Correction: Development of the Bristol Rabbit Pain Scale (BRPS): A multidimensional composite pain scale specific to rabbits (Oryctolagus cuniculus).

[This corrects the article DOI: 10.1371/journal.pone.0252417.].

The Mechanics of Building Functional Organs.

Organ morphogenesis is multifaceted, multiscale, and fundamentally a robust process. Despite the complex and dynamic nature of embryonic development, organs are built with reproducible size, shape, and function, allowing them to support organismal growth and life. This striking reproducibility of tissue form exists because morphogenesis is not entirely hardwired. Instead, it is an emergent product of mechanochemical information flow, operating across spatial and temporal scales-from local cellular deformations to organ-scale form and function, and back. In this review, we address the mechanical basis of organ morphogenesis, as understood by observations and experiments in living embryos. To this end, we discuss how mechanical information controls the emergence of a highly conserved set of structural motifs that shape organ architectures across the animal kingdom: folds and loops, tubes and lumens, buds, branches, and networks. Moving forward, we advocate for a holistic conceptual framework for the study of organ morphogenesis, which rests on an interdisciplinary toolkit and brings the embryo center stage.

Length dependent reversible off-on activation of photo-switchable relay anion transporters.

A homologous series of azobenzene-derived photo-switchable ion relay transporters is reported. We reveal that both the length and geometry of the relay strongly affect transport rate, allowing the relative activity of the E and Z isomers to be reversed and hence the wavelengths of light used for on and off switching to be exchanged.

Targeted non-invasive brain stimulation boosts attention and modulates contralesional brain networks following right hemisphere stroke.

Right hemisphere stroke patients frequently present with a combination of lateralised and non-lateralised attentional deficits characteristic of the neglect syndrome. Attentional deficits are associated with poor functional outcome and are challenging to treat, with non-lateralised deficits often persisting into the chronic stage and representing a common complaint among patients and families. In this study, we investigated the effects of non-invasive brain stimulation on non-lateralised attentional deficits in right-hemispheric stroke. In a randomised double-blind sham-controlled crossover study, twenty-two patients received real and sham transcranial Direct Current Stimulation (tDCS) whilst performing a non-lateralised attentional task. A high definition tDCS montage guided by stimulation modelling was employed to maximise current delivery over the right dorsolateral prefrontal cortex, a key node in the vigilance network. In a parallel study, we examined brain network response to this tDCS montage by carrying out concurrent fMRI during stimulation in healthy participants and patients. At the group level, stimulation improved target detection in patients, reducing overall error rate when compared with sham stimulation. TDCS boosted performance throughout the duration of the task, with its effects briefly outlasting stimulation cessation. Exploratory lesion analysis indicated that response to stimulation was related to lesion location rather than volume. In particular, reduced stimulation response was associated with damage to the thalamus and postcentral gyrus. Concurrent stimulation-fMRI revealed that tDCS did not affect local connectivity but influenced functional connectivity within large-scale networks in the contralesional hemisphere. This combined behavioural and functional imaging approach shows that brain stimulation targeted to surviving tissue in the ipsilesional hemisphere improves non-lateralised attentional deficits following stroke. This effect may be exerted via contralesional network effects.

Single-cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem-cell regulators.

Schistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. Here, single cell suspensions from S. mansoni mother sporocysts were produced and sequenced using the droplet-based 10X Genomics Chromium platform. Six cell clusters comprising two tegument, muscle, neuron, parenchyma and stem/germinal cell clusters were identified and validated by in situ hybridisation. Gene Ontology term analysis predicted key biological processes for each of the clusters, including three stem/germinal sub-clusters. Furthermore, putative transcription factors predicted for stem/germinal and tegument clusters may play key roles during parasite development and interaction with the intermediate host.

Responsive Anionophores with AND Logic Multi-Stimuli Activation.

Artificial ion transport systems have emerged as an important class of compounds that promise applications in chemotherapeutics as anticancer agents or to treat channelopathies. Stimulus-responsive systems that offer spatiotemporally controlled activity for targeted applications remain rare. Here we utilize dynamic hydrogen bonding interactions of a 4,6-dihydroxy-isophthalamide core to generate a modular platform enabling access to stimuli-responsive ion transporters that can be activated in response to a wide variety of external stimuli, including light, redox, and enzymes, with excellent OFF-ON activation profiles. Alkylation of the two free hydroxyl groups with stimulus-responsive moieties locks the amide bonds through intramolecular hydrogen bonding and hence makes them unavailable for anion binding and transport. Triggering using a particular stimulus to cleave both cages reverses the hydrogen bonding arrangement, to generate a highly preorganized anion binding cavity for efficient transmembrane transport. Integration of two cages that are responsive to orthogonal stimuli enables multi-stimuli activation, where both stimuli are required to trigger transport in an AND logic process. Importantly, the strategy provides a facile method to post-functionalize the highly active transporter core with a variety of stimulus-responsive moieties for targeted activation with multiple triggers.

Shrinking in the dark: Parallel endosymbiont genome erosions are associated with repeated host transitions to an underground life.

Microbial symbioses have had profound impacts on the evolution of animals. Conversely, changes in host biology may impact the evolutionary trajectory of symbionts themselves. Blattabacterium cuenoti is present in almost all cockroach species and enables hosts to subsist on a nutrient-poor diet. To investigate if host biology has impacted Blattabacterium at the genomic level, we sequenced and analyzed 25 genomes from Australian soil-burrowing cockroaches (Blaberidae: Panesthiinae), which have undergone at least seven separate subterranean, subsocial transitions from above-ground, wood-feeding ancestors. We find at least three independent instances of genome erosion have occurred in Blattabacterium strains exclusive to Australian soil-burrowing cockroaches. These shrinkages have involved the repeated inactivation of genes involved in amino acid biosynthesis and nitrogen recycling, the core role of Blattabacterium in the host-symbiont relationship. The most drastic of these erosions have occurred in hosts thought to have transitioned underground the earliest relative to other lineages, further suggestive of a link between gene loss in Blattabacterium and the burrowing behavior of hosts. As Blattabacterium is unable to fulfill its core function in certain host lineages, these findings suggest soil-burrowing cockroaches must acquire these nutrients from novel sources. Our study represents one of the first cases, to our knowledge, of parallel host adaptations leading to concomitant parallelism in their mutualistic symbionts, further underscoring the intimate relationship between these two partners.

Dating in the Dark: Elevated Substitution Rates in Cave Cockroaches (Blattodea: Nocticolidae) Have Negative Impacts on Molecular Date Estimates.

Rates of nucleotide substitution vary substantially across the Tree of Life, with potentially confounding effects on phylogenetic and evolutionary analyses. A large acceleration in mitochondrial substitution rate occurs in the cockroach family Nocticolidae, which predominantly inhabit subterranean environments. To evaluate the impacts of this among-lineage rate heterogeneity on estimates of phylogenetic relationships and evolutionary timescales, we analyzed nuclear ultraconserved elements (UCEs) and mitochondrial genomes from nocticolids and other cockroaches. Substitution rates were substantially elevated in nocticolid lineages compared with other cockroaches, especially in mitochondrial protein-coding genes. This disparity in evolutionary rates is likely to have led to different evolutionary relationships being supported by phylogenetic analyses of mitochondrial genomes and UCE loci. Furthermore, Bayesian dating analyses using relaxed-clock models inferred much deeper divergence times compared with a flexible local clock. Our phylogenetic analysis of UCEs, which is the first genome-scale study to include all 13 major cockroach families, unites Corydiidae and Nocticolidae and places Anaplectidae as the sister lineage to the rest of Blattoidea. We uncover an extraordinary level of genetic divergence in Nocticolidae, including two highly distinct clades that separated ~115 million years ago despite both containing representatives of the genus Nocticola. The results of our study highlight the potential impacts of high among-lineage rate variation on estimates of phylogenetic relationships and evolutionary timescales.

Coupling Photoresponsive Transmembrane Ion Transport with Transition Metal Catalysis.

Artificial ion transporters have been explored both as tools for studying fundamental ion transport processes and as potential therapeutics for cancer and channelopathies. Here we demonstrate that synthetic transporters may also be used to regulate the transport of catalytic metal ions across lipid membranes and thus control chemical reactivity inside lipid-bound compartments. We show that acyclic lipophilic pyridyltriazoles enable Pd(II) cations to be transported from the external aqueous phase across the lipid bilayer and into the interior of large unilamellar vesicles. In situ reduction generates Pd(0) species, which catalyze the generation of a fluorescent product. Photocaging the Pd(II) transporter allows for photoactivation of the transport process and hence photocontrol over the internal catalysis process. This work demonstrates that artificial transporters enable control over catalysis inside artificial cell-like systems, which could form the basis of biocompatible nanoreactors for applications such as drug synthesis and delivery or to mediate phototargeted catalyst delivery into cells.

Demystifying Dry Powder Inhaler Resistance with Relevance to Optimal Patient Care.

The selection of an inhaler device is a key component of respiratory disease management. However, there is a lack of clarity surrounding inhaler resistance and how it impacts inhaler selection. The most common inhaler types are dry powder inhalers (DPIs) that have internal resistance and pressurised metered dose inhalers (pMDIs) that use propellants to deliver the drug dose to the airways. Inhaler resistance varies across the DPIs available on the market, depending largely on the design geometry of the device but also partially on formulation parameters. Factors influencing inhaler choice include measures such as flow rate or pressure drop as well as inhaler technique and patient preference, both of which can lead to improved adherence and outcomes. For optimal disease outcomes, device selection should be individualised, inhaler technique optimised and patient preference considered. By addressing the common clinically relevant questions, this paper aims to demystify how DPI resistance should guide the selection of the right device for the right patient.

Selection of the right inhaler is important to ensure that patients with respiratory diseases get the most benefit from their treatment. Dry powder inhalers and pressurised metered dose inhalers are the most common inhaler types. Pressurised metered dose inhalers use propellants to deliver the drug to the lungs. In contrast, dry powder inhalers deliver the drug to the lungs by having internal resistance. This restricts the flow of air through the inhaler. As the patient inhales through the inhaler, the resistance against the air flow generates the power to separate the drug molecules and carry them to the lungs. While there are many factors to be considered for inhaler selection, there is often confusion around how resistance should guide selection of inhaler. With low-resistance devices, patients must inhale faster to generate the power to separate the drug molecules, which may be difficult in patients with poor lung function. With high-resistance devices, patients do not need to inhale as fast to separate the drug, and most patients can effectively use the inhaler. This article addresses the common clinically relevant questions to clarify how the internal resistance of the inhaler should be used to help guide the selection of the right device for the right patient.

Molecular Machines For The Control Of Transmembrane Transport.

Nature embeds some of its molecular machinery, including ion pumps, within lipid bilayer membranes. This has inspired chemists to attempt to develop synthetic analogues to exploit membrane confinement and transmembrane potential gradients, much like their biological cousins. In this perspective, we outline the various strategies by which molecular machines─molecular systems in which a nanomechanical motion is exploited for function─have been designed to be incorporated within lipid membranes and utilized to mediate transmembrane ion transport. We survey molecular machines spanning both switches and motors, those that act as mobile carriers or that are anchored within the membrane, mechanically interlocked molecules, and examples that are activated in response to external stimuli.

Exploiting the Catenane Mechanical Bond Effect for Selective Halide Anion Transmembrane Transport.

The first examples of [2]catenanes capable of selective anion transport across a lipid bilayer are reported. The neutral halogen bonding (XB) [2]catenanes were prepared via a chloride template-directed strategy in an unprecedented demonstration of using XB⋅⋅⋅anion interactions to direct catenane assembly from all-neutral components. Anion binding experiments in aqueous-organic solvent media revealed strong halide over oxoanion selectivity, and a marked enhancement in the chloride and bromide affinities of the catenanes relative to their constituent macrocycles. The catenanes additionally displayed an anti-Hofmeister binding preference for bromide over the larger iodide anion, illustrating the efficacy of employing sigma-hole interactions in conjunction with the mechanical bond effect to tune receptor selectivity. Transmembrane anion transport studies conducted in POPC LUVs revealed that the catenanes were more effective anion transporters than the constituent macrocycles, with high chloride over hydroxide selectivity, which is critical to potential therapeutic applications of anionophores. Remarkably these outperform existing acyclic halogen bonding anionophores with regards to this selectivity. Record chloride over nitrate anion transport selectivity was also observed. This represents a rare example of the direct translation of intrinsic anion binding affinities to anion transport behaviour, and demonstrates the key role of the catenane mechanical bond effect for enhanced anion transport selectivity.

Inter-Vesicle Signal Transduction Using a Photo-Responsive Zinc Ionophore.

Transmission of chemical information between cells and across lipid bilayer membranes is of profound significance in many biological processes. The design of synthetic signalling systems is a critical step towards preparing artificial cells with collective behaviour. Here, we report the first example of a synthetic inter-vesicle signalling system, in which diffusible chemical signals trigger transmembrane ion transport in a manner reminiscent of signalling pathways in biology. The system is derived from novel ortho-nitrobenzyl and BODIPY photo-caged ZnII transporters, in which cation transport is triggered by photo-decaging with UV or red light, respectively. This decaging reaction can be used to trigger the release of the cationophores from a small population of sender vesicles. This in turn triggers the transport of ions across the membrane of a larger population of receiver vesicles, but not across the sender vesicle membrane, leading to overall inter-vesicle signal transduction and amplification.

Halogen bonding relay and mobile anion transporters with kinetically controlled chloride selectivity.

Selective transmembrane transport of chloride over competing proton or hydroxide transport is key for the therapeutic application of anionophores, but remains a significant challenge. Current approaches rely on enhancing chloride anion encapsulation within synthetic anionophores. Here we report the first example of a halogen bonding ion relay in which transport is facilitated by the exchange of ions between lipid-anchored receptors on opposite sides of the membrane. The system exhibits non-protonophoric chloride selectivity, uniquely arising from the lower kinetic barrier to chloride exchange between transporters within the membrane, compared to hydroxide, with selectivity maintained across membranes with different hydrophobic thicknesses. In contrast, we demonstrate that for a range of mobile carriers with known high chloride over hydroxide/proton selectivity, the discrimination is strongly dependent on membrane thickness. These results demonstrate that the selectivity of non-protonophoric mobile carriers does not arise from ion binding discrimination at the interface, but rather through a kinetic bias in transport rates, arising from differing membrane translocation rates of the anion-transporter complexes.

Control of dynamic sp3-C stereochemistry.

Stereogenic sp3-hybridized carbon centres are fundamental building blocks of chiral molecules. Unlike dynamic stereogenic motifs, such as sp3-nitrogen centres or atropisomeric biaryls, sp3-carbon centres are usually fixed, requiring intermolecular reactions to undergo configurational changes. Here we report the internal enantiomerization of fluxional carbon cages and the consequences of their adaptive configurations for the transmission of stereochemical information. The sp3-carbon stereochemistry of the rigid tricyclic cages is inverted through strain-assisted Cope rearrangements, emulating the low-barrier configurational dynamics typical for sp3-nitrogen inversion or conformational isomerism. This dynamic enantiomerization can be stopped, restarted or slowed by external reagents, while the configuration of the cage is controlled by neighbouring, fixed stereogenic centres. As part of a phosphoramidite-olefin ligand, the fluxional cage acts as a conduit to transmit stereochemical information from the ligand while also transferring its dynamic properties to chiral-at-metal coordination environments, influencing catalysis, ion pairing and ligand exchange energetics.

Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores.

Synthetic supramolecular transmembrane anionophores have emerged as promising anticancer chemotherapeutics. However, key to their targeted application is achieving spatiotemporally controlled activity. Herein, we report a series of chalcogen-bonding diaryl tellurium-based transporters in which their anion binding potency and anionophoric activity are controlled through reversible redox cycling between Te oxidation states. This unprecedented in situ reversible multistate switching allows for switching between ON and OFF anion transport and is crucially achieved with biomimetic chemical redox couples.

Evidence that heat acclimation training may alter sleep and incidental activity.

This randomized cross-over study tested the hypothesis that heat acclimation training would detrimentally affect sleep variables and alter incidental physical activity compared to a thermoneutral training control condition. Eight recreationally trained males (V̇O2peak 49±4.9 mL.kg-1.min-1) completed two separate interventions separated by at least 31 days: 5 consecutive day training blocks of moderate-intensity cycling (60 min·day-1 at 50% peak power output) in a hot (34.9±0.7 °C and 53±4 % relative humidity) and a temperate (22.2±2.6 °C; 65±8 % relative humidity) environment. Wrist-mounted accelerometers were worn continuously for the length of the training blocks and recorded physical activity, sleep quality and quantity. Data were analysed in a Bayesian framework, with the results presented as the posterior probability that a coefficient was greater or less than zero. Compared to the temperate training environment, heat acclimation impaired sleep efficiency (Pr ß<0 = .979) and wake on sleep onset (Pr ß>0 = .917). Daily sedentary time was, on average, 35 min longer (Pr ß>0 = .973) and light physical activity time 18 min shorter (Pr ß>0 = .960) during the heat acclimation period. No differences were observed between conditions in sleep duration, subjective sleep quality, or moderate or vigorous physical activity. These findings may suggest that athletes and coaches need to be cognisant that heat acclimation training may alter sleep quality and increase sedentary behaviour.HighlightsFive consecutive days of heat training negatively affected some objective measures of sleep quality and incidental physical activity in recreationally trained athletes.Athletes and coaches need to be aware of the potential unintended consequences of using heat acclimation on sleep behaviours.

Estimating nitrate intake in the Australian diet: Design and validation of a food frequency questionnaire.

BACKGROUND: Dietary nitrates may play a role in mediating several key physiological processes impacting health and/or exercise performance. However, current methods for assessing dietary nitrate (NO3 - ) consumption are inadequate. The present study aimed to examine the dietary nitrate intake in a sample of 50 healthy adults, as well as test the validity of a purposefully developed food frequency questionnaire (FFQ). METHODS: Dietary nitrate intake was estimated over a week using (i) three 24-h dietary recalls; (ii) a short-term (7-day) FFQ; and (iii) a biomarker (urinary nitrate), in conjunction with a nitrate reference database. RESULTS: Daily dietary nitrate intake estimates were 130.94 mg (average of three 24-h recalls) and 180.62 mg (FFQ). The mean urinary NO3 - excretion was 1974.79 µmol day-1 (or 917.9 µmol L-1 ). Despite the difference between the two dietary assessment methods, there was a moderate positive correlation (r = 0.736, ρ < 0.001) between the two tools. There was also a positive correlation between urinary NO3 - and 24-h recall data (r = 0.632, ρ < 0.001), as well as between urinary NO3 - and FFQ (r = 0.579, ρ < 0.001). CONCLUSIONS: The ability to accurately estimate nitrate intakes depends on having suitable reference methods to estimate the concentrations of nitrate in the food supply, coupled with valid and reliable dietary assessment tools. Based on the findings from the present study, at an individual level, dietary recalls or records may be more accurate in estimating intakes of NO3 - . However, given the lower cost and time needed for administration relative to recalls, the FFQ has merit for estimating NO3 - intakes in health interventions, dietary surveys and surveillance programs.

A survey of handling and transportation of UK farmed deer.

Studies on the transport of deer (Cervidae), in the UK, were published > 15 years ago. A more recent study of deer transport is required to allow for assessments and improvements to the transport of farmed deer. Sixteen deer farmers participated in a survey describing their management practices related to transport. Their responses showed that most vehicles used to transport deer were designed for other livestock. Participating farmers estimated journey times to slaughter as 1-8 h, with an arithmetic mean of 4.8 (± 2.38) h. Specific concerns raised by the respondents, relating to the transport of deer, included a need for deer-specific vehicles, stop-off areas for long journeys, market locations and haulier experience. Furthermore, data were collected from two abattoirs between July 2019 and June 2020 comprising journey times, slaughter times, bruising, location of origin, vehicle type and the number of animals. In total, 4,922 deer were transported across 133 journeys (from farm to abattoir) from 61 farms. Median and range for journey length were 3.2 (0.4-9.8) h and 154.2 (7.1-462.2) km, whereas group size and time spent in the lairage were 24 (1-121) and 17.8 (10.2-68.9) h, respectively. Group size was found to be significantly associated with both the presence of bruising in a group and the amount of bruising per deer. This study provides a much-needed update on the transport of farmed deer in the UK and highlights key areas for future research including the welfare impact of transport in larger groups and for longer durations.