Objectives/Goals: Ischemic stroke treatments assist in restoring blood flow, but do not guarantee good outcomes. Since extracellular vesicles (EVs) able to cross the blood brain barrier, total (nonspecific) and astrocyte enriched EVs (TEVs, AEVs, respectively) from plasma may emerge as plasma biomarkers for prognostication and targeted therapeutics. Methods/Study Population: “Blood and Clot Thrombectomy Registry and Collaboration” (BACTRAC; NCT03153683) is a human stroke biobank at the University of Kentucky that collects samples at the time of mechanical thrombectomy during emergent large vessel occlusions (ELVO; ischemic stroke). EVs were isolated, via size exclusion chromatography, from unbanked plasma and concentrated resulting in TEVs. AEVs were immunoprecipitated with anti-EAAT1 (GLAST), an astrocyte-specific transmembrane glycoprotein. Isolated protein was sent to Olink and ran on their metabolic panel. Demographics and medical histories of the subjects were exported from REDcap and investigators were blinded during EV analysis. Results/Anticipated Results: ELVO subjects (8 females/ 5 males) were an average age of 71.1 ± 11.7 years. Lower TEV enolase 2, a neuronal glycolysis enzyme, associated with increased stroke severity (NIHSS; rs =  -0.7819, p = 0.0476). Higher systemically TEV quinoid dihydropteridine reductase (QDPR), essential co-factor enzyme, was associated with more severe strokes (NIHSS; rs =  0.8486, p = 0.0123) and lower cognition (MoCA; r2 =  0.7515, p = 0.0254). Interestingly, higher intracranial AEVs QDPR was associated with lower infarct volumes (rs =  -0.7333, p = 0.0202), less severe strokes (NIHSS; rs =  -0.6095, p = 0.0388), and better cognition (MoCA; r2 =  0.6095, p = 0.0388). Increased AEV nicotinamide adenine dinucleotide kinase another essential co-factor enzyme, intracranially also correlated to higher cognition (MoCA; rs =  0.8356, p = 0.0298). Discussion/Significance of Impact: Plasma TEV and AEV metabolic proteins correlate with the progression of stroke outcomes and should be investigated as target therapies during MT to improve outcomes.

Low vitamin D associated with high parathyroid hormone (PTH) is common in HIV infection. We determined the association between total 25(OH)D and PTH in adolescents living with HIV, in Zambia and Zimbabwe. Adolescents (11–19 years) perinatally infected with HIV and established on antiretroviral therapy for ≥ 6 months were recruited into a cross-sectional study. Socio-demographic and clinical characteristics were recorded, anthropometry measured and fasted serum concentrations of 1,25(OH)2D, total 25(OH)D and intact PTH measured. The association between total 25(OH)D and PTH was examined using natural cubic spline regression. 842 participants (female: 53·2%) with a median age of 15·5 (IQR: 13·2–17·9) years were enrolled. Median antiretroviral therapy duration was 9·8 (IQR: 6·3–12·3) years, and 165/841 had an HIV viral-load >60 copies/ml. Stunting (height-for-age z-score <–2) and underweight (weight-for-age z-score <–2) were observed in 29·9 and 30·0%, respectively. Three-quarters reported daily Ca intakes <150 mg/d. The mean (sd) concentrations of total 25(OH)D and 1,25(OH)2D were 66·1(16·5) nmol/l and 210·6 (70·4) pmol/l, respectively, and median PTH level was 4·3 (IQR: 3·3–5·5) pmol/l. There was an inverse non-linear relationship between total 25(OH)D and PTH, 25(OH)D levelling off at 74·6 nmol/l (95 % CI: 74·5, 75·2). Results were consistent in those taking tenofovir disoproxil fumarate and virally unsuppressed participants. In this population with extremely low habitual Ca intakes, the lack of association between 25(OH)D and PTH when 25(OH)D exceeded 75 nmol/l potentially suggests that levels of 25(OH)D >75 nmol/l may need to be achieved to improve bone health; investigation is needed in future research studies.

At low levels of hydration, exchangeable D+ in montmorillonite interacts with lattice OH groups and quantitatively converts AlMgOH groups to AlMgOD. Hydroxyl groups coordinated to two Al ions undergo a slower exchange, the extent of which is restricted by octahedral Fe3+ ions. The OH stretching vibration of AlMgOH groups in montmorillonite is assigned an unusually high frequency (3687 cm−1) compared with that of the same group in phengites (3602 cm−1).

Infrared and Mössbauer spectroscopy show that the extent of the reduction of nontronite is dependent on the chemical composition of the nontronite and on the nature of the reducing agent. Hydrazine reversibly reduces about 10% of the iron in all of the nontronites studied irrespective of composition and it is suggested that the resulting ferrous iron occurs only in distorted octahedral sites. Similar conclusions are reached for the dithionite reduction of the nontronites containing little tetrahedral iron, but for those with more than one in eight silicons replaced by iron, changes brought about by dithionite treatment are irreversible due to dissolution of appreciable quantities of iron. Results from both spectroscopic techniques suggest that iron in tetrahedral sites is preferentially dissolved and that up to 80% of the structural iron can be reduced.

Evidence is presented for the formation in these extensively reduced nontronites of a small amount of a mica-like phase resembling celadonite or glauconite, and, as dithionite is used for the pretreatment of soils, the implication of this observation is briefly discussed.

The use of deuterated hydrazine as a reducing agent has enabled the nontronite absorption band near 850 cm-1 to be assigned to a Si-O (apical) stretching vibration, which is inactive in the infrared for perfect hexagonal symmetry, but which is activated by distortions in the tetrahedral layer.

Mössbauer and i.r. spectra of a series of nontronites show that Fe3+ and Al3+ are distributed between tetrahedral and octahedral sites. The Mössbauer results have reaffirmed the occupation by Fe3+ of octahedral sites at which these ions are coordinated to pairs of OH groups in both cis and trans configurations. The distribution of Fe3+ between these two sites varies considerably but in all of the nontronites some Fe3+ occurs in the trans site in contrast to the all cis occupancy of the centro-symmetric structure proposed by Mering and Oberlin (1967). In one of the nontronites the distribution of Fe3+ between these two sites approaches that in the ideal non-centrosymmetric structure proposed for montmorillonite.

This study investigated sex differences in Fe status, and associations between Fe status and endurance and musculoskeletal outcomes, in military training. In total, 2277 British Army trainees (581 women) participated. Fe markers and endurance performance (2·4 km run) were measured at the start (week 1) and end (week 13) of training. Whole-body areal body mineral density (aBMD) and markers of bone metabolism were measured at week 1. Injuries during training were recorded. Training decreased Hb in men and women (mean change (–0·1 (95 % CI –0·2, –0·0) and –0·7 (95 % CI –0·9, –0·6) g/dl, both P < 0·001) but more so in women (P < 0·001). Ferritin decreased in men and women (–27 (95 % CI –28, –23) and –5 (95 % CI –8, –1) µg/l, both P ≤ 0·001) but more so in men (P < 0·001). Soluble transferrin receptor increased in men and women (2·9 (95 % CI 2·3, 3·6) and 3·8 (95 % CI 2·7, 4·9) nmol/l, both P < 0·001), with no difference between sexes (P = 0·872). Erythrocyte distribution width increased in men (0·3 (95 % CI 0·2, 0·4)%, P < 0·001) but not in women (0·1 (95 % CI –0·1, 0·2)%, P = 0·956). Mean corpuscular volume decreased in men (–1·5 (95 % CI –1·8, –1·1) fL, P < 0·001) but not in women (0·4 (95 % CI –0·4, 1·3) fL, P = 0·087). Lower ferritin was associated with slower 2·4 km run time (P = 0·018), sustaining a lower limb overuse injury (P = 0·048), lower aBMD (P = 0·021) and higher beta C-telopeptide cross-links of type 1 collagen and procollagen type 1 N-terminal propeptide (both P < 0·001) controlling for sex. Improving Fe stores before training may protect Hb in women and improve endurance and protect against injury.

In conceptualizing animal welfare, it is useful to distinguish among three types of concepts. ‘Type 1” are single, measurable attributes. ‘Type 2’ are single attributes that cannot be measured directly but can be estimated by correctly combining various contributing attributes. ‘Type 3’ are concepts involving multiple attributes which are grouped together because they serve some common function, and whose relative importance cannot be established in an entirely objective way. Individuals who treat animal welfare as a type 1 concept may propose single, objective measures of welfare, such as longevity or levels of stress-related hormones; however, this approach rests on judgements, which are not purely objective, about the relative importance of different factors for an animal's quality of life. Studies of animal preferences and motivation are sometimes seen as an objective way to weight different attributes according to the animals’ own priorities, and thus allow animal welfare to be treated as a type 2 concept. However, numerous technical and fundamental difficulties limit our ability to do this. Animal welfare is best seen as a type 3 concept incorporating multiple attributes, with considerable consensus over certain general principles (eg that a high level of welfare implies freedom from suffering) but with value-related disagreement over how these principles should be applied. Because the various attributes cannot be combined in a purely objective way, science is limited in its ability to determine the ‘overall’ welfare of an animal and to compare welfare in disparate environments. Instead of attempting to ‘measure’ animal welfare, the role of science should be seen as identifying, rectifying and preventing welfare problems.

In the social debate about animal welfare we can identify three different views about how animals should be raised and how their welfare should be judged: (1) the view that animals should be raised under conditions that promote good biological functioning in the sense of health, growth and reproduction, (2) the view that animals should be raised in ways that minimise suffering and promote contentment, and (3) the view that animals should be allowed to lead relatively natural lives. When attempting to assess animal welfare, different scientists select different criteria, reflecting one or more of these value-dependent views. Even when ostensibly covering all three views, scientists may differ in what they treat as inherently important versus only instrumentally important, and their selection of variables may be further influenced by a desire to use measures that are scientifically respected and can be scored objectively. Value assumptions may also enter animal welfare assessment at the farm and group level (1) when empirical data provide insufficient guidance on important issues, (2) when we need to weigh conflicting interests of different animals, and (3) when we need to weigh conflicting evidence from different variables. Although value assumptions cannot be eliminated from animal welfare assessment, they can be made more explicit as the first step in creating animal welfare assessment tools. Different value assumptions could lead to different welfare assessment tools, each claiming validity within a given set of assumptions.

Scientific research on ‘animal welfare’ began because of ethical concerns over the quality of life of animals, and the public looks to animal welfare research for guidance regarding these concerns. The conception of animal welfare used by scientists must relate closely to these ethical concerns if the orientation of the research and the interpretation of the findings is to address them successfully.

At least three overlapping ethical concerns are commonly expressed regarding the quality of life of animals: (1) that animals should lead natural lives through the development and use of their natural adaptations and capabilities, (2) that animals should feel well by being free from prolonged and intense fear, pain, and other negative states, and by experiencing normal pleasures, and (3) that animals should function well, in the sense of satisfactory health, growth and normal functioning of physiological and behavioural systems. Various scientists have proposed restricted conceptions of animal welfare that relate to only one or other of these three concerns. Some such conceptions are based on value positions about what is truly important for the quality of life of animals or about the nature of human responsibility for animals in their care. Others are operational claims: (1) that animal welfare research must focus on the functioning of animals because subjective experiences fall outside the realm of scientific enquiry, or (2) that studying the functioning of animals is sufficient because subjective experiences and functioning are closely correlated. We argue that none of these positions provides fully satisfactory guidance for animal welfare research.

We suggest instead that ethical concerns about the quality of life of animals can be better captured by recognizing three classes of problems that may arise when the adaptations possessed by an animal do not fully correspond to the challenges posed by its current environment. (I) If animals possess adaptations that no longer serve a significant function in the new environment, then unpleasant subjective experiences may arise, yet these may not be accompanied by significant disruption to biological functioning. Thus, a bucket-fed calf may experience a strong, frustrated desire to suck, even though it obtains adequate milk. (2) If the environment poses challenges for which the animal has no corresponding adaptation, then functional problems may arise, yet these may not be accompanied by significant effects on subjective feelings. Thus, a pig breathing polluted air may develop lung damage without appearing to notice or mind the problem. (3) Where animals have adaptations corresponding to the kinds of environmental challenges they face, problems may still arise if the adaptations prove inadequate. For example, an animal's thermoregulatory adaptations may be insufficient in a very cold environment such that the animal both feels poorly and functions poorly. We propose that all three types of problems are causes of ethical concern over the quality of life of animals and that they together define the subject matter of animal welfare science.

As adult cats can often be difficult to re-home, they may spend long periods in rescue shelters where barren housing and inconsistent handling can reduce their welfare. In this study, 165 adult cats in an animal shelter in Vancouver, Canada, were assigned to four treatments. The Basic Single treatment reflected typical conditions in that particular shelter, with cats handled in an inconsistent manner by various staff and housed singly in relatively barren cages. Three alternative treatments involved more consistent, positive handling by only the experimenter and research assistants, plus three housing conditions: Enriched Single (individual cages with opportunities to perch and hide), Basic Communal (group housing with opportunity for each cat to have personal space), and Enriched Communal (group housing enriched to encourage play and cat – cat interaction). The Basic Single treatment had the lowest percentage adopted in 21 days (45% versus 69-76% for other treatment, and higher stress scores than other treatments. The three alternative treatments did not differ significantly on any measure. Cats euthanised for poor health showed higher stress levels when alive than other cats. In a questionnaire, most adopters cited certain behavioural/emotional traits (‘friendly’, ‘playful’, ‘happy’) as reasons for selecting cats; these were generally associated with lower stress scores. The results suggest that consistent handling combined with a range of improved housing options can improve the chances of adoption for adult cats, perhaps by reducing fear-related behaviours that make cats less attractive to adopters.