Use of vaccines and factors associated with their uptake variability in dogs, cats and rabbits attending a large sentinel network of veterinary practices across Great Britain.

Vaccination remains a mainstay of companion animal population health. However, how vaccine use at a population level complies with existing guidelines is unknown. Here we use electronic health records to describe vaccination in dogs, cats and rabbits attending a large sentinel network of UK veterinary practices. In total, 77.9% (95% CI: 77.6-78.1) of animals had recorded vaccinations. The percentage of animals with recorded vaccinations was higher in dogs, neutered animals, in insured dogs and cats and in purebred dogs. Vaccination rates varied in different regions of Great Britain in all species. Dogs and cats belonging to owners living in less deprived areas of England and Scotland were more likely to be recorded as vaccinated. In the vaccinated population, cats received more core vaccines per year of life (0.86) than dogs (0.75), with feline leukaemia vaccines almost as frequent as core vaccines. In dogs, leptospira vaccines were more frequent than core vaccines. This descriptive study suggests a substantial proportion of animals are not benefiting from vaccine protection. For the first time, we identify potential factors associated with variations in recorded vaccination frequency, providing a critical baseline against which to monitor future changes in companion animal vaccination and evidence to inform future targeted health interventions.

A multinational survey of companion animal veterinary clinicians: How can antimicrobial stewardship guidelines be optimised for the target stakeholder?

Antimicrobial stewardship initiatives are widely regarded as a cornerstone for ameliorating the global health impact of antimicrobial resistance. Within companion animal health, such efforts have largely focused on development and dissemination of antimicrobial stewardship guidelines (ASGs). However, there have been few attempts to understand veterinarian attitudes towards and knowledge of ASGs or to determine how awareness regarding ASGs might best be increased. An online survey regarding ASGs was formulated for veterinarians who treat companion animals. The survey was distributed across 46 European and associated countries between 12 January and 30 June, 2022. In total, 2271 surveys were completed, with 64.9% of respondents (n = 1474) reporting awareness and usage of at least one ASG. Respondents from countries with greater awareness of ASGs tended to report more appropriate use of antimicrobials (Spearman's rank coefficient = 0.6084, P ≤ 0.001), with respondents from countries with country-specific ASGs tending to score highest across both awareness and appropriate use domains. Respondents prioritised guidance around antimicrobial choice (82.0%, n = 1863), duration of treatment (66.0%, n = 1499), and dosage (51.9%, n = 1179) for inclusion in future ASGs, with 78.0% (n = 1776) of respondents preferring ASGs to be integrated into their patient management system. Awareness of ASGs and their use in companion animal veterinary practice appears to be greater than previously reported, with respondents tending to report antimicrobial prescription decision making broadly in line with current clinical recommendations. However, further initiatives aimed at maximising accessibility to ASGs both within countries and individual veterinary practices are recommended.

Efficacy of antimicrobial and nutraceutical treatment for canine acute diarrhoea: A systematic review and meta-analysis for European Network for Optimization of Antimicrobial Therapy (ENOVAT) guidelines.

Systemic antimicrobial treatments are commonly prescribed to dogs with acute diarrhoea, while nutraceuticals (prebiotics, probiotics, and synbiotics) are frequently administered as an alternative treatment. The aim of this systematic review and meta-analysis was to assess the effectiveness of antimicrobials and nutraceutical preparations for treatment of canine acute diarrhoea (CAD). The results of this study will be used to create evidence-based treatment guidelines. PICOs (population, intervention, comparator, and outcome) were generated by a multidisciplinary expert panel taking into account opinions from stakeholders (general practitioners and dog owners). The Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to evaluate the certainty of the evidence. The systematic search yielded six randomised controlled trials (RCT) for antimicrobial treatment and six RCTs for nutraceutical treatment meeting the eligibility criteria. Categories of disease severity (mild, moderate, and severe) were created based on the presence of systemic signs and response to fluid therapy. Outcomes included duration of diarrhoea, duration of hospitalization, progression of disease, mortality, and adverse effects. High certainty evidence showed that antimicrobial treatment did not have a clinically relevant effect on any outcome in dogs with mild or moderate disease. Certainty of evidence was low for dogs with severe disease. Nutraceutical products did not show a clinically significant effect in shortening the duration of diarrhoea (based on very low to moderate certainty evidence). No adverse effects were reported in any of the studies.

Approach to initial management of canine generalised epileptic seizures in primary-care veterinary practices in the United Kingdom.

OBJECTIVES: To investigate how primary care clinicians in the UK approach initial management of canine generalised epileptic seizures, including factors potentially associated with prescription and choice of anti-seizure drugs. MATERIALS AND METHODS: Electronic health records concerning 3,150,713 consultations (917,373 dogs) were collected from 224 veterinary practices by the Small Animal Veterinary Surveillance Network. Free-text clinical narratives were reviewed to identify those consistent with generalised epileptic seizure activity, including only those recording the first presentation for seizures. Dogs older than 6 years were excluded. RESULTS: Five hundred and seventeen cases were included. Sixty-seven dogs (13.0%) received anti-seizure drugs at first presentation; this was significantly more likely in dogs presented with cluster seizures (odds ratio 13.8, 95% confidence interval 7.3 to 26.1). Phenobarbital (n=36) and imepitoin (n=29) were the most frequently chosen anti-seizure drugs. Presentation for a single epileptic seizure occurred in 321 dogs; seven were prescribed anti-seizure drugs. Eighty-six dogs were presented with cluster seizures; 38 were prescribed anti-seizure drugs, most frequently imepitoin (n= 19) and phenobarbital (n=17). Of the dogs presenting with a single seizure and at least 6-month follow-up (n=165), 33 (20%) did not have subsequent seizures recorded. CLINICAL SIGNIFICANCE: Primary care clinicians rarely prescribed anti-seizure drugs following a single epileptic seizure in accordance with International Veterinary Epilepsy Task Force recommendations. Less than half of dogs initially presenting with cluster seizures were prescribed anti-seizure drugs. Imepitoin was frequently selected in the treatment of cluster seizures despite no authorisation for this purpose. These findings may ultimately contribute to improved cohesion in the management of canine epileptic seizures between primary care and referral institutions.

Analysis of canine cardiovascular therapeutic agent prescriptions using electronic health records in primary care veterinary practices in the United Kingdom.

INTRODUCTION/OBJECTIVES: Canine cardiovascular (CV) diseases are often managed in primary care settings. The objectives were to describe CV therapeutic agent (CVTA) prescribing patterns in primary care practices in the United Kingdom (UK) and to evaluate recorded clinical signs, diagnostic tests and justifications for use of torasemide, a recently marketed and authorised loop-diuretic in the UK. ANIMALS, MATERIALS AND METHODS: Electronic health records (EHRs) describing 3,579,420 consultations (1,043,042 unique dogs) were collated (1 April 2014 and 31 December 2018) by the Small Animal Veterinary Surveillance Network from 270 veterinary practices. Consultations prescribing at least one CVTA were identified. Annual variation in individual CVTA prescriptions was analysed using mixed-effects binomial regression models. Free-text clinical narratives were manually read to determine the first-prescribing event for torasemide. RESULTS: Twenty-nine thousand and seven consultations (0.81% of all consultations, 95% confidence interval [CI], 0.76-0.86) prescribed CVTA in 14,148 (1.36%) dogs. Furosemide (52.8% of CV-prescribing consultations, 95% CI 50.7-54.9) and pimobendan (51.9%, 95% CI 50.1-53.7) were most prescribed. Longitudinal analysis (2014-2018) showed a significant negative temporal trend for angiotensin-converting enzyme inhibitors (p < 0.001), and furosemide (p = 0.003) and a positive temporal trend for pimobendan (p = 0.020) and torasemide (p < 0.001). First prescriptions of torasemide were identified in 16.5% of torasemide-prescribing consultations. Where justification for prescription of torasemide was identified (32.5%), furosemide resistance was the most common (92.0%). CONCLUSIONS: EHRs can be used to temporally monitor prescribing habits, including responses to market authorisations. Despite authorisation in the UK for torasemide use as a first-line diuretic, it was most commonly prescribed after furosemide resistance.

A mechanistic model of developing immunity to Teladorsagia circumcincta infection in lambs.

Acquired immunity influences the severity of parasitic disease, but modelling the effects of acquired immunity in helminth infections has proved challenging. This may be due to a lack of suitable immunological data, or to the perceived complexity of modelling the immune response. We have developed a model of T. circumcincta infection in domestic sheep that incorporates the effects of acquired immunity on parasite establishment and fecundity. A large data set from commercially managed populations of Scottish Blackface sheep was used, which included relationships between IgA activity and worm length, and between worm length and fecundity. Use was also made of a recently published meta-analysis of parasite establishment rates. This realistic but simple model of nematode infection emulates observed patterns of faecal egg counts. The end-of-season faecal egg counts are remarkably robust to perturbations in the majority of the parameters, possibly because of priming of the immune system early in the season, reducing parasite establishment and growth and, therefore, faecal egg counts. Lowering the amount of early infection leads to higher end-of-season egg counts. The periparturient rise in egg counts in ewes appears to have an important role in supplying infection for the priming of the immune response. This feedback in the immune priming suggests that nematode infections may be difficult to eliminate.

An evolutionary perspective on gastrointestinal nematodes of sheep.

The purpose of this paper was to discuss from an evolutionary perspective the interaction between domestic sheep (Ovis aries) and their gastrointestinal nematodes. Although evolution is the central theme of biology, there has been little attempt to consider how evolutionary forces have shaped and continue to shape the relationships between domestic animals and their parasite community. Mathematical modelling of the host-parasite relationship indicated that the system is remarkably robust to perturbations in its parameters. This robustness may be a consequence of the long coevolution of host and parasites. Although nematodes can potentially evolve faster than the host, coevolution is not dominated by the parasite and there are several examples where breeds of cattle or sheep have evolved high levels of resistance to disease. Coevolution is a more equal partnership between host and nematode than is commonly assumed. Coevolution between parasites and the host immune system is often described as an arms race where both host immune response genes and parasite proteins evolve rapidly in response to each other. However, initial results indicate that nematode antigens are not evolving rapidly; the arms race between the immune system and nematodes, if it exists, is happening very slowly. Fisher's fundamental theorem of natural selection states that genes with positive effects on fitness will be fixed by natural selection. Consequently, heritable variation in fitness traits is expected to be low. Contrary to this argument, there is considerable genetic variation in resistance to nematode infection. In particular, the heritabilities of nematode-specific IgA and IgE activity are moderate to high. The reasons for this apparent violation of the fundamental theorem of natural selection are not clear but several possible explanations are explored. Faecal nematode egg counts increase at the beginning of the grazing season - a phenomenon known as the periparturient rise. This increase benefits host and parasite and appears to be a consequence of coevolution. In conclusion, an evolutionary perspective can shed light on many aspects of the host-parasite relationship in domestic animals.

Human monocytes can produce tissue-type plasminogen activator.

Evidence has previously been presented that monocytes and macrophages produce urokinase-type plasminogen activator. We have shown for the first time that human monocytes, when stimulated appropriately in vitro, can produce tissue type-plasminogen activator (t-PA) of 70 kD. Detection of t-PA mRNA was consistent with the biochemical and immunological characterization of t-PA produced by human monocytes.

Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants.

To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transport-defective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A)+ RNA at 37 degrees C. At 37 degrees C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splicing mutants, these strains do not interrupt splicing of pre-mRNA at 37 degrees C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins in the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A)+ RNA in characteristic patterns in the nucleus.

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins.

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

New approaches to pharmacosurveillance for monitoring prescription frequency, diversity, and co-prescription in a large sentinel network of companion animal veterinary practices in the United Kingdom, 2014-2016.

Pharmaceutical agents (PAs) are commonly prescribed in companion animal practice in the United Kingdom. However, little is known about PA prescription on a population-level, particularly with respect to PAs authorised for human use alone prescribed via the veterinary cascade; this raises important questions regarding the efficacy and safety of PAs prescribed to companion animals. This study explored new approaches for describing PA prescription, diversity and co-prescription in dogs, cats and rabbits utilising electronic health records (EHRs) from a sentinel network of 457 companion animal-treating veterinary sites throughout the UK over a 2-year period (2014-2016). A novel text mining-based identification and classification methodology was utilised to semi-automatically map practitioner-defined product descriptions recorded in 918,333 EHRs from 413,870 dogs encompassing 1,242,270 prescriptions; 352,730 EHRs from 200,541 cats encompassing 491,554 prescriptions, and 22,526 EHRS from 13,398 rabbits encompassing 18,490 prescriptions respectively. PA prescription as a percentage of booked consultations was 65.4% (95% confidence interval, CI, 64.6-66.3) in dogs; in cats it was 69.1% (95% CI, 67.9-70.2) and in rabbits, 56.3% (95% CI, 54.7-57.8). Vaccines were the most commonly prescribed PAs in all three species, with antibiotics, antimycotics, and parasiticides also commonly prescribed. PA prescription utilising products authorised for human use only (hence, 'human-authorised') comprised 5.1% (95% CI, 4.7-5.5) of total canine prescription events; in cats it was 2.8% (95% CI, 2.6-3.0), and in rabbits, 7.8% (95% CI, 6.5-9.0). The most commonly prescribed human-authorised PA in dogs was metronidazole (antibiotic); in cats and rabbits it was ranitidine (H2 histamine receptor antagonist). Using a new approach utilising the Simpson's Diversity Index (an ecological measure of relative animal, plant etc. species abundance), we identified differences in prescription based on presenting complaint and species, with rabbits generally exposed to a less diverse range of PAs than dogs or cats, potentially reflecting the paucity of authorised PAs for use in rabbits. Finally, through a novel application of network analysis, we demonstrated the existence of three major co-prescription groups (preventive health; treatment of disease, and euthanasia); a trend commonly observed in practice. This study represents the first time PA prescription has been described across all pharmaceutical families in a large population of companion animals, encompassing PAs authorised for both veterinary and human-only use. These data form a baseline against which future studies could be compared, and provides some useful tools for understanding PA comparative efficacy and risks when prescribed in the varied setting of clinical practice.

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.

The secretory carrier membrane protein family: structure and membrane topology.

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

Patterns of antimicrobial agent prescription in a sentinel population of canine and feline veterinary practices in the United Kingdom.

Antimicrobial resistance is an increasingly important global health threat and the use of antimicrobial agents is a key risk factor in its development. This study describes antimicrobial agent prescription (AAP) patterns over a 2year period using electronic health records (EHRs) from booked consultations in a network of 457 sentinel veterinary premises in the United Kingdom. A semi-automated classification methodology was used to map practitioner defined product codes in 918,333 EHRs from 413,870 dogs and 352,730 EHRs from 200,541 cats, including 289,789 AAPs. AAP as a proportion of total booked consultations was more frequent in dogs (18.8%, 95% confidence interval, CI, 18.2-19.4) than cats (17.5%, 95% CI 16.9-18.1). Prescription of topical antimicrobial agents was more frequent in dogs (7.4%, 95% CI 7.2-7.7) than cats (3.2%, 95% CI 3.1-3.3), whilst prescription of systemic antimicrobial agents was more frequent in cats (14.8%, 95% CI 14.2-15.4) than dogs (12.2%, 95% CI 11.7-12.7). A decreasing temporal pattern was identified for prescription of systemic antimicrobial agents in dogs and cats. Premises which prescribed antimicrobial agents frequently for dogs also prescribed frequently for cats. AAP was most frequent during pruritus consultations in dogs and trauma consultations in cats. Clavulanic acid potentiated amoxicillin was the most frequently prescribed antimicrobial agent in dogs (28.6% of prescriptions, 95% CI 27.4-29.8), whereas cefovecin, a third generation cephalosporin, was the most frequently prescribed antimicrobial agent in cats (36.2%, 95% CI 33.9-38.5). This study demonstrated patterns in AAP over time and for different conditions in a population of companion animals in the United Kingdom.

Canine babesiosis and tick activity monitored using companion animal electronic health records in the UK.

Recent publications highlighting autochthonous Babesia canis infection in dogs from Essex that have not travelled outside the UK are a powerful reminder of the potential for pathogen emergence in new populations. Here the authors use electronic health data collected from two diagnostic laboratories and a network of 392 veterinary premises to describe canine Babesia cases and levels of Babesia concern from January 2015 to March 2016, and the activity of ticks during December 2015-March 2016. In most areas of the UK, Babesia diagnosis in this population was rare and sporadic. In addition, there was a clear focus of Babesia cases in the affected area in Essex. Until February 2016, analysis of health records indicated only sporadic interest in Babesia largely in animals coming from overseas. Following media coverage in March 2016, there was a spike in owner concern that was geographically dispersed beyond the at-risk area. Tick activity (identified as ticks being removed from animals in veterinary consultations) was consistent but low during the period preceding the infections (<5 ticks/10,000 consultations), but increased in March. This highlights the use of electronic health data to describe rapidly evolving risk and concern that follows the emergence of a pathogen.

Recombinant human interleukin-1 stimulates human articular cartilage to undergo resorption and human chondrocytes to produce both tissue- and urokinase-type plasminogen activator.

Cytokines capable of stimulating cartilage resorption have frequently been identified as 'interleukin-1 (IL-1)-like' peptides. In this study for the first time we have employed homogeneous recombinant IL-1 alpha and IL-1 beta in an all-human culture system to define the effects of IL-1 on articular cartilage and chondrocytes in culture. Recombinant IL-1 (10-100 U/ml) could stimulate cartilage resorption, although the maximum degree of tissue breakdown rarely reached the levels obtained when cartilage was treated with crude mononuclear-cell conditioned medium or all-trans retinoic acid (1 microM) over a similar time course. Levels of plasminogen activator (PA) activity, a neutral proteinase which may contribute to cartilage destruction in arthritis, increased markedly in the cartilage/chondrocyte culture supernatants and in the chondrocyte cell layers in response to the stimulation of cultures with recombinant IL-1 (1-100 U/ml). Elevated levels of PA activity were detectable after 4-8 h stimulation of the chondrocytes with IL-1 while characterization of the PA activities indicated that both types of PA activity were expressed, viz. urokinase-type PA (u-PA) and tissue-type PA (t-PA). Both IL-1 alpha and IL-1 beta could elicit these responses and their effects were comparable for a given dose. These studies show definitively that pure IL-1, free from contaminating cytokines, is capable of inducing human cartilage resorption and stimulating the expression of two types of PA activity by chondrocytes. In contrast to IL-1, retinoic acid increased the detectable levels of only u-PA in the chondrocyte cell layers. Chondrocyte u-PA may have an important role in cartilage degradative processes since it is one of the few neutral proteinases now known to be increased in activity in retinoid-stimulated cartilage.

Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis.

Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.

Novel, potent aldose reductase inhibitors: 3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl] methyl]-1-phthalazineacetic acid (zopolrestat) and congeners.

A new working hypothesis that there is a hitherto unrecognized binding site on the aldose reductase (AR) enzyme with strong affinity for benzothiazoles was pursued for the design of novel, potent aldose reductase inhibitors (ARIs). The first application of this hypothesis led to a novel series of 3,4-dihydro-4-oxo-3-(benzothiazolylmethyl)-1-phthalazineacetic+ + + acids. The parent of this series (207) was a potent inhibitor of AR from human placenta (IC50 = 1.9 x 10(-8) M) and was orally active in preventing sorbitol accumulation in rat sciatic nerve, in an acute test of diabetic complications (ED50 = 18.5 mg/kg). Optimization of this lead through medicinal chemical rationale, including analogy from other drug series, led to more potent congeners of 207 and culminated in the design of 3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl] methyl]-1-phthalazineacetic acid (216, CP-73,850, zopolrestat). Zopolrestat was found to be more potent than 207, both in vitro and in vivo. Its IC50 against AR and ED50 in the acute test were 3.1 x 10(-9)M and 3.6 mg/kg, respectively. Its ED50s in reversing already elevated sorbitol accumulation in rat sciatic nerve, retina, and lens in a chronic test were 1.9, 17.6, and 18.4 mg/kg, respectively. It was well absorbed in diabetic patients, resulting in high blood level, showed a highly favorable plasma half-life (27.5 h), and is undergoing further clinical evaluation. An assortment of synthetic methods used for the construction of benzothiazoles, including an efficient synthesis of zopolrestat, is described. Structure-activity relationships in the new series are discussed.

Quantitative structure-activity relationships of antitumor guanidinothiazolecarboxamides with survival enhancement for therapy in the 3LL Lewis lung carcinoma model.

Guanidinothiazolecarboxamides (GTCs) are a novel class of antitumor agents found to be systemically active against experimental pulmonary metastases of 3LL Lewis lung carcinoma. A series of substituted benzothiazole GTCs were found to produce enhancement of survival in this model by using 8 days of intraperitoneal dosing initiated 2 days after intravenous tumor challenge. Quantitative structure-activity relationships have been discovered in the GTC series with survival enhancement correlated to substituent parameters. Optimal correlations were found between the probit transform of the drug-induced increased lifespan (ILS) and field and pi parameters. Among the most effective analogues in this series was N-(5-fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxam ide.

13C and 2H kinetic isotope effects and the mechanism of bromination of 1-pentene under synthetic conditions.

[formula: see text] The 13C and 2H kinetic isotope effects for the bromination of 1-pentene with Br2 in CCl4 were determined and interpreted with the aid of calculationally predicted isotope effects. The isotope effects observed are consistent with rate-limiting bromonium ion formation and do not fit with either rate-limiting production of a pi complex or reaction of a reversibly formed bromonium ion. This rules out some of the mechanistic complexities suggested for other brominations, though the identity of the brominating reagent(s) under these synthetic conditions remains uncertain.