Development, initial validation and reliability testing of a web-based, generic feline health-related quality of life instrument

Objectives: The objective of this study was to develop a valid, reliable web-based, generic feline health-related quality-of-life (HRQoL) questionnaire instrument to measure the affective impact of chronic disease. Methods: A large initial item pool, obtained through interviews with cat owners, was reduced using predetermined criteria, survey scores for relevance and clarity, and the ability of individual items to discriminate between healthy and sick cats when owners completed a prototype questionnaire. Using these data, factor analysis was used to derive a scoring algorithm and provide evidence for factorial validity. Validity was demonstrated further in a field trial using a ‘known groups’ approach (sick vs healthy cats will have a different HRQoL profile, and the HRQoL profile of cats will deteriorate as comorbidities increase). Test–retest reliability was assessed using intra-class correlation coefficients (ICC). Results: In total, 165 items were reduced to 20 and, on the basis of a factor analysis that explained 72.3% of the variation in scores input by 71 owners of 30 healthy and 41 sick cats using the prototype, these were allocated to three domains (vitality, comfort and emotional wellbeing [EWB]) with a scoring algorithm derived using item loadings. Subsequently, the owners of 36 healthy and 58 sick cats completed one or two (n = 48) assessments. Median scores (healthy vs sick) for all domains were significantly different (P <0.001), 78% of cats were correctly classified as healthy or sick and for comorbidities the correlation coefficients were moderate (vitality 0.64; comfort 0.63; EWB 0.50). Test–retest reliability was good (ICC vitality 0.635; comfort 0.716; EWB 0.853). Conclusions and relevance: This study provides initial evidence for the validity and reliability of a novel HRQoL instrument to aid the assessment and management of chronic diseases of cats

Unravelling the specific site preference in doping of calcium hydroxyapatite with strontium from ab initio investigations and Rietveld analyses

Strontium can be substituted into the calcium sublattice of hydroxyapatite without a solubility limit. However, recent ab initio simulations carried out at 0 K report endothermic nature of this process. There is also striking discrepancy between experimentally observed preference of Sr doping at Ca-II sites and the first principles calculations, which indicate that a Ca-I site is preferred energetically for the Sr substitution. In this paper we combine insights from Density Functional Theory simulations and regular configurational entropy calculations to determine the site preference of Sr doping in the range of 0-100 at% at finite temperatures. In addition, samples of Sr-HA are synthesized and refinement of the relevant structural information provides benchmark information on the experimental unit cell parameters of Sr-HA. We find that the contribution of the entropy of mixing can efficiently overcome the endothermic excess energy at a temperature typical of the calcining step in the synthesis route of hydroxyapatite (700-950 degrees C). We observe that the most preferential substitution pattern is mixed substitution of Sr regardless of the concentration. For a wet chemical method, carried out at a moderate temperature (90 degrees C), the mixed doping is still slightly favourable at higher Sr-concentrations, except the range at 20% Sr, where Site II substitution is not restricted energetically and equally possible as the mixed doping. We observe a close correspondence between our theoretical results and available experimental data. Hence it should be possible to apply this theory to other divalent dopants in HA, such as Zn2+, Mg2+, Pb2+, Cu2+, Ba2+, Cd2+ etc

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits

The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5–41 × higher rates of editing than homology-directed repair or prime editing

Co-induction of cyclooxyenase-2 and early growth response gene (Egr-1) in spinal cord in a clinical model of persistent inflammation and hyperalgesia

BackgroundThis study characterised the effects of persistent peripheral inflammation of the foot on pain and spinal cord expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) and early growth response gene 1 (Egr-1), known markers of neuronal plasticity, in a clinical model of naturally-occurring inflammatory disease and hyperalgesia in sheep ('footrot'), before and after routine treatment (parenteral treatment with antibiotics and antiseptic footbathing). The temporal pattern of expression of COX-1, COX-2 and Egr-1 mRNA and protein were analysed using real-time PCR and Western blotting. ResultsAnimals affected with persistent peripheral inflammation displayed significant hyperalgesia and lameness (a proxy indicator of spontaneous pain) restricted to the inflamed limb. Hyperalgesia and lameness were significantly attenuated 1 day after treatment, and resolved further by day 7 and day 3, respectively. COX-2 but not COX-1, protein expression was up-regulated in spinal cord from lame animals on day 0, before treatment. Following treatment and attenuation of pain behaviours, levels of COX-2 returned to control levels. Significant induction of Egr-1 mRNA and protein were observed in spinal cord from lame animals. Three days after treatment, levels of Egr-1 mRNA returned to control levels, however, Egr-1 protein remained elevated. ConclusionElevated levels of spinal COX-2 and Egr-1 protein correlate with the presence of pain and hyperalgesia, and may underlie persistent pain, although a direct causal link has still to be established. Understanding the temporal pattern of expression of key mediators in clinical pain states may lead to better strategies to manage pai

Evaluation of facial expression in acute pain in cats

OBJECTIVESTo describe the development of a facial expression tool differentiating pain-free cats from those in acute pain.METHODSObservers shown facial images from painful and pain-free cats were asked to identify if they were in pain or not. From facial images, anatomical landmarks were identified and distances between these were mapped. Selected distances underwent statistical analysis to identify features discriminating pain-free and painful cats. Additionally, thumbnail photographs were reviewed by two experts to identify discriminating facial features between the groups.RESULTSObservers (n?=?68) had difficulty in identifying pain-free from painful cats, with only 13% of observers being able to discriminate more than 80% of painful cats. Analysis of 78 facial landmarks and 80 distances identified six significant factors differentiating pain-free and painful faces including ear position and areas around the mouth/muzzle. Standardised mouth and ear distances when combined showed excellent discrimination properties, correctly differentiating pain-free and painful cats in 98% of cases. Expert review supported these findings and a cartoon-type picture scale was developed from thumbnail images.CLINICAL SIGNIFICANCEInitial investigation into facial features of painful and pain-free cats suggests potentially good discrimination properties of facial images. Further testing is required for development of a clinical too

Leukotoxin Diols from Ground Corncob Bedding Disrupt Estrous Cyclicity in Rats and Stimulate MCF-7 Breast Cancer Cell Proliferation

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (isoleukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [(3)H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents

Development of a behaviour-based measurement tool with defined intervention level for assessing acute pain in cats

OBJECTIVES: To develop a Composite Measure Pain Scale - Feline (CMPS-F) tool to assess acute pain in cats and derive an intervention score. METHODS: To develop the prototype CMPS-F, words describing painful cats were collected, grouped into behavioural categories and ranked. To assess prototype validity two observers independentlyassigned CMPS-F and numerical rating scale (NRS) scores to 25 hospitalised cats before and afteranalgesic treatment. Following interim analysis the prototype was revised (rCMPS-F). To determine intervention score two observers independently assigned rCMPS-F and NRS scores to 116 cats. Afurther observer, a veterinarian, stated whether analgesia was necessary. Statistical tests includedWilcoxon, Mann-Whitney, 95% confidence intervals (CI), general linear model ANOVA and linear discriminant analysis (p < 0.05).RESULTS: Mean ± SD decrease in rCMPS-F and NRS scores following analgesia were 2.4 ± 2.87 and 1.9 ± 2.34, respectively (95% CI for mean change in rCMPS-F between 1.21 and 3.6). Changesin rCMPS-F and NRS were significantly correlated (r = 0.8) (

V3 Loop Truncations in HIV-1 Envelope Impart Resistance to Coreceptor Inhibitors and Enhanced Sensitivity to Neutralizing Antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria