Amyloid formation and depolymerization of tumor suppressor p16INK4a are regulated by a thiol-dependent redox mechanism.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Characterizing the amyloid core region of the tumor suppressor protein p16INK4a using a limited proteolysis and peptide-based approach.

The human tumor suppressor p16INK4a is a small monomeric protein that can form amyloid structures. Formation of p16INK4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all α-helical structure into ß-sheet fibrils. Currently, structural insights into p16INK4a amyloid fibrils are lacking. Here, we investigate the amyloid-forming regions of this tumor suppressor using isotope-labeling limited-digestion mass spectrometry analysis. We discover two key regions that likely form the structured core of the amyloid. Further investigations using thioflavin-T fluorescence assays, electron microscopy, and solution nuclear magnetic resonance spectroscopy of shorter peptide regions confirm the self-assembly of the identified sequences that include methionine and leucine repeat regions. This work describes a simple approach for studying protein motifs involved in the conversion of monomeric species into aggregated fibril structures. It provides insight into the polypeptide sequence underlying the core structure of amyloid p16INK4a formed after a unique oxidation-driven structural transition.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

2022 ISFM/AAFP Cat Friendly Veterinary Environment Guidelines.

PRACTICAL RELEVANCE: The '2022 ISFM/AAFP Cat Friendly Veterinary Environment Guidelines' (hereafter the 'Cat Friendly Veterinary Environment Guidelines') describe how the veterinary clinic environment can be manipulated to minimise feline patient distress. Many components of a veterinary clinic visit or stay may result in negative experiences for cats. However, much can be done to improve a cat's experience by making the veterinary clinic more cat friendly. Exposure to other cats and other species can be reduced, and adjustments made with consideration of the feline senses and species-specific behaviour. Caregivers can prepare cats for a clinic visit with appropriate advice. Waiting rooms, examination rooms, hospital wards and other clinic areas can be designed and altered to reduce stress and hence encourage positive emotions. Changes need not be structural or expensive in order to be effective and make a difference to the cats and, in turn, to cat caregivers and the veterinary team. Moreover, by improving the all-round experience at the veterinary clinic, there are positive effects on preventive healthcare, identification of and recovery from illness, and compliance with treatment. CLINICAL CHALLENGES: Good feline healthcare necessitates visiting the veterinary clinic, which, simply by being outside of a cat's territory and familiar surroundings, may lead to negative experiences. Such experiences can trigger negative (protective) emotions and associated physiological stress, which can result in misleading clinical findings, patient distress, prolonged recovery from illness, further difficulties with handling at subsequent visits and potential veterinary personnel injury. There may be a mistaken belief that veterinary clinics must undergo significant renovation or building work to become cat friendly, and that, if species cannot be separated, then clinics cannot improve their care of cats. These Guidelines aim to dispel any such misconceptions and provide detailed practical advice. EVIDENCE BASE: These Guidelines have been created by a Task Force of experts convened by the International Society of Feline Medicine and American Association of Feline Practitioners, based on an extensive literature review and, where evidence is lacking, the authors' experience. Endorsements: These Guidelines have been endorsed by a number of groups and organisations, as detailed on page 1161 and at icatcare.org/cat-friendly-guidelines and catvets.com/environment.

2022 AAFP/ISFM Cat Friendly Veterinary Interaction Guidelines: Approach and Handling Techniques.

PRACTICAL RELEVANCE: The '2022 AAFP/ISFM Cat Friendly Veterinary Interaction Guidelines: Approach and Handling Techniques' (hereafter the 'Cat Friendly Veterinary Interaction Guidelines') support veterinary professionals with feline interactions and handling to reduce the impact of fear and other protective (negative) emotions, in so doing enhancing feline welfare and In implementing these Guidelines, team satisfaction and cat caregiver confidence in the veterinary team will increase as the result of efficient examinations, better experience, more reliable diagnostic testing and improved feline wellbeing. Veterinary professionals will learn the importance of understanding and appropriately responding to the current emotional state of the cat and tailoring each visit to the individual. CLINICAL CHALLENGES: Cats have evolved with emotions and behaviors that are necessary for their survival as both a predator and prey species. A clinical setting and the required examinations and procedures to meet their physical health needs can result in behavioral responses to protective emotions. Cat friendly interactions require understanding, interpreting and appropriately responding to cats' emotional states and giving them a perceived sense of control while performing the required assessment. EVIDENCE BASE: These Guidelines have been created by a Task Force of experts convened by the American Association of Feline Practitioners and the International Society of Feline Medicine, based on an extensive literature review and, where evidence is lacking, the authors' experience. ENDORSEMENTS: These Guidelines have been endorsed by a number of groups and organizations, as detailed on page 1127 and at catvets.com/interactions and icatcare.org/cat-friendly-guidelines.

Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

Formation of a U(VI)-Persulfide Complex during Environmentally Relevant Sulfidation of Iron (Oxyhydr)oxides.

Uranium is a risk-driving radionuclide in both radioactive waste disposal and contaminated land scenarios. In these environments, a range of biogeochemical processes can occur, including sulfate reduction, which can induce sulfidation of iron (oxyhydr)oxide mineral phases. During sulfidation, labile U(VI) is known to reduce to relatively immobile U(IV); however, the detailed mechanisms of the changes in U speciation during these biogeochemical reactions are poorly constrained. Here, we performed highly controlled sulfidation experiments at pH 7 and pH 9.5 on U(VI) adsorbed to ferrihydrite and investigated the system using geochemical analyses, X-ray absorption spectroscopy (XAS), and computational modeling. Analysis of the XAS data indicated the formation of a novel, transient U(VI)-persulfide complex as an intermediate species during the sulfidation reaction, concomitant with the transient release of uranium to the solution. Extended X-ray absorption fine structure (EXAFS) modeling showed that a persulfide ligand was coordinated in the equatorial plane of the uranyl moiety, and formation of this species was supported by computational modeling. The final speciation of U was nanoparticulate U(IV) uraninite, and this phase was evident at 2 days at pH 7 and 1 year at pH 9.5. Our identification of a new, labile U(VI)-persulfide species under environmentally relevant conditions may have implications for U mobility in sulfidic environments pertinent to radioactive waste disposal and contaminated land scenarios.

Common feline problem behaviours: Unacceptable indoor elimination

PRACTICAL RELEVANCE: One of the reasons why cats enjoy such a high level of popularity as domestic pets is the fact that they are clean. When there is a breakdown in this fastidious behaviour and elimination occurs outside of the litter box or tray, the strain on the cat-owner bond and on human relationships within the household can be considerable. EVIDENCE BASE: Indoor elimination behaviour is one of the most common reasons for cat owners to seek professional advice and there is a wide range of articles, book chapters and research papers that reference it. In many cases the topic of urination and defecation in unacceptable indoor locations is considered in combination with the deposition of urine or faeces as a marker, but this review focuses purely on problematic elimination. An accompanying article in this special issue discusses urine spraying. CLINICAL CHALLENGES: Indoor elimination problems necessitate a clinical approach combining knowledge from the fields of physical and emotional health and an understanding of normal feline behaviour. They also require comprehensive history-taking skills as well as effective communication skills and a degree of empathy for owners who are often finding their pet's behaviour very distressing. GLOBAL IMPORTANCE: Early diagnosis of physical health disorders that are associated with indoor elimination is extremely important in terms of safeguarding feline welfare. As we become more aware of the interplay between physical and emotional health, the significance of identifying suboptimal social and physical environments in terms of optimising the welfare of domestic cats is also being recognised. The potential for indoor elimination problems to cause considerable human distress highlights the importance of the concepts of One Health and One Welfare.

Feline hyperaesthesia syndrome with self-trauma to the tail: retrospective study of seven cases and proposal for an integrated multidisciplinary diagnostic approach.

CASE SERIES SUMMARY: This was a retrospective study on the clinical features and response to treatment in seven cats with feline hyperaesthesia syndrome (FHS) and tail mutilation. FHS is a poorly understood disorder characterised by skin rippling over the dorsal lumbar area, episodes of jumping and running, excessive vocalisation, and tail chasing and self-trauma. The majority of the cats were young, with a median age of 1 year at the onset of clinical signs, male (n = 6) and with access to the outdoors (n = 5). Multiple daily episodes of tail chasing and self-trauma were reported in five cats, with tail mutilation in four cats. Vocalisation during the episodes (n = 5) and rippling of lumbar skin (n = 5) were also reported. Haematology, serum biochemistry, Toxoplasma gondii and feline immunodeficiency virus/feline leukaemia virus serology, MRI scans of brain, spinal cord and cauda equina, cerebrospinal fluid analysis and electrodiagnostic tests did not reveal any clinically significant abnormalities. A definitive final diagnosis was not reached in any of the cats, but hypersensitivity dermatitis was suspected in two cases. A variety of medications was used alone or in combination, including gabapentin (n = 6), meloxicam (n = 4), antibiotics (n = 4), phenobarbital (n = 2), prednisolone (n = 2) and topiramate (n = 2); ciclosporin, clomipramine, fluoxetine, amitriptyline and tramadol were used in one cat each. Clinical improvement was achieved in six cases; in five cats complete remission of clinical signs was achieved with gabapentin alone (n = 2), a combination of gabapentin/ciclosporin/amitriptyline (n = 1), gabapentin/prednisolone/phenobarbital (n = 1) or gabapentin/topiramate/meloxicam (n = 1). RELEVANCE AND NOVEL INFORMATION: This is the first retrospective study on a series of cats with FHS. The diagnostic work-up did not reveal any significant abnormalities of the central or peripheral nervous system; dermatological and behavioural problems could not be ruled out. We propose an integrated multidisciplinary diagnostic pathway to be used for the management of clinical cases and for future prospective studies.

Understanding feline emotions: and their role in problem behaviours.

Practical relevance: Despite its importance, emotional health is a subject that is sadly neglected in the context of companion animals. Understanding emotions is at the heart of veterinary behavioural medicine and is key to preventing, managing and treating reported behavioural problems in domestic cats. Clinical challenges: On a daily basis, veterinary practices are presented with the physical health impact of emotional health and with emotionally motivated behaviours that are undesirable to owners and/or detrimental to the cat. Emotional health is of equal importance to physical health and lies at the very core of veterinary medicine. Clinically, the emotional motivation for a behaviour must be identified before an assessment is made of whether the motivation is contextually appropriate and whether the cat's response is justified and normal, or abnormal in the circumstances. Evidence base: The majority of referenced evidence for our understanding of emotional motivations in mammals has come from the human field, but recently there has been increasing interest in the emotional health of non-human animals and a resulting growth in research. This review draws on the published literature and the author's personal experience to explore how emotions can influence feline behaviours. Global importance: Understanding the importance of emotional health is a major factor in ensuring positive welfare for cats, wherever they are kept as companion animals. It impacts on their physical health and their quality of life, and also on the relationship between cat and owner.

AAFP and ISFM Guidelines for diagnosing and solving house-soiling behavior in cats.

RATIONALE: These Guidelines have been developed by the American Association of Feline Practitioners (AAFP) and the International Society of Feline Medicine (ISFM) as a resource for veterinary practitioners who want to better understand and manage the important clinical condition of house-soiling in their feline patients. The Guidelines offer straightforward, practical solutions that, in most cases, will help veterinarians and cat owners prevent, manage or entirely remediate feline house-soiling behavior. EVIDENCE BASE: The Guidelines include scientifically documented information when it is available. However, because research is often lacking, some recommendations reflect the accumulated clinical experience of the authors.

AAFP and ISFM feline environmental needs guidelines.

GUIDELINES RATIONALE: A cat's level of comfort with its environment is intrinsically linked to its physical health, emotional wellbeing and behavior. Having a basic understanding of the cat's species-specific environmental needs and how cats interact with their environment will provide a foundation for addressing these fundamental requirements. ENVIRONMENTAL NEEDS: Addressing environmental needs is essential (not optional) for optimum wellbeing of the cat. Environmental needs include those relating not only to the cat's physical surroundings (indoors or outdoors; in the home environment or at the veterinary practice) but also those affecting social interaction, including responses to human contact. FIVE 'PILLARS' FRAMEWORK: The authorship panel has organized the Guidelines around five primary concepts ('pillars') that provide the framework for a healthy feline environment. Understanding these principles and the unique environmental needs of the cat will help veterinarians, cat owners and care-givers to reduce stress, the incidence of stress-related disorders, and unwanted behavior in their feline patients and pets. The recommendations in the Guidelines apply to all pet cats, regardless of lifestyle.

The effect of EDTA on the groundwater transport of thorium through sand.

The effect of the anthropogenic complexing agent EDTA on thorium transport in groundwater has been studied using sand-packed columns and flow rates in the range of 20-100 m y⁻¹. The concentrations injected into the columns were in the range of 0.4-4 mM for Th and 4-40 mM for EDTA, and with EDTA:Th ratios in the range 1:1 to 10:1. The results show that EDTA can significantly increase Th transport, but two very different behaviours are observed at Th concentrations of 0.4 and 4 mM. At the lower concentration, Th breakthrough is retarded with respect to a conservative tracer, with a peak width that is consistent with a single K(d) value, followed by a longer tail, and the behaviour is very sensitive to the flow rate. However at 4 mM Th, the breakthrough peak appears near to that of the tracer, and the width of the peak is consistent with a distribution of K(d) values and/or a larger dispersivity than the tracer. Speciation and transport modelling have been used to interpret the data, and a model was developed that could explain the 0.4 mM behaviour. This suggests that ternary surface complexes are important in these systems, with at least two different species involved, although the complexity of Th speciation in these systems leads to significant uncertainty in the values of the equilibrium and kinetic parameters. For the 4 mM systems, the rapid transport observed could not be explained by a simple chemical model; instead it is likely that EDTA plays an important role in stabilising and transporting thorium colloids and clusters.

AAFP and ISFM feline-friendly handling guidelines.

BACKGROUND: The number of pet cats is increasing in most countries, often outnumbering pet dogs, yet cats receive less veterinary care than their canine counterparts.(1) Clients state the difficulty of getting the cat into a carrier at home, driving to the clinic, and dealing with the fearful cat at the veterinary clinic as reasons for fewer visits.(2) Educating and preparing the client and the veterinary team with regard to respectful feline handling is necessary in order to avoid stress and accomplish the goal of good health care. Without such preparation, feline stress may escalate into fear or fear-associated aggression. The resulting stress may alter results of the physical examination and laboratory tests, leading to incorrect diagnoses (eg, diabetes mellitus) and unnecessary treatments.(3-5) Without compassionate and respectful handling by the veterinary team, clients may feel the team lacks skills and compassion, or does not understand cats. Injury may occur to the cat, client and/or veterinary team.(6) Clients who want to avoid stress for their cat may avoid veterinary visits or choose another practice instead. GOALS: The use of feline-friendly handling techniques should reduce these problems. Handling is most successful when the veterinary team adapts the approach to each individual cat and situation. The goal of these guidelines is to provide useful information for handling cats that can lead to: ✜ Reduced fear and pain for the cat. ✜ Reinforced veterinarian-client-cat bond, trust and confidence, and thus better lifelong medical care for the cat. ✜ Improved efficiency, productivity and job satisfaction for the veterinary team. ✜ Increased client compliance. ✜ Timely reporting and early detection of medical and behavioral concerns. ✜ Fewer injuries to clients and the veterinary team. ✜ Reduced anxiety for the client.

Feline orofacial pain syndrome (FOPS): a retrospective study of 113 cases.

Feline orofacial pain syndrome (FOPS) is a pain disorder of cats with behavioural signs of oral discomfort and tongue mutilation. This report describes the findings from a case series of 113 cats including 100 Burmese. FOPS is suspected to be a neuropathic pain disorder and the predominance within the Burmese cat breed suggests an inherited disorder, possibly involving central and/or ganglion processing of sensory trigeminal information. The disease is characterised by an episodic, typically unilateral, discomfort with pain-free intervals. The discomfort is triggered, in many cases, by mouth movements. The disease is often recurrent and with time may become unremitting - 12% of cases in this series were euthanased as a consequence of the condition. Sensitisation of trigeminal nerve endings as a consequence of oral disease or tooth eruption appears to be an important factor in the aetiology - 63% of cases had a history of oral lesions and at least 16% experienced their first sign of discomfort during eruption of permanent teeth. External factors can also influence the disease as FOPS events could be directly linked to a situation causing anxiety in 20% of cats. FOPS can be resistant to traditional analgesics and in some cases successful management required anti-convulsants with an analgesic effect.

Novel dietary strategies can improve the outcome of weight loss programmes in obese client-owned cats.

A randomised, single-blinded, positively controlled, field trial for weight loss in obese client-owned cats was undertaken to look at novel diets and dietary strategies that could improve owner compliance and, therefore, success of feline weight loss programmes. Three dietary strategies were evaluated: strategy A used a novel dry high fibre ration; strategy B used ready-prepared portions of dry and moist food; strategy C used an existing commercial dry high fibre ration fed with a measuring cup. Cats were assessed at weeks 4, 12 and 20, and adjustments to the energy allocation made if required. Mean weight loss at 20 weeks (A: 11.0+/-1.3%, B: 10.9+/-1.2%, C: 11.9+/-1.7%) and mean energy allocation (A: 31.0, B: 28.5 and C: 32.2 kcal/kg of target body weight/day) were similar amongst strategies. However, owners' subjective hunger score was significantly (P<0.001) higher with strategy C than with strategies A and B. Further, owner satisfaction was lowest with strategy C, and more owners also regarded food allowance as insufficient with this strategy. Novel diets and feeding strategies may improve outcome in feline weight loss programmes.

Reactions of benzene oxide with thiols including glutathione.

S-Phenylmercapturic acid is a minor metabolite of benzene used as a biomarker for human benzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, the initial metabolite of benzene, is presumed to give 1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which undergoes dehydration to S-phenylglutathione, the precursor of S-phenylmercapturic acid. To validate the proposed route to S-phenylglutathione, reactions of benzene oxide-oxepin with glutathione and other sulfur nucleophiles have been studied. The reaction of benzene oxide with an excess of aqueous sodium sulfide, followed by acetylation, gave bis-(6-trans-5-acetoxycyclohexa-1,3-dienyl)sulfide, the structure of which was proved by X-ray crystallography. Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO were monitored by 1H NMR spectroscopy. In the absence of glutathione, the half-life of benzene oxide-oxepin was ca. 34 min at 25 degrees C and pD 7.0. The half-life was not affected in the range of 2-15 mM glutathione in the presence and absence of a commercial sample of human glutathione S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(S-glutathionyl)-cyclohexa-3,5-diene-2-ol was identified in these reaction mixtures, especially at higher pH, by mass spectrometry and by its acid-catalyzed decomposition to S-phenylglutathione. Incubation of benzene oxide with N-acetyl-L-cysteine at 37 degrees C and pH 10.0 and subsequent mass spectrometric analysis of the mixture showed formation of pre-S-phenylmercapturic acid and the dehydration product, S-phenylmercapturic acid. The data validate the premise that benzene oxide-oxepin can be captured by glutathione to give (1R,2R)- and/or (1S,2S)-1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which dehydrate to S-phenylglutathione. The capture is a relatively inefficient process at pH 7 that is accelerated at higher pH. These studies account for the observation that the metabolism of benzene is dominated by the formation of phenol. The pathway leading to S-phenylmercapturic acid is necessarily minor on account of the low efficiency of benzene oxide capture by glutathione at pH 7 vs spontaneous rearrangement to phenol.