Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Primate veterinarians' knowledge and attitudes regarding pain in macaques.

BACKGROUND: Assessment of pain in macaques is challenging. The aims of this study were (1) to investigate current knowledge and attitudes of primate veterinarians concerning acute pain in macaques; (2) to synthesise current knowledge and opinion to facilitate pain assessment. A primary question of interest was whether more confident individuals differ in their knowledge and attitudes from less-confident individuals. METHODS: An online survey was conducted amongst primate veterinarians serving both laboratories and zoos/sanctuaries. The questionnaire consisted of demographic information, attitudes towards pain, pain rating and analgesics, pain recognition and confidence in recognising pain and sources of information used. RESULTS AND CONCLUSIONS: There was generally good use of analgesia by respondents. More confident individuals reported that they recognise pain both behaviourally and in facial expressions, rated all pain signs more highly and used more analgesics. Specialist support networks aimed at increasing veterinarian confidence in macaque pain assessment could be beneficial.

Emerging and legacy brominated flame retardants in the breast milk of first time Irish mothers suggest positive response to restrictions on use of HBCDD and Penta- and Octa-BDE formulations.

The brominated flame retardants (BFRs) hexabromocyclododecane (HBCDD), eight polybrominated diphenyl ethers (PBDEs), and decabromodiphenyl ethane (DBDPE) were measured in 16 pools of human milk from Ireland. Concentrations of BDEs-47, -99, -100, -153, and HBCDD were significantly lower (p < 0.05) than those in Irish human milk collected in 2011. In contrast, concentrations of BDE-209 in our study exceeded those in 2011, and while decabromodiphenyl ethane (DBDPE) was not detected in 2011 it was detected in 3 of our samples. This suggests increased use of DBDPE and that while restrictions on the Penta- and Octa-BDE formulations are reducing human exposure, those on Deca-BDE use have yet to reduce body burdens. Estimated exposures for nursing infants to all target BFRs do not suggest a health concern. A one compartment pharmacokinetic model was used to predict body burdens arising from BFR intakes via air, dust and diet. While for most targeted BFRs, predicted and observed body burdens derived from our human milk data compared reasonably well; predicted BDE-209 and DBDPE values were substantially lower than observed. This suggests exposure pathways not included in the model like dermal uptake from fabrics may be important, and highlights knowledge gaps about the human half-lives and bioavailability of these contaminants.

Concentrations of Brominated Flame Retardants in Indoor Air and Dust from Ireland Reveal Elevated Exposure to Decabromodiphenyl Ethane.

Concentrations of decabromodiphenyl ethane (DBDPE), 13 polybrominated diphenyl ethers (PBDEs), and hexabromocyclododecane (HBCDD) were measured in indoor air and dust collected from Irish homes, cars, offices, and primary schools during 2016-2017. Median concentrations of DBDPE in air (88 pg/m3) and dust (6500 ng/g) significantly exceed those previously reported internationally, with concentrations highest in offices and schools, suggesting that DBDPE is widely used in Ireland. Median concentrations of BDE-209 in air (340 pg/m3) and dust (7100 ng/g) exceed or are within the range of concentrations reported recently for the same microenvironments in the U.K., and exceed those reported in many other countries. Concentrations of BDE-209 in cars exceeded significantly (p < 0.05) those in other microenvironments. HBCDD was detected in all dust samples (median: 580 ng/g), and in 81% of air samples (median: 24 pg/m3) at concentrations similar to those reported recently for the U.K. and elsewhere. Estimates of exposure to DBDPE of Irish adults (92 ng/day) and toddlers (210 ng/day) as well as to BDE-209 (220 ng/day and 650 ng/day for adults and toddlers, respectively) substantially exceed those reported for the U.K. population. Moreover, our estimates of exposure of the Irish population to Σtrideca-PBDEs exceed previous estimates for Ireland via dietary exposure.

Perfluoroalkyl Substances in Drinking Water, Indoor Air and Dust from Ireland: Implications for Human Exposure.

Perfluoroalkyl substances (PFASs) were measured in air and dust from cars, homes, offices, and school classrooms in Ireland, along with drinking water from homes and offices. Perfluorooctanoic acid (PFOA) dominated air and drinking water, while perfluorobutane sulfonate (PFBS) dominated dust. This is the first report of PFOA, perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), PFBS, and perfluorononanoic acid (PFNA) in air inside cars and school classrooms. PFOS concentrations in classroom air exceeded significantly (p ≤ 0.05) those in homes. Atmospheric concentrations of PFOA, PFNA, and methyl perfluorooctane sulfonamido ethanol (MeFOSE) (p ≤ 0.05) were significantly higher in cars containing child car seats than in cars without. PFOS, PFOA, PFBS, and PFHxS were all detected frequently in drinking water, but concentrations of PFASs were low, and although ΣPFASs were 64 ng/L in one bottled water sample, this fell below a Swedish Action Level of 90 ng ΣPFASs/L. The Irish population's exposure to PFOS and PFOA via non-dietary sources is well below estimates of dietary exposure elsewhere in Europe. Moreover, even under a high-end exposure scenario, it falls below the European Food Safety Authority's (EFSA) provisional tolerable weekly intakes for PFOS and PFOA.

Interaction between amino-functionalized inorganic nanoshells and acid-autocatalytic reactions.

Amino-functionalized inorganic silica nanoshells with a diameter of 511 ± 57 nm are efficiently used as hydrogen ion binders with a base dissociation constant of (1.2 ± 0.1) × 10-4. The hydrogen removal has been shown to produce reaction-diffusion fronts of constant propagation velocity in the autocatalytic chlorite-tetrathionate reaction when it is run in thin planar slices of nanoshell-containing agarose gel to exclude all convection related effects. By controlling the exact amount of amino-functionalized hollow nanospheres in the gel matrix it is possible to finely tune the propagation velocity of the chemical front in the 0.1-10 cm h-1 range. Remarkably, this can be achieved with very low amino-functionalized hollow inorganic nanosphere loadings between 0.1-0.01 (m V-1)%. The front width has also been determined experimentally, which increases by a factor of two with one magnitude decrease in the front velocity.

How Does Autologous Breast Reconstruction Impact Downtime?

BACKGROUND: Although autologous breast reconstruction is technically quite demanding, it offers the best outcomes in terms of durable results, patient perceptions, and postoperative pain. Many studies have focused on clinical outcomes and technical aspects of such procedures, but few have addressed the impact of various flaps on patient recovery times. This particular investigation entailed an assessment of commonly used flaps, examining the periods of time required to resume daily activities. METHODS: Multiple choice questionnaires were administered to 121 patients after recovery from autologous reconstruction to determine the times required in returning to specific physical activities. To analyze results, the analysis of variance F-test was applied, and odds ratios (ORs) were determined. RESULTS: Among the activities surveyed, recovery time was not always a function of free-flap surgery. Additional treatments and psychological effects also contributed. Adjuvant chemotherapy increased average downtime by 2 weeks, and postoperative irradiation prolonged recovery as much as 4 weeks. Patient downtime was unrelated to flap type, ranging from 2.9 to 21.3 weeks for various activities in question. Deep inferior epigastric perforator (DIEP) flaps yielded the highest OR and transverse upper gracilis (TUG) flaps the lowest. CONCLUSION: Compared with superior gluteal artery perforator and TUG flaps, the DIEP flap was confirmed as the gold standard in autologous breast reconstruction, conferring the shortest recovery times. All adjuvant therapies served to prolong patient recovery as well. Surgical issues, patient lifestyles, and donor-site availability are other important aspects of flap selection.

Dual-energy micro-CT with a dual-layer, dual-color, single-crystal scintillator.

A wide range of X-ray imaging applications demand micrometer spatial resolution. In material science and biology especially, there is a great interest in material determination and material separation methods. Here we present a new detector design that allows the recording of a low- and a high-energy radiography image simultaneously with micrometer spatial resolution. The detector system is composed of a layered scintillator stack, two CCDs and an optical system to image the scintillator responses onto the CCDs. We used the detector system with a standard laboratory microfocus X-ray tube to prove the working principle of the system and derive important design characteristics. With the recorded and registered dual-energy data set, the material separation and determination could be shown at an X-ray tube peak energy of up to 160 keV with a spatial resolution of 12 µm. The detector design shows a great potential for further development and a wide range of possible applications.

The influence of dog ownership on objective measures of free-living physical activity and sedentary behaviour in community-dwelling older adults: a longitudinal case-controlled study.

BACKGROUND: There is some evidence to suggest that dog ownership may improve physical activity (PA) among older adults, but to date, studies examining this, have either depended on self-report or incomplete datasets due to the type of activity monitor used to record physical activity. Additionally, the effect of dog ownership on sedentary behaviour (SB) has not been explored. The aim of the current study was to address these issues by using activPAL monitors to evaluate the influence of dog ownership on health enhancing PA and SB in a longitudinal study of independently-mobile, community-dwelling older adults. METHODS: Study participants (43 pairs of dog owners and non-dog owners, matched on a range of demographic variables) wore an activPAL monitor continuously for three, one-week data collection periods over the course of a year. Participants also reported information about their own and their dog demographics, caring responsibilities, and completed a diary of wake times. Diary data was used to isolate waking times, and outcome measures of time spent walking, time spent walking at a moderate cadence (>100 steps/min), time spent standing, time spent sitting, number of sitting events (continuous periods of sitting), and the number of and of time spent sitting in prolonged events (>30 min). For each measure, a linear mixed effects model with dog ownership as a fixed effect, and a random effects structure of measurement point nested in participant nested in pair was used to assess the effect of dog ownership. RESULTS: Owning a dog indicated a large, potentially health improving, average effect of 22 min additional time spent walking, 95%CI (12, 34), and 2760 additional steps per day, 95%CI (1667, 3991), with this additional walking undertaken at a moderate intensity cadence. Dog owners had significantly fewer sitting events. However, there were no significant differences between the groups for either the total time spent sitting, or the number or duration of prolonged sedentary events. CONCLUSIONS: The scale of the influence of dog ownership on PA found in this study, indicates that future research regarding PA in older adults should assess and report dog ownership and/or dog walking status.

CT of Anatomic Variants of the Paranasal Sinuses and Nasal Cavity: Poor Correlation With Radiologically Significant Rhinosinusitis but Importance in Surgical Planning.

OBJECTIVE: The purpose of this study was to determine the incidence of sinonasal anatomic variants and to assess their relation to sinonasal mucosal disease. MATERIALS AND METHODS: A retrospective evaluation of 192 sinus CT examinations of patients with a clinical history of rhinosinusitis was conducted. The CT scans were evaluated for the presence of several anatomic variants of the sinonasal cavities, and the prevalence of each variant was calculated. Prevalences of all sinonasal anatomic variants were compared between patients who had minimal to no apparent imaging evidence of rhinosinusitis and those who had radiologic evidence of clinically significant rhinosinusitis. RESULTS: The most common normal variants were nasal septal deviation, Agger nasi cells, and extension of the sphenoid sinuses into the posterior nasal septum. We found no statistically significant difference in the prevalence of any of the studied anatomic variants between patients with minimal and those with clinically significant paranasal sinus or nasal cavity disease. CONCLUSION: Analysis of every routine CT scan of the paranasal sinuses obtained for sinusitis or rhinitis for the presence of different anatomic variants is of questionable value unless surgery is planned.

Could Vulnerability to Motion Sickness and Chronic Pain Coexist within a Sensorimotor Phenotype? Insights from over 500 Pre-Pain Motion Sickness Reports.

BACKGROUND: The sensorimotor incongruence theory proposes that certain instances of pain result from conflicts in the brain's sensorimotor networks. Indeed, injuries may cause abnormalities in afferent and cortical signaling resulting in such conflicts. Motion sickness also occurs in instances of incongruent sensorimotor data. It is possible that a sensory processing phenotype exists that predisposes people to both conditions. AIM: The aim of this study was to investigate whether participants with chronic pain recall greater susceptibility to motion sickness before chronic pain onset. METHOD: Data were collected via an online LimeSurvey. A self-report tendency toward motion sickness was measured using the Motion Sickness Susceptibility Questionnaire. Group differences were analysed using analysis of covariance methods. RESULTS: 530 patients (low back pain, n = 198; neck pain, n = 59; whiplash-associated disorder, n = 72; fibromyalgia syndrome, n = 114; Migraine, n = 41) and 165 pain-free controls were surveyed. ANCOVA analysis, using sex and anxiety as covariates, suggested that childhood motion sickness susceptibility scores differed by group (F = 2.55 (6, 615), p = 0.019, (ηp2) = 0.024). Planned comparisons, with corrected p-values, suggested that childhood motion sickness was not statistically greater for low back pain, rheumatoid arthritis, migraine, neck pain or whiplash-associated disorder (ps > 0.05), although scores were on average 27%, 42%, 47%, 48% and 58% higher, respectively. Childhood susceptibility was statistically higher in people with FMS (p = 0.018), with scores on average 83% higher than controls. ANCOVA analysis, using sex and anxiety as covariates, suggested that adult motion sickness susceptibility scores did not differ by group (F = 1.86 (6, 613), p = 0.086), although average scores were, on average, at least 33% higher in persistent pain groups. CONCLUSIONS: According to retrospective reporting, greater susceptibility to motion sickness appears to pre-date persistent pain in some conditions. This supports the possibility that motion sickness and chronic pain may, in some cases, have overlapping mechanisms related to the handling of incongruent sensorimotor data.

Rapid Determination of Selected PFAS in Textiles Entering the Waste Stream.

Due to new European legislation, products entering the waste stream containing some perfluoro alkyl substances (PFAS) are subject to "low persistent organic pollutant concentration limits". Concentrations of restricted PFAS must be below this limit for them to be legally recycled or disposed of. A rapid extraction and clean-up method was developed for the determination of 21 PFAS in various polymers used in soft furnishings and upholstery. The optimised method used vortexing and ultrasonication in methanol (0.1% NH4OH), followed by a dilution and syringe filter clean-up step. PFAS were subsequently determined via UPLC-TripleTOF/MS. Good recoveries (80-120%) of target analytes were obtained with tall and narrow chromatogram peaks. The method was validated using control matrix samples spiked with target analytes. Repeated measurements of concentrations of target compounds showed good agreement with the spiked concentrations demonstrating good accuracy and precision. The resultant extracts provided low noise levels resulting in low limits of quantification ranging from 0.1 to 0.4 mg/kg. The developed method was applied successfully to real consumer products and it provided various advantages over traditional methods, including a substantially reduced analysis time, consumables and solvent consumption, and a high sample throughput which is critical to comply with implemented and proposed legislation.

Quality by Design for enabling RNA platform production processes.

RNA-based products have emerged as one of the most promising and strategic technologies for global vaccination, infectious disease control, and future therapy development. The assessment of critical quality attributes (CQAs), product-process interactions, relevant process analytical technologies, and process modeling capabilities can feed into a robust Quality by Design (QbD) framework for future development, design, and control of manufacturing processes. QbD implementation will help the RNA technology reach its full potential and will be central to the development, pre-qualification, and regulatory approval of rapid response, disease-agnostic RNA platform production processes.

Exposure, risk and predictors of hexabromocyclododecane and Tetrabromobisphenol-A in house dust from urban, rural and E-waste dismantling sites in Thailand.

In this study, for the first time, we determined concentrations of hexabromocyclododecane (HBCDD) and tetrabromobisphenol-A (TBBPA) in house dust and estimated human exposure to these substances in houses located in an e-waste dismantling site and in urban and rural residential areas of Thailand. The median HBCDD concentration in urban residential houses (2.10 ng g-1) was similar to that in houses in an e-waste dismantling site (2.05 ng g-1, p > 0.05) and slightly higher than that in rural residential houses (1.11 ng g-1, p > 0.05). In contrast, significantly higher TBBPA concentrations were present in house dust from an e-waste dismantling site (median = 720 ng g-1; range = 44-2300 ng g-1) compared to those in urban (68.6 ng g-1; 3.5-300 ng g-1, p < 0.001) and rural residential areas (17 ng g-1; 2.0-201 ng g-1, p < 0.001). TBBPA concentrations increased with the increasing presence of electronic devices and a decreasing distance to the e-waste dismantling site. These results suggest that e-waste dismantling activities may contribute to TBBPA contamination of house dust. The median estimated daily intake (EDI) of HBCDD and TBBPA through dust ingestion for toddlers exceeded that for children and adults. However, EDI values for HBCDD and TBBPA from all age groups were below the oral reference dose guideline value suggested by the US National Research Council and National Toxicology Program (NTP).

Domestic dogs (Canis familiaris) grieve over the loss of a conspecific.

Behavioural reactions towards a dead conspecific have been observed rarely in wild canids and there is no documented scientific evidence of grief in pet dogs. A quantitative analysis of grief-related responses in both dogs and owners was conducted, using the validated online Mourning Dog Questionnaire. The survey was completed by 426 Italian adults who had owned at least two dogs, one of whom died while the other was still alive. This research aims to explore whether, how and what a dog may experience over the loss of a companion dog. Multiple logistic regression indicates that both a friendly or parental relationship between two dogs but also the fact that dogs used to share food and the owner's grief and anger are principal predictors of negative behavioural changes. According to dog owners' answers, the surviving dog after the death of the companion dog changed both in terms of activities ("playing", "sleeping", and "eating") and emotions (fearfulness), which occurred as a function of the quality of the relationship between the two animals. By contrast, the time the two dogs had spent together had no effect on the behaviours of surviving dog. Owner perceptions about their dog's reactions and emotions were not related to the memory or suffering of the event that tended to diminish over time. These findings indicate that a dog may show grief-related behavioural and emotional patterns when a close conspecific dies, with aspects of the latter possibly related to the owner's emotional status.

Introducing a Controlled Outdoor Environment Impacts Positively in Cat Welfare and Owner Concerns: The Use of a New Feline Welfare Assessment Tool.

There is much debate over the pros and cons of allowing cats to roam freely as opposed to keeping them confined indoors. We surveyed owners who implemented a commercial physical containment system to the outdoors to evaluate their characteristics and the apparent impact of this system on cat welfare and owner perceptions. As part of the latter aim, we also developed a new feline welfare assessment tool based on the mathematical relationship between different measures. The survey was circulated to customers over the preceding 2 years of ProtectaPet® between May and June 2019 and gathered 446 responses. Univariate analyses compared changes following installation on factors such as the amount of time the cat spent outside, other cats entering the owner's garden and owners' concerns about their cat outside. Principal component analysis was used to reduce 21 potential indicators of feline welfare into fewer variables. This resulted in 4 subscales, 2 relating to positive welfare and 2 relating to negative welfare. The effects of installation of the containment system and significant predictors of these four welfare subscales were assessed. The majority of respondents lived in an urban environment with a relatively small garden, had multiple cats and a history of feline trauma associated with a road traffic accident. As expected, the time spent outside significantly increased, while the frequency of other cats entering the garden and owner concern about leaving their cats outside significantly decreased. The 4 welfare subscales grouped into positivity, maintenance behaviors, health issues and fearfulness; installation of the system was associated with significant improvements across all of these. Time spent outside after installation had a significant effect on positivity and, to a lesser extent, maintenance behaviors. Overall, installation was associated with positive changes in both owner and cat quality of life, which seem to be particularly associated with an increased sense of security. This suggests that housing cats within a controlled outside environment with physical barriers can provide a practical solution for many of the problems associated with cats being allowed out.

Cats vs. Dogs: The Efficacy of Feliway FriendsTM and AdaptilTM Products in Multispecies Homes.

Seven percent of UK households are estimated to own both a cat and a dog, despite a popular view that the two do not live well together. This is the first study to evaluate the effects of pheromone products Feliway FriendsTM and AdaptilTM on cat-dog interactions, in homes where owners perceived the potential for improvement in the relationship between their cat and dog. A blinded parallel randomized trial design over a 6-week period was used to evaluate the effect of each of the two products, with 17 participants in each group completing the trial. Owners reported weekly on the frequency of 10 specific undesirable interactions and seven specific desirable interactions. Total undesirable and desirable interaction scores both showed significant linear contrasts over time (undesirable score decreased; desirable score increased). Undesirable interaction scores were significantly lower (with a very large effect size) during treatment compared with baseline. There were no significant differences between the two pheromone products in relation to these outcome measures. AdaptilTM and Feliway FriendsTM were both associated with a significant decrease in: dog chasing cat/cat runs away; cat hiding from dog; cat/dog staring at the other; and dog barking at cat. With AdaptilTM a significant increase was also seen in: friendly greeting and times spent relaxed in the same room. From baseline (Week 2) to the end of the study (Week 6) there was a significant improvement in owners' perception of dog relaxation in those participants who received AdaptilTM and of cat relaxation in those participants who received Feliway FriendsTM. Similarity in the core chemical structure of the appeasing pheromones might explain the main effects, whilst different species-specific additions may explain the product-appropriate species-specific increases in relaxation scores. Specific behavioral improvements seen with AdaptilTM may reflect a greater calming of dogs in this group, reducing their interest in seeking interaction with cats in the same home and the tension in the cat as a result. In conclusion, both products appear to improve the cat-dog relationship and it would be beneficial to further study their use in combination and against placebo. If selecting one product AdaptilTM may be preferable, unless there is a particular need to increase the cat's relaxation.

Potential Risk Factors for Aggression and Playfulness in Cats: Examination of a Pooling Fallacy Using Fe-BARQ as an Example.

Using a popular method of behaviour evaluation which rates the intensity of behaviour in different contexts, we demonstrate how pooling item scores relating to a given construct can reveal different potential risk factors for the dependent variable depending on how the total score is constructed. We highlight how similar simple total scores can be constructed through very different combinations of constituent items. We argue for the importance of examining individual item score distributions, and the results from different intensity thresholds before deciding on the preferred method for calculating a meaningful dependent variable. We consider simply pooling individual item scores which conflate context with intensity to calculate an average score and assuming this represents a biologically meaningful measure of trait intensity is a fallacy. Specifically using four items that describe intercat aggression and eleven that describe playfulness in cats in Fe-BARQ, we found sex and neuter status, social play and fearfulness were consistently significant predictors for intercat aggression scores; and age, age when obtained, social play and fearfulness were significant predictors of playfulness scores. However, the significance of other factors such as scratching varied with the threshold used to calculate to the total score. We argue that some of these inconsistent variables may be biologically and clinically important and should not be considered random error. Instead they need to be evaluated in the context of other available evidence.

Concentrations of perfluoroalkyl substances in human milk from Ireland: Implications for adult and nursing infant exposure.

Concentrations of 10 perfluoroalkyl substances (PFASs) were measured in 16 pools of human milk from Ireland. Only four PFASs were detected (PFOA, PFNA, PFHxS and PFOS), with concentrations dominated by PFOA which was detected in all samples at a median of 0.10 ng/mL. Concentrations and the relative abundance of PFASs in human milk from Ireland are within the range reported for other countries. Estimated exposures for nursing infants to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) do not suggest a health concern. A one compartment pharmacokinetic model was used to predict the intakes of PFOS and PFOA required to support the observed concentrations in human milk. This suggests current adult exposure in Ireland to PFOS is below the provisional tolerable weekly intake (TWI) proposed by EFSA. In contrast, the model predicts that the maximum concentration detected in human milk in this study, implies a level of adult exposure that would exceed EFSA's provisional TWI for PFOA. As exposure of the Irish population to PFASs via drinking water, indoor air and dust is well-characterised, current understanding suggests that the major contributor to overall exposure of the Irish population is via the diet and/or less well-studied pathways like dermal uptake from PFAS-containing fabrics and cosmetics.