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Background: Glomerular endothelial cell (GEnC) fenestrations are recognised as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease.Methods: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular ultrafiltration coefficient and glomerular filtration rate in diabetic mice (BTBR ob-/ob-). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explore its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells.Results: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knock-down b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation.Conclusions: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease

Funder: Biotechnology and Biological Sciences Research Council, Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (-)-adaline, found in the 2-spot (

Objective As well as being an established oncoprotein and therapeutic target in cancer, proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is implicated in human autoimmunity. This study was undertaken to investigate Pim-1 and its family members as potential therapeutic targets in early rheumatoid arthritis (RA). Methods A flow cytometry assay for PIM1 transcript measurement in peripheral blood mononuclear cells from patients with early arthritis was validated and applied as a biomarker of Pim-1 activity at the cellular level. Synovial protein expression was similarly determined by multiplex immunofluorescence in tissue samples from untreated RA patients and non-RA disease controls. Functional consequences of Pim kinase family manipulation in freshly isolated CD4+ T cells from these individuals were ascertained, along with the impact of Pim inhibition on mice with collagen-induced arthritis (CIA). Results The percentage of circulating CD4+ T cells positive for PIM1 transcript by flow cytometry proved a faithful surrogate for gene expression and was significantly higher in patients with early RA than in those with other diseases. Pim-1 protein levels were similarly up-regulated in synovial CD4+ T cells from patients with early RA. Ex vivo, exposure of T cell receptor-stimulated early RA CD4+ T cells to Pim kinase inhibitors restrained their activation and proliferative capacity. Diminished production of proinflammatory cytokines (interferon-γ and interleukin-17) and an expanded CD25high FoxP3+ Treg cell fraction were also observed in exposed versus unexposed cells. Finally, administration of Pim inhibitors robustly limited arthritis progression and cartilage destruction in CIA. Conclusion Our findings indicate that Pim kinases are plausible therapeutic targets in a readily identifiable subgroup of patients with early RA. Repurposing of Pim inhibitors for this disease should be considered.

Plasmids are mobile elements that can carry genes encoding traits of clinical concern, including antimicrobial resistance (AMR) and virulence. Population-level studies of

Background:Genetic variations indicating loss of function in theDLG2gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability.DLG2encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear.Methods:Hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygousDlg2+/−rat model usingex vivopatch-clamp electrophysiology, pharmacology, and computational modelling.Results:Dlg2+/−rats had increased NMDA receptor-mediated synaptic currents and, conversely, impaired associative long-term potentiation. This impairment resulted from an increase in potassium channel function leading to a decrease in input resistance and reduced supra-linear dendritic integration during induction of associative long-term potentiation. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in theDlg2+/−rats.Conclusions:Despite increasing synaptic NMDA receptor currents, the combined impact of reduced DLG2 impairs synaptic integration in dendrites resulting in disrupted associative synaptic plasticity. This biological phenotype can be reversed by compound classes used clinically such as muscarinic M1 receptor agonists and is therefore a potential target for therapeutic intervention.

Stimulator of interferon genes (STING)-mediated innate immune activation plays a key role in tumor- and self-DNA-elicited antitumor immunity and autoimmunity. However, STING can also suppress tumor immunity and autoimmunity. STING signaling in host nonhematopoietic cells was reported to either protect against or promote graft-versus-host disease (GVHD), a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Host hematopoietic antigen-presenting cells (APCs) play key roles in donor T-cell priming during GVHD initiation. However, how STING regulates host hematopoietic APCs after allo-HCT remains unknown. We utilized murine models of allo-HCT to assess the role of STING in hematopoietic APCs. STING-deficient recipients developed more severe GVHD after major histocompatibility complex-mismatched allo-HCT. Using bone marrow chimeras, we found that STING deficiency in host hematopoietic cells was primarily responsible for exacerbating the disease. Furthermore, STING on host CD11c(+) cells played a dominant role in suppressing allogeneic T-cell responses. Mechanistically, STING deficiency resulted in increased survival, activation, and function of APCs, including macrophages and dendritic cells. Consistently, constitutive activation of STING attenuated the survival, activation, and function of APCs isolated from STING V154M knock-in mice. STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression, and migration into intestinal tissues, resulting in accelerated/exacerbated GVHD. Using pharmacologic approaches, we demonstrated that systemic administration of a STING agonist (bis-(3′-5′)-cyclic dimeric guanosine monophosphate) to recipient mice before transplantation significantly reduced GVHD mortality. In conclusion, we revealed a novel role of STING in APC activity that dictates T-cell allogeneic responses and validated STING as a potential therapeutic target for controlling GVHD after allo-HCT.

Genetic studies implicate disruption to the DLG2 gene in copy number variants as increasing risk for schizophrenia, autism spectrum disorders and intellectual disability. To investigate psychiatric endophenotypes associated with DLG2 haploinsufficiency (and concomitant PSD-93 protein reduction) a novel clinically relevant Dlg2+/- rat was assessed for abnormalities in anxiety, sensorimotor gating, hedonic reactions, social behaviour, and locomotor response to the N-Methyl-D-aspartic acid receptor antagonist phencyclidine. Dlg gene and protein expression were also investigated to assess model validity. Reductions in PSD-93 messenger RNA and protein were observed in the absence of compensation by other related genes or proteins. Behaviourally Dlg2+/- rats show potentiated locomotor response to phencyclidine, as is typical of psychotic disorder models, in the absence of deficits in the other behavioural phenotypes assessed here. This shows that the behavioural effects of Dlg2 haploinsufficiency may specifically relate to psychosis vulnerability but are subtle, providing a contrast to the gross deficits in Dlg2 homozygous models (Winkler, et al., 2018; Yoo et al., 2020a) which do not so specifically model the single chromosome DLG2 deletion in carriers of risk-associated copy number variants.

Clinical response to immunotherapy is closely associated with the immunosuppressive tumour microenvironment (TME), and influenced by the dynamic interaction between tumour cells and lymphatic endothelial cells (LECs). Here, we show that high levels of miR-142-5p positively correlate with indoleamine 2,3-dioxygenase (IDO) expression in tumour-associated lymphatic vessels in advanced cervical squamous cell carcinoma (CSCC). The miR-142-5p is transferred by CSCC-secreted exosomes into LECs to exhaust CD8+ T cells via the up-regulation of lymphatic IDO expression, which was abrogated by an IDO inhibitor. Mechanistically, miR-142-5p directly down-regulates lymphatic AT-rich interactive domain-containing protein 2 (ARID2) expression, inhibits DNA methyltransferase 1 (DNMT1) recruitment to interferon (IFN)-γ promoter, and enhances IFN-γ transcription by suppressing promoter methylation, thereby leading to elevated IDO activity. Furthermore, increased serum exosomal miR-142-5p levels and the consequent IDO activity positively correlate with CSCC progression. In conclusion, exosomes secreted by CSCC cells deliver miR-142-5p to LECs and induce IDO expression via ARID2–DNMT1–IFN-γ signalling to suppress and exhaust CD8+ T cells. Our study suggests that LECs act as an integral component of the immune checkpoint(s) in the TME and may serve as a potential new target for CSCC diagnosis and treatment.

Release of the neuromodulator noradrenaline signals salience during wakefulness, flagging novel or important experiences to reconfigure information processing and memory representations in the hippocampus. Noradrenaline is therefore expected to enhance hippocampal responses to synaptic input; however, noradrenergic agonists have been found to have mixed and sometimes contradictory effects on Schaffer collateral synapses and the resulting CA1 output. Here, we examine the effects of endogenous, optogenetically driven noradrenaline release on synaptic transmission and spike output in mouse hippocampal CA1 pyramidal neurons. We show that endogenous noradrenaline release enhances the probability of CA1 pyramidal neuron spiking without altering feedforward excitatory or inhibitory synaptic inputs in the Schaffer collateral pathway. β-adrenoceptors mediate this enhancement of excitation-spike coupling by reducing the charge required to initiate action potentials, consistent with noradrenergic modulation of voltage-gated potassium channels. Furthermore, we find the likely effective concentration of endogenously released noradrenaline is sub-micromolar. Surprisingly, although comparable concentrations of exogenous noradrenaline cause robust depression of slow afterhyperpolarization currents, endogenous release of noradrenaline does not, indicating that endogenous noradrenaline release is targeted to specific cellular locations. These findings provide a mechanism by which targeted endogenous release of noradrenaline can enhance information transfer in the hippocampus in response to salient events.

Rationale Reward-related impairments are common in major depressive disorder (MDD) and may contribute to the loss of interest in pleasurable activities. A novel approach to studying reward-related decision-making are effort-based tasks; however, direct comparisons between delayed-onset and rapid-acting antidepressants (ADs) have not yet been carried out. Objectives To investigate the effects of conventional delayed-onset ADs versus rapid-acting ADs, ketamine and scopolamine, on effort-related choice behaviour. Methods Female Lister hooded rats were trained in an operant effort for reward task (EfRT) where animals choose between working for a high value-high effort reward and consuming low value-low effort chow. Using a within-subject study design, animals were then tested following acute treatment with different monoaminergic ADs, and the rapid-acting ADs ketamine or scopolamine. Results Consistent with previous findings, we found choice behaviour was sensitive to dopaminergic manipulations. We observed that pre-feeding altered choice behaviour and that the use of high or low value reward differentially affected behaviour. Monoamine re-uptake inhibitors and rapid-acting ADs resulted in similar, general patterns of reduced motivation without any evidence for specific effects, and we did not observe any clear differences between these classes of antidepressant. Conclusions Motivational changes induced by dopaminergic manipulations and pre-feeding differentially affect effort choice behaviour. However, both conventional delayed-onset ADs and ketamine and scopolamine appear to have detrimental effects on motivation in this task at the higher doses tested without any evidence of specificity for effort-related choice behaviour, in contrast to their specificity in tasks which look at more cognitive aspects of reward processing.

BackgroundThe discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability.MethodsHomozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene’s function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing.ResultsHomozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum–anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits.ConclusionsThese data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.

Significance Multiple representatives of eulipotyphlan mammals (shrews, hedgehogs, moles, and solenodons) are venomous, but little is known about the evolutionary history and composition of their oral venom systems. Herein we characterized venom from the endangered Hispaniolan solenodon (Solenodon paradoxus) and find that it consists of hypotensive proteins likely used to facilitate vertebrate prey capture. We demonstrate that venom has evolved independently on at least 4 occasions in eulipotyphlans, and that molecular components of these venoms have also evolved convergently, with kallikrein-1 proteins coopted as toxins in both solenodons and shrews following their divergence over 70 million years ago. Our findings present an elegant example of convergent molecular evolution and highlight that mammalian venom systems may be subjected to evolutionary constraints., Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Australia relies on periodic antimicrobial resistance (AMR) surveys to determine trends and changes in AMR in animal production systems. This study is a follow-up to a survey of Escherichia coli from healthy cattle at slaughter conducted in 2013, which provided baseline data on AMR prevalence across cattle groups and production practices. In this study, 591 beef cattle, 194 dairy cattle, and 216 veal calf fecal samples were collected from 25 beef and veal processing establishments in Australia, representing approximately 77% of total export volume. A total of 969 matrix-assisted laser desorption-ionization results confirmed commensal E. coli isolates from 574 beef cattle, 186 dairy cattle, and 209 veal calves were recovered, and antimicrobial susceptibility testing was carried out by microbroth dilution to 16 drugs from 10 classes interpreted against epidemiological cutoff breakpoints. Overall, a high proportion of E. coli isolates (83.8%) were wild type for all antimicrobials assessed. In addition, isolates that were non-wild type (NWT) for three or more classes of antimicrobial did not exceed 4% for any of the cattle groups. The prevalence of E. coli that were NWT for antimicrobials that are critical or of high importance to human health was very low, with 1.4% of all isolates tested determined to be NWT for fluoroquinolones, third-generation cephalosporins, or polymyxins. Genomic analysis of NWT isolates identified one beef cattle isolate (ST-10) harboring blaCMY-2 and a dairy isolate (ST-58) and two veal calf isolates (ST-58 and ST-394) that had qnrS1, which confer resistance to extended-spectrum cephalosporins and fluoroquinolones, respectively. The low levels of AMR reported in this study confirm previous Australian studies, which indicated that there is minimal evidence that specific production practices lead to widespread disproportionate development of NWT isolates.

The authors also thank funding sources: the Natural Environment Research Council (NERC; UK National Capability award number NE/R016429/1 as part of the UKSCaPE programme); Joint Nature Conservatio Committee (JNCC); Environment and Climate Change Canada; Natural Resources Canada; New Bedford Harbor Trustee Council; The Norwegian Environment Agency (and its predecessors), the SEAPOP programme (www.seapop.no) and its key institutions: The Norwegian Institute for Nature Research, The Norwegian Polar Institute and Tromsø University Museum and the French Polar Institute. 1. Timing of breeding, an important driver of fitness in many populations, is widely studied in the context of global change, yet despite considerable efforts to identify environmental drivers of seabird nesting phenology, for most populations we lack evidence of strong drivers. Here we adopt an alternative approach, examining the degree to which different populations positively covary in their annual phenology to infer whether phenological responses to environmental drivers are likely to be (i) shared across species at a range of spatial scales, (ii) shared across populations of a species, or (iii) idiosyncratic to populations. 2. We combined 51 long-term datasets on breeding phenology spanning 50 years from nine seabird species across 29 North Atlantic sites and examined the extent to which different populations share early versus late breeding seasons depending on a hierarchy of spatial scales comprising breeding site, small-scale region, large-scale region and the whole North Atlantic. 3. In about a third of cases we found laying dates of populations of different species sharing the same breeding site or small-scale breeding region were positively correlated, which is consistent with the hypothesis that they share phenological responses to the same environmental conditions. In comparison we found no evidence for positive phenological covariation among populations across species aggregated at larger spatial scales. 4. In general we found little evidence for positive phenological covariation between populations of a single species, and in many instances the inter-year variation specific to a population was substantial, consistent with each population responding idiosyncratically to local environmental conditions. Black-legged kittiwake (Rissa tridactyla) was the exception, with populations exhibiting positive covariation in laying dates that decayed with the distance between breeding sites, suggesting that populations may be responding to a similar driver. 5. Our approach sheds light on the potential factors that may drive phenology in our study species, thus furthering our understanding of the scales at which different seabirds interact with interannual variation in their environment. We also identify additional systems and phenological questions to which our inferential approach could be applied. Postprint

The aim of this study was to evaluate phenotypic and genotypic AMR characteristics of Salmonella enterica isolates from Australian cattle collected through a structured national survey utilizing 1001 faecal samples collected from healthy cattle at slaughter. A total of 184 Salmonella isolates were subsequently derived and subjected to microbroth dilution to 16 drugs from 11 classes with interpretation of minimum inhibitory concentrations (MICs) using epidemiological cut off (ECOFF) values to distinguish between wild-type and non-wild-type populations. Most isolates were susceptible (wild type) to all antimicrobials tested, with no resistance (non-wild type) detected for colistin, nalidixic acid, meropenem, gentamicin, florfenicol or chloramphenicol. Low rates of resistance were detected for ampicillin (2.2%), cefoxitin (2.2%), ceftiofur (2.2%), ceftriaxone (2.2%), ciprofloxacin (0.5%), streptomycin (3.3%) and tetracycline (0.5%). Isolates resistant to ceftriaxone (a critically important antimicrobial, CIA) carried the extended spectrum cephalosporin gene bla

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 μM at −75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 μM at −75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 μM at −75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at −75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Levodopa (L-DOPA) is an oral Parkinson's Disease drug that generates the active metabolite - dopamine (DA) in vivo. However, oral L-DOPA exhibits low oral bioavailability, limited brain uptake, peripheral DA-mediated side effects and its poor brain bioavailability can lead to long-term complications. Here we show that L-DOPA forms stable (for at least 5 months) 300 nm nanoparticles when encapsulated within N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ). A nano-in-microparticle GCPQ-L-DOPA formulation (D

Understanding why some species thrive in captivity, while others struggle to adjust, can suggest new ways to improve animal care. Approximately half of all Psittaciformes, a highly threatened order, live in zoos, breeding centres and private homes. Here, some species are prone to behavioural and reproductive problems that raise conservation and ethical concerns. To identify risk factors, we analysed data on hatching rates in breeding centres (115 species, 10 255 pairs) and stereotypic behaviour (SB) in private homes (50 species, 1378 individuals), using phylogenetic comparative methods (PCMs). Small captive population sizes predicted low hatch rates, potentially due to genetic bottlenecks, inbreeding and low availability of compatible mates. Species naturally reliant on diets requiring substantial handling were most prone to feather-damaging behaviours (e.g. self-plucking), indicating inadequacies in the composition or presentation of feed (often highly processed). Parrot species with relatively large brains were most prone to oral and whole-body SB: the first empirical evidence that intelligence can confer poor captive welfare. Together, results suggest that more naturalistic diets would improve welfare, and that intelligent psittacines need increased cognitive stimulation. These findings should help improve captive parrot care and inspire further PCM research to understand species differences in responses to captivity.

Continuous accurate attainment of the body temperature of foals is important to detect early stages of severe heat stress or fever due to a systemic illness. Among a number of methods to measure body temperature, measuring rectal temperature with a digital thermometer is most frequently used due to being relatively fast and simple method. It is also comparatively accurate and correlates well with the core body temperature. However, this method requires restraining the foal for a few seconds to obtain the temperature, and it can be dangerous for the handling person. Percutaneous thermal sensing microchips (PTSMs) are a means of monitoring the body temperature of horses, which offers a non-invasive, hygienic, quick, and accurate way to measure body temperature and provide an identification number for each individual, once it is implanted. This study tested the hypothesis that PTSM has a strong relationship with a conventional body temperature measurement, i.e., measuring rectal temperature with a digital thermometer of foals during summer seasons. Thirty-two foals in three consecutive foaling seasons (2018, 2019, and 2020 season) were implanted a PTSM into the right pectoral muscle, the right splenius muscle, the right gluteal muscle, and the nuchal ligament as early as two weeks after birth. The four PTSM temperatures, rectal temperature, and climate conditions (air temperature, relative humidity, and wet-bulb globe temperature) were obtained simultaneously during the three summer seasons and paired for comparison analysis. Among the PTSM temperatures, the pectoral muscle had the highest correlation and the least differences with rectal temperature. Using PTSM was safe, easy, and reliable for attaining body temperature in foals.

Neuropathic pain is one of the most important clinical consequences of injury to the somatosensory system. Nevertheless, the critical pathophysiological mechanisms involved in neuropathic pain development are poorly understood. In this study, we found that neuropathic pain is abrogated when the kynurenine metabolic pathway (KYNPATH) initiated by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is ablated pharmacologically or genetically. Mechanistically, it was found that IDO1-expressing dendritic cells (DCs) accumulated in the dorsal root leptomeninges and led to an increase in kynurenine levels in the spinal cord. In the spinal cord, kynurenine was metabolized by kynurenine-3-monooxygenase-expressing astrocytes into the pronociceptive metabolite 3-hydroxykynurenine. Ultimately, 3-hydroxyanthranilate 3,4-dioxygenase-derived quinolinic acid formed in the final step of the canonical KYNPATH was also involved in neuropathic pain development through the activation of the glutamatergic N-methyl-D-aspartate receptor. In conclusion, these data revealed a role for DCs driving neuropathic pain development through elevation of the KYNPATH. This paradigm offers potential new targets for drug development against this type of chronic pain.

Han et al. study the effect of primary cardiac hypertrophy on cardiac mechano-energetics. Using isolated cardiac tissues, they show that despite impaired contractile performance, cardiac mechanical efficiency is preserved in hypertrophic hearts., Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.

Case History: Two calves delivered following elective caesarean section showed behaviour typical of neonatal maladjustment syndrome described in foals, i.e., indifference to environmental stimuli, ...

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 non-selectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here we examined the action of seventeen analogues of PhTX-343 against ganglionic (?3?4) and brain (?4?2) nAChRs expressed in Xenopus oocytes by using two-electrode voltage-clamp at -100 mV. IC50 values for PhTX-343 inhibition of ?3?4 and ?4?2 receptors were 7.7 nM and 80 nM, respectively. All of the studied analogues had significantly higher potency at ?3?4 nAChRs with IC50 values as low as 0.16 nM and with up to a 91-fold selectivity for ?3?4 over ?4?2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for ?3?4 nAChRs. 2

Women with metastatic breast cancer have a disheartening 5-year survival rate of only 28%. CREB3L1 (cAMP-responsive element binding protein 3 like 1) is a metastasis suppressor that functions as a transcription factor, and in an estrogen-dependent model of rat breast cancer, it repressed the expression of genes that promote breast cancer progression and metastasis. In this report, we set out to determine the expression level of CREB3L1 across different human breast cancer subtypes and determine whether CREB3L1 functions as a metastasis suppressor, particularly in triple negative breast cancers (TNBCs). CREB3L1 expression was generally increased in luminal A, luminal B and HER2 breast cancers, but significantly reduced in a high proportion (75%) of TNBCs. Two luminal A (HCC1428, T47D) and two basal TNBC (HCC1806, HCC70) CREB3L1-deficient breast cancer cell lines were characterized as compared to their corresponding HA-CREB3L1-expressing counterparts. HA-CREB3L1 expression significantly reduced both cell migration and anchorage-independent growth in soft agar but had no impact on cell proliferation rates as compared to the CREB3L1-deficient parental cell lines. Restoration of CREB3L1 expression in HCC1806 cells was also sufficient to reduce mammary fat pad tumor formation and lung metastases in mouse xenograft models of breast cancer as compared to the parental HCC1806 cells. These results strongly support a metastasis suppressor role for CREB3L1 in human luminal A and TNBCs. Further, the ability to identify the subset of luminal A (7%) and TNBCs (75%) that are CREB3L1-deficient provides opportunities to stratify patients that would benefit from additional treatments to treat their more metastatic disease.

BackgroundObesity, metabolic disease and some psychiatric conditions are associated with changes to relative abundance of bacterial species and specific genes in the fecal microbiome. Little is known about the impact of pharmacologically induced weight loss on distinct gut microbiome species and their respective gene programs in obese individuals.ResultsUsing shotgun metagenomics, the composition of the microbiome was obtained for two cohorts of obese female Wistar rats (n=10-12, total of 82) maintained on a high fat diet before and after a 42-day treatment with a panel of four investigatory or approved anti-obesity drugs (tacrolimus/FK506, bupropion, naltrexone and sibutramine), alone or in combination. We found that sibutramine treatment induced consistent weight loss through reducing food intake. Decreased weight loss in sibutramine-treated rats was associated with changes to the gut microbiome that included increased beta-diversity, increased Bacteroides/Firmicutes ratio and increased relative abundance of multiple Bacteroides species. In addition, the relative abundance of multiple genes was found to be differentially abundant, including significant reductions in components of flagellum and genes involved in flagellum assembly.ConclusionsThis study provides a large resource comprising complete shotgun metagenomics datasets of the fecal microbiome coupled with weight change and food intake at day 3, day 15 and day 42 from 82 obese rats treated with a range of compounds used for weight loss, which are available to the community for detailed analysis. Furthermore, by conducting a detailed analysis of the microbiome associated with sibutramine-induced weight loss, we have identified multiple weight-loss associated microbial taxa and pathways. These include a reduction in components of flagellum and the flagellum assembly pathway that points to a potential role of sibutramine-induced weight-loss on regulating bacterially driven anti-inflammatory responses.

There is a need for the development of effective treatments for focal articular cartilage injuries. We previously developed a multiphasic 3D-bioplotted osteochondral scaffold design that can drive site-specific tissue formation when seeded with adipose-derived stem cells (ASC). The objective of this study was to evaluate this scaffold in a large animal model. Osteochondral defects were generated in the trochlear groove of Yucatan minipigs and repaired with scaffolds that either contained or lacked an electrospun tidemark and were either unseeded or seeded with ASC. Implants were monitored via computed tomography (CT) over the course of 4 months of in vivo implantation and compared to both open lesions and autologous explants. ICRS II evaluation indicated that defects with ASC-seeded scaffolds had healing that most closely resembled the aulogous explant. Scaffold-facilitated subchondral bone repair mimicked the structure of native bone tissue, but cartilage matrix staining was not apparent within the scaffold. The open lesions had the highest volumetric infill detected using CT analysis (p < 0.05), but the repair tissue was largely disorganized. The acellular scaffold without a tidemark had significantly more volumetric filling than either the acellular or ASC seeded groups containing a tidemark (p < 0.05), suggesting that the tidemark limited cell infiltration into the cartilage portion of the scaffold. Overall, scaffold groups repaired the defect more successfully than an open lesion but achieved limited repair in the cartilage region. With further optimization, this approach holds potential to treat focal cartilage lesions in a highly personalized manner using a human patient's own ASC cells.

Background Evidence of global heterochromatin decay and aberrant gene expression in models of physiological and premature ageing have long supported the “heterochromatin loss theory of ageing”, which proposes that ageing is aetiologically linked to, and accompanied by, a progressive, generalised loss of repressive epigenetic signatures. However, the remarkable plasticity of chromatin conformation suggests that the re-establishment of such marks could potentially revert the transcriptomic architecture of animal cells to a “younger” state, promoting longevity and healthspan. To expand our understanding of the ageing process and its connection to chromatin biology, we screened an RNAi library of chromatin-associated factors for increased longevity phenotypes. Results We identified the lysine demethylases jmjd-3.2 and utx-1, as well as the lysine methyltransferase mes-2 as regulators of both lifespan and healthspan in C. elegans. Strikingly, we found that both overexpression and loss of function of jmjd-3.2 and utx-1 are all associated with enhanced longevity. Furthermore, we showed that the catalytic activity of UTX-1, but not JMJD-3.2, is critical for lifespan extension in the context of overexpression. In attempting to reconcile the improved longevity associated with both loss and gain of function of utx-1, we investigated the alternative lifespan pathways and tissue specificity of longevity outcomes. We demonstrated that lifespan extension caused by loss of utx-1 function is daf-16 dependent, while overexpression effects are partially independent of daf-16. In addition, lifespan extension was observed when utx-1 was knocked down or overexpressed in neurons and intestine, whereas in the epidermis, only knockdown of utx-1 conferred improved longevity. Conclusions We show that the regulation of longevity by chromatin modifiers can be the result of the interaction between distinct factors, such as the level and tissue of expression. Overall, we suggest that the heterochromatin loss model of ageing may be too simplistic an explanation of organismal ageing when molecular and tissue-specific effects are taken into account.

Diabetes is associated with cardiac metabolic disturbances and increased heart failure risk. Plasma fructose levels are elevated in diabetic patients. A direct role for fructose involvement in diabetic heart pathology has not been investigated. The goals of this study were to clinically evaluate links between myocardial fructose and sorbitol (a polyol pathway fructose precursor) levels with evidence of cardiac dysfunction, and to experimentally assess the cardiomyocyte mechanisms involved in mediating the metabolic effects of elevated fructose. Fructose and sorbitol levels were increased in right atrial appendage tissues of type 2 diabetic patients (2.8- and 1.5-fold increase respectively). Elevated cardiac fructose levels were confirmed in type 2 diabetic rats. Diastolic dysfunction (increased E/e’, echocardiography) was significantly correlated with cardiac sorbitol levels. Elevated myocardial mRNA expression of the fructose-specific transporter, Glut5 (43% increase), and the key fructose-metabolizing enzyme, Fructokinase-A (50% increase) was observed in type 2 diabetic rats (Zucker diabetic fatty rat). In neonatal rat ventricular myocytes, fructose increased glycolytic capacity and cytosolic lipid inclusions (28% increase in lipid droplets/cell). This study provides the first evidence that elevated myocardial fructose and sorbitol are associated with diastolic dysfunction in diabetic patients. Experimental evidence suggests that fructose promotes the formation of cardiomyocyte cytosolic lipid inclusions, and may contribute to lipotoxicity in the diabetic heart.

Synaptic plasticity has classically been characterized to involve the NMDA and AMPA subtypes of glutamate receptors, with NMDA receptors providing the key trigger for the induction of long-term plasticity leading to changes in AMPA receptor expression. Here we review the more subtle roles played by kainate receptors, which contribute critical postsynaptic signalling as well as playing major presynaptic auto-receptor roles. We focus on two research areas: plasticity of kainate receptors themselves and the contribution they make to the plasticity of synaptic transmission. This article is part of the special issue on Glutamate Receptors - Kainate receptors.

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of − 3 to − 4 at a concentration of 10–100 μg/ mL (p p 50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

In the hippocampus, episodic memories are thought to be encoded by the formation of ensembles of synaptically coupled CA3 pyramidal cells driven by sparse but powerful mossy fiber inputs from dentate gyrus granule cells. The neuromodulators acetylcholine and noradrenaline are separately proposed as saliency signals that dictate memory encoding but it is not known if they represent distinct signals with separate mechanisms. Here, we show experimentally that acetylcholine, and to a lesser extent noradrenaline, suppress feed-forward inhibition and enhance Excitatory–Inhibitory ratio in the mossy fiber pathway but CA3 recurrent network properties are only altered by acetylcholine. We explore the implications of these findings on CA3 ensemble formation using a hierarchy of models. In reconstructions of CA3 pyramidal cells, mossy fiber pathway disinhibition facilitates postsynaptic dendritic depolarization known to be required for synaptic plasticity at CA3-CA3 recurrent synapses. We further show in a spiking neural network model of CA3 how acetylcholine-specific network alterations can drive rapid overlapping ensemble formation. Thus, through these distinct sets of mechanisms, acetylcholine and noradrenaline facilitate the formation of neuronal ensembles in CA3 that encode salient episodic memories in the hippocampus but acetylcholine selectively enhances the density of memory storage., Author summary How the brain decides which experiences to encode to memory and which to discard is a fundamental question in neuroscience. The neuromodulators acetylcholine and noradrenaline are believed to separately play a central role in determining what is encoded but the mechanisms by which they act are mostly unknown and there have been no direct comparisons made between these two critical neuromodulators. In this study, we investigate the effects of acetylcholine and noradrenaline on a key circuit responsible for the encoding of memories, namely, the dentate gyrus–CA3 microcircuit in the hippocampus. Using slice electrophysiology, we measure the effects of acetylcholine and noradrenaline on key synaptic and cellular nodes within this neuronal network. We then explore the network level implications of these findings on neuronal ensemble formation using a hierarchy of computational models. Based on the observed physiological effects of acetylcholine and noradrenaline, our models predict that acetylcholine facilitates efficient formation of ensembles within CA3 with a high degree of overlap whereas noradrenaline has more limited effects and no impact on the efficiency or overlap of ensemble formation.

BackgroundActivating the Stimulator of Interferon Genes (STING) adaptor incites antitumor immunity against immunogenic tumors in mice, prompting clinical trials to test STING activators. However, STING signaling in the tumor microenvironment (TME) during development of Lewis lung carcinoma (LLC) suppresses antitumor immunity to promote tumor growth. We hypothesized that local immune balance favoring suppression of antitumor immunity also attenuates antitumor responses following STING activation. The purpose of this study was to evaluate how STING activation impacts antitumor responses in mice bearing LLC tumors.MethodsMice bearing established LLC tumors were treated with synthetic cyclic diadenyl monophosphate (CDA) to activate STING. Mice were monitored to assess LLC tumor growth, survival and protective antitumor immunity. Transcriptional and metabolic analyses were used to identify pathways responsive to CDA, and mice were co-treated with CDA and drugs that disrupt these pathways.ResultsCDA slowed LLC tumor growth but most CDA-treated mice (77%) succumbed to tumor growth. No evidence of tumor relapse was found in surviving CDA-treated mice at experimental end points but mice were not immune to LLC challenge. CDA induced rapid increase in immune regulatory pathways involving programmed death-1 (PD-1), indoleamine 2,3 dioxygenase (IDO) and cyclooxygenase-2 (COX2) in the TME. PD-1 blockade enhanced antitumor responses to CDA and increased mouse survival but mice did not eliminate primary tumor burdens. Two IDO inhibitor drugs had little or no beneficial effects on antitumor responses to CDA. A third IDO inhibitor drug synergized with CDA to enhance tumor control and survival but mice did not eliminate primary tumor burdens. In contrast, co-treatments with CDA and the COX2-selective inhibitor celecoxib controlled tumor growth, leading to uniform survival without relapse, and mice acquired resistance to LLC re-challenge and growth of distal tumors not exposed directly to CDA. Thus, mice co-treated with CDA and celecoxib acquired stable and systemic antitumor immunity.ConclusionsSTING activation incites potent antitumor responses and boosts local immune regulation to attenuate antitumor responses. Blocking STING-responsive regulatory pathways synergizes with CDA to enhance antitumor responses, particularly COX2 inhibition. Thus, therapy-induced resistance to STING may necessitate co-treatments to disrupt regulatory pathways responsive to STING in patients with cancer.

The process of molting, known alternatively as ecdysis, is a feature integral in the life cycles of species across the arthropod phylum. Regulation occurs as a function of the interaction of ecdysteroid hormones with the arthropod nuclear ecdysone receptor—a process preceding the triggering of a series of downstream events constituting an endocrine signaling pathway highly conserved throughout environmentally prevalent insect, crustacean, and myriapod organisms. Inappropriate ecdysone receptor binding and activation forms the essential molecular initiating event within possible adverse outcome pathways relating abnormal molting to mortality in arthropods. Definition of the characteristics of chemicals liable to stimulate such activity has the potential to be of great utility in mitigation of hazards posed toward vulnerable species. Thus the aim of the present study was to develop a series of rule‐sets, derived from the key structural and physicochemical features associated with identified ecdysone receptor ligands, enabling construction of Konstanz Information Miner (KNIME) workflows permitting the flagging of compounds predisposed to binding at the site. Data describing the activities of 555 distinct chemicals were recovered from a variety of assays across 10 insect species, allowing for formulation of KNIME screens for potential binding activity at the molecular initiating event and adverse outcome level of biological organization. Environ Toxicol Chem 2020;39:1438–1450. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Summary Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [ 1 ]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins—RhoQ and RhoJ [ 2 ]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [ 3 , 4 ]. RhoJ is required for angiogenesis [ 5 , 6 ] and has multiple roles in this process [ 7 , 8 ]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [ 9 ]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5β1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5β1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.

Aim Altered organisation of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. Methods We exploited the 'formamide-detubulation' technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilisation by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. Results Following structural disorganisation from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. Conclusions These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.

Antigen-specific stem-like memory CD8+ T cells (Tscm) have a series of stem cell characteristics, including long-term survival, self-renewal, anti-apoptosis and persistent differentiation into cytotoxic T cells. The effective induction of tumor-specific CD8+ Tscm could persistently eradicate tumor in pro-tumor hostile microenvironment. This study was to investigate the role of CD40 in HPV16-specific CD8+ Tscm induction and its anti-tumor function. We found that CD40 activation accelerated vaccine-induced HPV16 E7-specific CD8+ Tscm formation. Comparing to other HPV-specific CD8+ T cells, CD8+ Tscm were found to be stronger and long-term anti-tumor function, in vivo and in vitro, even in the adoptive cellular transferring model. Furthermore, high frequencies of Tscm might prevent the HPV infection to move on to the development of cancer. And the CD40 effect on Tscm involved Wnt/β-catenin activation. Our study suggest that CD40 activation supports the generation of tumor-specific CD8+ Tscm, thus providing new insight into cancer immunotherapy.

Many high-consequence human and animal pathogens persist in wildlife reservoirs. An understanding of the dynamics of these pathogens in their reservoir hosts is crucial to inform the risk of spill-over events, yet our understanding of these dynamics is frequently insufficient. Viral persistence in a wild bat population was investigated by combining empirical data and in-silico analyses to test hypotheses on mechanisms for viral persistence. A fatal zoonotic virus, European Bat lyssavirus type 2 (EBLV-2), in Daubenton’s bats (Myotis daubentonii) was used as a model system. A total of 1839 M. daubentonii were sampled for evidence of virus exposure and excretion during a prospective nine year serial cross-sectional survey. Multivariable statistical models demonstrated age-related differences in seroprevalence, with significant variation in seropositivity over time and among roosts. An Approximate Bayesian Computation approach was used to model the infection dynamics incorporating the known host ecology. The results demonstrate that EBLV-2 is endemic in the study population, and suggest that mixing between roosts during seasonal swarming events is necessary to maintain EBLV-2 in the population. These findings contribute to understanding how bat viruses can persist despite low prevalence of infection, and why infection is constrained to certain bat species in multispecies roosts and ecosystems.

This review outlines the processes followed by New Zealand Thoroughbred Racing (NZTR) when developing its Thoroughbred Welfare Assessment Guidelines. It accepted that guidance on welfare management must be based on up-to-date knowledge of how animal welfare is understood scientifically. NZTR established an expert panel to facilitate this process. First, major changes in animal welfare science thinking over the last 40 years were considered. For example, the separate biological function and affective state orientations were later accepted as dynamically interacting elements within the body operating as an integrated whole entity; conceptual problems with the Five Freedoms framework led to the formulation of the Five Provisions and Welfare Aims paradigm and development of the Five Domains Model for assessing nutritional, environmental, health, behavioural and mental facets of animal welfare; and the initial major focus on negative experiences evolved to include both negative and positive experiences. The Five Domains Model was very effective for illustrating up-to-date understanding of animal welfare and its use demonstrated how comprehensive animal welfare assessments may be conducted. The NZTR panel followed a sequential approach that included an update on animal welfare thinking and the Five Provisions and Welfare Aims paradigm; the generic Five Domains Model was refocused specifically on equids; a detailed model assessment of equine welfare practices was conducted; enhanced equine welfare practices were emphasised by comparing them to inadequate welfare practices; guidelines were framed in terms which provide domain-specific advice on provisions that achieve positive welfare; other domain-specific guidelines were focused on welfare-compromising consequences of inadequate provisions; and welfare-appropriate conditions were clarified for all stages of a Thoroughbred's life cycle (in work and rest) to facilitate exercising a life-long duty of care. Finally, the guidelines were expressed in general terms to avoid them becoming overly detailed and unwieldy. They therefore do not address specific welfare issues such as use of whips, bits, spurs and tight nosebands, however the Five Domains Model may also be used for these specific purposes. The guidelines, and the way they were formulated, provide an example of one approach which other organisations may find immediately useful, or which may stimulate them to devise their own approaches when progressing such equine welfare initiatives.