The power of effective study design in animal experimentation: Exploring the statistical and ethical implications of asking multiple questions of a data set.

One of the chief advantages of using highly standardised biological models including model organisms is that multiple variables can be precisely controlled so that the variable of interest is more easily studied. However, such an approach often obscures effects in sub-populations resulting from natural population heterogeneity. Efforts to expand our fundamental understanding of multiple sub-populations are in progress. However, such stratified or personalised approaches require fundamental modifications of our usual study designs that should be implemented in Brain, Behavior and Immunity (BBI) research going forward. Here we explore the statistical feasibility of asking multiple questions (including incorporating sex) within the same experimental cohort using statistical simulations of real data. We illustrate and discuss the large explosion in sample numbers necessary to detect effects with appropriate power for every additional question posed using the same data set. This exploration highlights the strong likelihood of type II errors (false negatives) for standard data and type I errors when dealing with complex genomic data, where studies are too under-powered to appropriately test these interactions. We show this power may differ for males and females in high throughput data sets such as RNA sequencing. We offer a rationale for the use of alternative experimental and statistical strategies based on interdisciplinary insights and discuss the real-world implications of increasing the complexities of our experimental designs, and the implications of not attempting to alter our experimental designs going forward.

Gender-specific effects of COVID-19 lockdowns on scientific publishing productivity: Impact and resilience.

RATIONALE: The SARS-CoV2 pandemic led to drastic social restrictions globally. Early data suggest that women in science have been more adversely affected by these lockdowns than men, with relatively fewer scientific articles authored by women. However, these observations test broad populations with many potential causes of disparity. Australia presents a natural experimental condition where several states of similar demographics and disease impact had differing approaches in their social isolation strategies. The state of Victoria experienced 280 days of lockdowns from 2020 to 2021, whereas the comparable state of New South Wales experienced 107 days, most of these in 2021, and other states even fewer restrictions. OBJECTIVE AND METHODS: To assess how the gender balance changed in Australian biomedical publishing with the lockdowns, we created a custom workflow to analyse PubMed data from more than 120,000 published articles submitted in 2019-2021 from Australian authors. RESULTS: Broadly, Australian women have been incredibly resilient to the challenges faced by the lockdowns. There was an increase in the number of published articles submitted in 2020 that was equally due to women as men, including from Victoria. On the other hand, articles specifically addressing COVID-19 were significantly less likely to be authored by women than those on other topics, a finding not likely due to particular gender imbalance in virology or viral epidemiology, since publications on HIV followed similar patterns to previous years. By 2021, this imbalance had reversed, with more COVID-19-related papers authored by women than men. CONCLUSIONS: These data suggest women from Victoria were less able to rapidly transition to new research early in the pandemic but had accommodated to the new conditions by 2021. This work indicates we need strategies to support women in science as the pandemic continues and to continue to monitor the situation for its impact on vulnerable groups.

Longitudinal study of Salmonella 1,4,[5],12:i:- shedding in five Australian pig herds.

The shedding patterns of Salmonella spp. and MLVA profiles of Salmonella enterica subspecies enterica (I) serotype 1,4,[5],12:i:- were monitored in a 12-month longitudinal observational study of five pig herds to inform management; provide indications of potential hazard load at slaughter; and assist evaluation of MLVA for use by animal and public health practitioners. Twenty pooled faecal samples, stratified by age group, were collected quarterly. When Salmonella was cultured, multiple colonies were characterized by serotyping and where S. Typhimurium-like serovars were confirmed, isolates were further characterized by phage typing and multiple locus variable number tandem repeat analysis (MLVA). Salmonella was detected in 43% of samples. Salmonella 1,4,[5],12:i- was one of several serovars that persisted within the herds and was found among colonies from each production stage. Virtually all Salmonella 1,4,[5],12:i:- isolates were phage type 193, but exhibited 12 different, closely-related MLVA profiles. Salmonella 1,4,[5],12:i:- diversity within herds was low and MLVA profiles were stable indicating colonization throughout the herds and suggesting each farm had an endemic strain. High prevalence of S. 1,4,[5],12:i:- specific shedding among terminal animals indicated high hazard load at slaughter, suggesting that primary production may be an important pathway of S. 1,4,[5],12:i:- into the human food chain, this has implications for on-farm management and the application and targeting control measures and further evidence of the need for effective process control procedures to be in place during slaughter and in pork boning rooms. These findings have implications for animal health and food safety risk mitigation and risk management.

The effects of air pollution on asthma hospital admissions in Adelaide, South Australia, 2003-2013: time-series and case-crossover analyses.

BACKGROUND: Air pollution can have adverse health effects on asthma sufferers, but the effects vary with geographic, environmental and population characteristics. There has been no long time-series study in Australia to quantify the effects of environmental factors including pollen on asthma hospitalizations. OBJECTIVES: This study aimed to assess the seasonal impact of air pollutants and aeroallergens on the risk of asthma hospital admissions for adults and children in Adelaide, South Australia. METHODS: Data on hospital admissions, meteorological conditions, air quality and pollen counts for the period 2003-2013 were sourced. Time-series analysis and case-crossover analysis were used to assess the short-term effects of air pollution on asthma hospitalizations. For the time-series analysis, generalized log-linear quasi-Poisson and negative binomial regressions were used to assess the relationships, controlling for seasonality and long-term trends using flexible spline functions. For the case-crossover analysis, conditional logistic regression was used to compute the effect estimates with time-stratified referent selection strategies. RESULTS: A total of 36,024 asthma admissions were considered. Findings indicated that the largest effects on asthma admissions related to PM2.5 , NO2 , PM10 and pollen were found in the cool season for children (0-17 years), with the 5-day cumulative effects of 30.2% (95% CI: 13.4-49.6%), 12.5% (95% CI: 6.6-18.7%), 8.3% (95% CI: 2.5-14.4%) and 4.2% (95% CI: 2.2-6.1%) increases in risk of asthma hospital admissions per 10 unit increments, respectively. The largest effect for ozone was found in the warm season for children with the 5-day cumulative effect of an 11.7% (95% CI: 5.8-17.9%) increase in risk of asthma hospital admissions per 10 ppb increment in ozone level. CONCLUSION: Findings suggest that children are more vulnerable and the associations between exposure to air pollutants and asthma hospitalizations tended to be stronger in the cool season compared to the warm season, with the exception of ozone. This study has important public health implications and provides valuable evidence for the development of policies for asthma management.

Body temperature measurements in pigs during general anaesthesia.

The aim was to compare rectal, pharyngeal and oesophageal temperature measurements in anaesthetized pigs. Data were compared using the Bland-Altman method, and correlation coefficients and error measures were calculated. Sixty-six sets of data were collected from 16 pigs weighing 16.2 ± 4.2 kg. The bias (and 95% limit of agreement) for rectal and pharyngeal compared with oesophageal temperature were 0.69 (-1.18 to 2.57) ℃ and 0.22 (-0.84 to 1.28) ℃, respectively. The correlation coefficients for rectal and pharyngeal compared with oesophageal temperature were 0.47 and 0.87, respectively. The absolute error for rectal and pharyngeal compared with oesophageal temperature was 0.7 ± 0.9℃ and 0.2 ± 0.5℃, respectively. Pharyngeal temperature measurement may be more suitable than rectal temperature measurement for estimation of oesophageal temperature during general anaesthesia of pigs.

Codeine-induced hyperalgesia and allodynia: investigating the role of glial activation.

Chronic morphine therapy has been associated with paradoxically increased pain. Codeine is a widely used opioid, which is metabolized to morphine to elicit analgesia. Prolonged morphine exposure exacerbates pain by activating the innate immune toll-like receptor-4 (TLR4) in the central nervous system. In silico docking simulations indicate codeine also docks to MD2, an accessory protein for TLR4, suggesting potential to induce TLR4-dependent pain facilitation. We hypothesized codeine would cause TLR4-dependent hyperalgesia/allodynia that is disparate from its opioid receptor-dependent analgesic rank potency. Hyperalgesia and allodynia were assessed using hotplate and von Frey tests at days 0, 3 and 5 in mice receiving intraperitoneal equimolar codeine (21 mg kg(-1)), morphine (20 mg kg(-1)) or saline, twice daily. This experiment was repeated in animals with prior partial nerve injury and in TLR4 null mutant mice. Interventions with interleukin-1 receptor antagonist (IL-1RA) and glial-attenuating drug ibudilast were assessed. Analyses of glial activation markers (glial fibrillary acid protein and CD11b) in neuronal tissue were conducted at the completion of behavioural testing. Despite providing less acute analgesia (P=0.006), codeine induced similar hotplate hyperalgesia to equimolar morphine vs saline (-9.5 s, P<0.01 and -7.3 s, P<0.01, respectively), suggesting codeine does not rely upon conversion to morphine to increase pain sensitivity. This highlights the potential non-opioid receptor-dependent nature of codeine-enhanced pain sensitivity-although the involvement of other codeine metabolites cannot be ruled out. IL-1RA reversed codeine-induced hyperalgesia (P<0.001) and allodynia (P<0.001), and TLR4 knock-out protected against codeine-induced changes in pain sensitivity. Glial attenuation with ibudilast reversed codeine-induced allodynia (P<0.001), and thus could be investigated further as potential treatment for codeine-induced pain enhancement.

Gene profiling for determining pluripotent genes in a time course microarray experiment.

In microarray experiments, it is often of interest to identify genes which have a prespecified gene expression profile with respect to time. Methods available in the literature are, however, typically not stringent enough in identifying such genes, particularly when the profile requires equivalence of gene expression levels at certain time points. In this paper, the authors introduce a new methodology, called gene profiling, that uses simultaneous differential and equivalent gene expression level testing to rank genes according to a prespecified gene expression profile. Gene profiling treats the vector of true gene expression levels as a linear combination of appropriate vectors, for example, vectors that give the required criteria for the profile. This gene profile model is fitted to the data, and the resulting parameter estimates are summarized in a single test statistic that is then used to rank the genes. The theoretical underpinnings of gene profiling (equivalence testing, intersection-union tests) are discussed in this paper, and the gene profiling methodology is applied to our motivating stem-cell experiment.

Evaluation of the triple tibial osteotomy. A new technique for the management of the canine cruciate-deficient stifle.

The triple tibial osteotomy (TTO) is a technique which combines the features of tibial tuberosity advancement and wedge osteotomy for the treatment of complete and partial cruciate ligament injuries in dogs. In this paper, the technique is described and the results of a prospective study of 64 consecutive cases are presented. TTO provided a satisfactory clinical outcome in a very high percentage of cases. The technique is relatively easy to learn and has a low post-operative complication rate.

Fingers on the red button.

General practitioners are sometimes reluctant to pass information about patients to social workers. Author looks at communication between doctors and social workers.