia during November. While ACCESS-S1 accurately predicted the strength of La Niña over the Niño3.4 region, it over-predicted the ocean warming to the north of Australia and under-predicted the strength of the November MJO event, leading to an over-prediction of the Australian spring rainfall and especially the November-mean rainfall.

Abstract Increasing the efficiency of current forage breeding programs through adoption of new technologies, such as genomic selection (GS) and phenomics (Ph), is challenging without proof of concept demonstrating cost effective genetic gain (∆G). This paper uses decision support software DeltaGen (tactical tool) and QU-GENE (strategic tool), to model and assess relative efficiency of five breeding methods. The effect on ∆G and cost ($) of integrating GS and Ph into an among half-sib (HS) family phenotypic selection breeding strategy was investigated. Deterministic and stochastic modelling were conducted using mock data sets of 200 and 1000 perennial ryegrass HS families using year-by-season-by-location dry matter (DM) yield data and in silico generated data, respectively. Results demonstrated short (deterministic)- and long-term (stochastic) impacts of breeding strategy and integration of key technologies, GS and Ph, on ∆G. These technologies offer substantial improvements in the rate of ∆G, and in some cases improved cost-efficiency. Applying 1% within HS family GS, predicted a 6.35 and 8.10% ∆G per cycle for DM yield from the 200 HS and 1000 HS, respectively. The application of GS in both among and within HS selection provided a significant boost to total annual ∆G, even at low GS accuracy rA of 0.12. Despite some reduction in ∆G, using Ph to assess seasonal DM yield clearly demonstrated its impact by reducing cost per percentage ∆G relative to standard DM cuts. Open-source software tools, DeltaGen and QuLinePlus/QU-GENE, offer ways to model the impact of breeding methodology and technology integration under a range of breeding scenarios.

Abstract Lung cancer is the most frequent cause of cancer death worldwide. It affects more men than women, and men generally have worse survival outcomes. We compared gene co-expression networks in affected and unaffected lung tissue from 126 consecutive patients with Stage IA–IV lung cancer undergoing surgery with curative intent. We observed marked degradation of a sex-associated transcription network in tumour tissue. This disturbance, detected in 27.7% of male tumours in the discovery dataset and 27.3% of male tumours in a further 123-sample replication dataset, was coincident with partial losses of the Y chromosome and extensive autosomal DNA hypomethylation. Central to this network was the epigenetic modifier and regulator of sexually dimorphic gene expression, KDM5D. After accounting for prognostic and epidemiological covariates including stage and histology, male patients with tumour KDM5D deficiency showed a significantly increased risk of death (Hazard Ratio [HR] 3.80, 95% CI 1.40–10.3, P = 0.009). KDM5D deficiency was confirmed as a negative prognostic indicator in a further 1100 male lung tumours (HR 1.67, 95% CI 1.4–2.0, P = 1.2 × 10–10). Our findings identify tumour deficiency of KDM5D as a prognostic marker and credible mechanism underlying sex disparity in lung cancer.

Abstract Ethanol-induced transcriptional changes underlie important physiological responses to ethanol that are likely to contribute to the addictive properties of the drug. We examined the transcriptional responses of Caenorhabditis elegans across a timecourse of ethanol exposure, between 30 min and 8 h, to determine what genes and genetic pathways are regulated in response to ethanol in this model. We found that short exposures to ethanol (up to 2 h) induced expression of metabolic enzymes involved in metabolizing ethanol and retinol, while longer exposure (8 h) had much more profound effects on the transcriptome. Several genes that are known to be involved in the physiological response to ethanol, including direct ethanol targets, were regulated at 8 h of exposure. This longer exposure to ethanol also resulted in the regulation of genes involved in cilia function, which is consistent with an important role for the effects of ethanol on cilia in the deleterious effects of chronic ethanol consumption in humans. Finally, we found that food deprivation for an 8-h period induced gene expression changes that were somewhat ameliorated by the presence of ethanol, supporting previous observations that worms can use ethanol as a calorie source.

Abstract The cytochrome P450 (CYP) superfamily is a diverse and important enzyme family, playing a central role in chemical defense and in synthesis and metabolism of major biological signaling molecules. The CYPomes of four cnidarian genomes (Hydra vulgaris, Acropora digitifera, Aurelia aurita, Nematostella vectensis) were annotated; phylogenetic analyses determined the evolutionary relationships amongst the sequences and with existing metazoan CYPs. 155 functional CYPs were identified and 90 fragments. Genes were from 24 new CYP families and several new subfamilies; genes were in 9 of the 12 established metazoan CYP clans. All species had large expansions of clan 2 diversity, with H. vulgaris having reduced diversity for both clan 3 and mitochondrial clan. We identified potential candidates for xenobiotic metabolism and steroidogenesis. That each genome contained multiple, novel CYP families may reflect the large evolutionary distance within the cnidarians, unique physiology in the cnidarian classes, and/or different ecology of the individual species.

Abstract Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Abstract The identification of cell surface markers specific to pancreatic beta cells is important for both the study of islet biology and for investigating the pathophysiology of diseases in which this cell type is lost or damaged. Following analysis of publicly available RNAseq data, we identified specific integrin subunits, integrin αv and integrin β5, that were expressed in beta cells. This finding was further elaborated using immunofluorescence analysis of histological sections derived from donor human pancreas. Despite the broad expression of specific integrin subunits, we found that expression of integrin αvβ5 heterodimers was restricted to beta cells and that this complex persisted in islet remnants of some type 1 diabetic individuals from which insulin expression had been lost. This study identifies αvβ5 heterodimers as a novel cell surface marker of human pancreatic beta cells, a finding that will aid in the identification and characterisation of this important cell type.

Abstract Ancient DNA analysis of human oral microbial communities within calcified dental plaque (calculus) has revealed key insights into human health, paleodemography, and cultural behaviors. However, contamination imposes a major concern for paleomicrobiological samples due to their low endogenous DNA content and exposure to environmental sources, calling into question some published results. Decontamination protocols (e.g. an ethylenediaminetetraacetic acid (EDTA) pre-digestion or ultraviolet radiation (UV) and 5% sodium hypochlorite immersion treatments) aim to minimize the exogenous content of the outer surface of ancient calculus samples prior to DNA extraction. While these protocols are widely used, no one has systematically compared them in ancient dental calculus. Here, we compare untreated dental calculus samples to samples from the same site treated with four previously published decontamination protocols: a UV only treatment; a 5% sodium hypochlorite immersion treatment; a pre-digestion in EDTA treatment; and a combined UV irradiation and 5% sodium hypochlorite immersion treatment. We examine their efficacy in ancient oral microbiota recovery by applying 16S rRNA gene amplicon and shotgun sequencing, identifying ancient oral microbiota, as well as soil and skin contaminant species. Overall, the EDTA pre-digestion and a combined UV irradiation and 5% sodium hypochlorite immersion treatment were both effective at reducing the proportion of environmental taxa and increasing oral taxa in comparison to untreated samples. This research highlights the importance of using decontamination procedures during ancient DNA analysis of dental calculus to reduce contaminant DNA.

Abstract Creutzfeldt–Jakob Disease (CJD) is a fatal, currently incurable, neurodegenerative disease. The search for candidate treatments would be greatly facilitated by the availability of human cell-based models of prion disease. Recently, an induced pluripotent stem cell derived human cerebral organoid model was shown to take up and propagate human CJD prions. This model offers new opportunities to screen drug candidates for the treatment of human prion diseases in an entirely human genetic background. Here we provide the first evidence that human cerebral organoids can be a viable model for CJD drug screening by using an established anti-prion compound, pentosan polysulfate (PPS). PPS delayed prion propagation in a prophylactic-like treatment paradigm and also alleviated propagation when applied following establishment of infection in a therapeutic-like treatment paradigm. This study demonstrates the utility of cerebral organoids as the first human 3D cell culture system for screening therapeutic drug candidates for human prion diseases.

Abstract Glacier ice is an extreme environment in which most animals cannot survive. Here we report the colonization of high elevation, climate-threatened glaciers along New Zealand’s southwestern coast by species of Arthropoda, Nematoda, Platyhelminthes, Rotifera and Tardigrada. Based on DNA barcoding and haplotype-inferred evidence for deep genetic variability, at least 12 undescribed species are reported, some of which have persisted in this niche habitat throughout the Pleistocene. These findings identify not only an atypical biodiversity hotspot but also highlight the adaptive plasticity of microinvertebrate Animalia.

Abstract Rates and extents of mineral precipitation in porous media are difficult to predict, in part because laboratory experiments are problematic. It is similarly challenging to implement numerical methods that model this process due to the need to dynamically evolve the interface of solid material. We developed a multiphase solver that implements a micro-continuum simulation approach based on the Darcy–Brinkman–Stokes equation to study mineral precipitation. We used the volume-of-fluid technique in sharp interface implementation to capture the propagation of the solid mineral surface. Additionally, we utilize an adaptive mesh refinement method to improve the resolution of near interface simulation domain dynamically. The developed solver was validated against both analytical solution and Arbitrary Lagrangian–Eulerian approach to ensure its accuracy on simulating the propagation of the solid interface. The precipitation of barite (BaSO4) was chosen as a model system to test the solver using variety of simulation parameters: different geometrical constraints, flow conditions, reaction rate and ion diffusion. The growth of a single barite crystal was simulated to demonstrate the solver’s capability to capture the crystal face specific directional growth.

Abstract Remote technologies are producing leapfrog advances in identifying the routes and connectivity of migratory species, which are still unknown for hundreds of taxa, especially Asian ones. Here, we used GPS-telemetry to uncover the migration routes and breeding areas of the massive population of migratory Black-eared kites wintering around the megacity of Delhi-India, which hosts the largest raptor concentration of the world. Kites migrated for 3300–4800 km along a narrow corridor, crossing the Himalayas at extremely high elevations (up to > 6500 m a.s.l.) by the K2 of the Karakoram Range and travelled long periods at elevations above 3500 m. They then crossed/circumvented the Taklamakan Desert and Tian Shan Range to reach their unknown breeding quarters at the intersection between Kazakhstan, Russia, China and Mongolia. Route configuration seemed to be shaped by dominant wind support and barrier avoidance. Wintering ranges were smaller than breeding ranges and concentrated around Delhi, likely in response to massive human food-subsidies. Our results illustrate that high-elevation crossings by soaring migrants may be more common than previously appreciated and suggest the delineation of a hitherto poorly-appreciated “Central Asian Flyway”, which must funnel hundreds of thousands of migrants from central Asia into the Indian subcontinent via multiple modes of the Himalayan crossing.

Abstract The Arabian horse, one of the world’s oldest breeds of any domesticated animal, is characterized by natural beauty, graceful movement, athletic endurance, and, as a result of its development in the arid Middle East, the ability to thrive in a hot, dry environment. Here we studied 378 Arabian horses from 12 countries using equine single nucleotide polymorphism (SNP) arrays and whole-genome re-sequencing to examine hypotheses about genomic diversity, population structure, and the relationship of the Arabian to other horse breeds. We identified a high degree of genetic variation and complex ancestry in Arabian horses from the Middle East region. Also, contrary to popular belief, we could detect no significant genomic contribution of the Arabian breed to the Thoroughbred racehorse, including Y chromosome ancestry. However, we found strong evidence for recent interbreeding of Thoroughbreds with Arabians used for flat-racing competitions. Genetic signatures suggestive of selective sweeps across the Arabian breed contain candidate genes for combating oxidative damage during exercise, and within the “Straight Egyptian” subgroup, for facial morphology. Overall, our data support an origin of the Arabian horse in the Middle East, no evidence for reduced global genetic diversity across the breed, and unique genetic adaptations for both physiology and conformation.

Abstract Previous work using non-invasive radiofrequency field treatment (RFT) in cancer has demonstrated its therapeutic potential as it can increase intratumoral blood perfusion, localization of intravenously delivered drugs, and promote a hyperthermic intratumoral state. Despite the well-known immunologic benefits that febrile hyperthermia can induce, an investigation of how RFT could modulate the intra-tumoral immune microenvironment had not been studied. Thus, using an established 4T1 breast cancer model in immune competent mice, we demonstrate that RFT induces a transient, localized, and T-cell dependent intratumoral inflammatory response. More specifically we show that multi- and singlet-dose RFT promote an increase in tumor volume in immune competent Balb/c mice, which does not occur in athymic nude models. Further leukocyte subset analysis at 24, 48, and 120 hours after a single RFT show a rapid increase in tumoral trafficking of CD4+ and CD8+ T-cells 24 hours post-treatment. Additional serum cytokine analysis reveals an increase in numerous pro-inflammatory cytokines and chemokines associated with enhanced T-cell trafficking. Overall, these data demonstrate that non-invasive RFT could be an effective immunomodulatory strategy in solid tumors, especially for enhancing the tumoral trafficking of lymphocytes, which is currently a major hindrance of numerous cancer immunotherapeutic strategies.

Abstract The removal of macroalgal biomass is critical to the health of coral reef ecosystems. Previous studies on relatively intact reefs with diverse and abundant fish communities have quantified rapid removal of macroalgae by herbivorous fishes, yet how these findings relate to degraded reef systems where fish diversity and abundance are markedly lower and algal biomass substantially higher, is unclear. We surveyed roving herbivorous fish communities and quantified their capacity to remove the dominant macroalga Sargassum ilicifolium on seven reefs in Singapore; a heavily degraded urbanized reef system. The diversity and abundance of herbivorous fishes was extremely low, with eight species and a mean abundance ~1.1 individuals 60 m−2 recorded across reefs. Consumption of S. ilicifolium varied with distance from Singapore’s main port with consumption being 3- to 17-fold higher on reefs furthest from the port (Pulau Satumu: 4.18 g h−1; Kusu Island: 2.38 g h−1) than reefs closer to the port (0.35–0.78 g h−1). Video observations revealed a single species, Siganus virgatus, was almost solely responsible for removing S. ilicifolium biomass, accounting for 83% of the mass-standardized bites. Despite low herbivore diversity and intense urbanization, macroalgal removal by fishes on some Singaporean reefs was directly comparable to rates reported for other inshore Indo-Pacific reefs.

Abstract Hendra virus (HeV) is an emerging zoonotic pathogen harbored by Australian mainland flying foxes. HeV infection can cause lethal disease in humans and horses, and to date all cases of human HeV disease have resulted from contact with infected horses. Currently, diagnosis of acute HeV infections in horses relies on the productive phase of infection when virus shedding may occur. An assay that identifies infected horses during the preclinical phase of infection would reduce the risk of zoonotic viral transmission during management of HeV outbreaks. Having previously shown that the host microRNA (miR)-146a is upregulated in the blood of HeV-infected horses days prior to the detection of viremia, we have profiled miRNAs at the transcriptome-wide level to comprehensively assess differences between infected and uninfected horses. Next-generation sequencing and the miRDeep2 algorithm identified 742 mature miRNA transcripts corresponding to 593 miRNAs in whole blood of six horses (three HeV-infected, three uninfected). Thirty seven miRNAs were differentially expressed in infected horses, two of which were validated by qRT-PCR. This study describes a methodology for the transcriptome-wide profiling of miRNAs in whole blood and supports the notion that measuring host miRNA expression levels may aid infectious disease diagnosis in the future.

Abstract This study evaluated the impact of calcium and magnesium on the in vitro degradation and in vivo clearance of oxaliplatin. Intact oxaliplatin and Pt(DACH)Cl2 were measured in incubation solutions by HPLC-UV. A clinical study determined changes in plasma concentrations of calcium and magnesium in cancer patients and their impact on oxaliplatin clearance. Kinetic analyses modelled oxaliplatin degradation reactions in vitro and contributions to oxaliplatin clearance in vivo. Calcium and magnesium accelerated oxaliplatin degradation to Pt(DACH)Cl2 in chloride-containing solutions in vitro. Kinetic models based on calcium and magnesium binding to a monochloro-monooxalato ring-opened anionic oxaliplatin intermediate fitted the in vitro degradation time-course data. In cancer patients, calcium and magnesium plasma concentrations varied and were increased by giving calcium gluconate and magnesium sulfate infusions, but did not alter or correlate with oxaliplatin clearance. The intrinsic in vitro clearance of oxaliplatin attributed to chloride-, calcium- and magnesium-mediated degradation predicted contributions of

Abstract Accurate pH measurements in polar waters and sea ice brines require pH indicator dyes characterized at near-zero and below-zero temperatures and high salinities. We present experimentally determined physical and chemical characteristics of purified meta-Cresol Purple (mCP) pH indicator dye suitable for pH measurements in seawater and conservative seawater-derived brines at salinities (S) between 35 and 100 and temperatures (T) between their freezing point and 298.15 K (25 °C). Within this temperature and salinity range, using purified mCP and a novel thermostated spectrophotometric device, the pH on the total scale (pHT) can be calculated from direct measurements of the absorbance ratio R of the dye in natural samples as $${\boldsymbol{p}}{{\boldsymbol{H}}}_{{\boldsymbol{T}}}{\boldsymbol{=}}{\boldsymbol{-}}{\bf{log}}({{\boldsymbol{k}}}_{{\bf{2}}}^{{\boldsymbol{T}}}{{\boldsymbol{e}}}_{{\bf{2}}}){\boldsymbol{+}}\,{\bf{log}}(\frac{{\boldsymbol{R}}{\boldsymbol{-}}{{\boldsymbol{e}}}_{{\bf{1}}}}{{\bf{1}}{\boldsymbol{-}}{\boldsymbol{R}}\frac{{{\boldsymbol{e}}}_{{\bf{3}}}}{{{\boldsymbol{e}}}_{{\bf{2}}}}})$$ p H T = − log ( k 2 T e 2 ) + log ( R − e 1 1 − R e 3 e 2 ) Based on the mCP characterization in these extended conditions, the temperature and salinity dependence of the molar absorptivity ratios and − $${\bf{log}}({{\boldsymbol{k}}}_{{\bf{2}}}^{{\boldsymbol{T}}}{{\boldsymbol{e}}}_{{\bf{2}}})$$ log ( k 2 T e 2 ) of purified mCP is described by the following functions: e 1 = −0.004363 + 3.598 × 10−5 T, e 3/e 2 = −0.016224 + 2.42851 × 10−4 T + 5.05663 × 10−5(S − 35), and − $${\bf{log}}({{\boldsymbol{k}}}_{{\bf{2}}}^{{\boldsymbol{T}}}{{\boldsymbol{e}}}_{{\bf{2}}})$$ log ( k 2 T e 2 ) = −319.8369 + 0.688159 S −0.00018374 S 2 + (10508.724 − 32.9599 S + 0.059082S 2) T−1 + (55.54253 − 0.101639 S) ln T −0.08112151T. This work takes the characterisation of mCP beyond the currently available ranges of 278.15 K ≤ T ≤ 308.15 K and 20 ≤ S ≤ 40 in natural seawater, thereby allowing high quality pHT measurements in polar systems.