Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring.

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng µL-1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80-120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

Suicide-Related Out-of-Hospital Cardiac Arrests in Queensland, Australia: Temporal Trends of Characteristics and Outcomes over 14 Years.

BACKGROUND: Research into suicide-related out-of-hospital cardiac arrests (OHCA) using OHCA registries is scant. A more complete understanding of methods, patient characteristics, and outcomes is essential to inform prehospital management strategies and public health interventions. METHODS: Included were all OHCA attended by Queensland Ambulance Service (Australia) paramedics between 1 January 2007 and 31 December 2020, where suicide-related causes could be identified. Age- and sex-standardized incidence rates were calculated. Suicide methods, patient characteristics, and survival outcomes were described. Factors associated with survival outcomes were investigated. RESULTS: Seven thousand three hundred and fifty-six suicide-related OHCA cases were included. The incidence rates increased from 9.0 per 100,000 population in 2007 to 12.4 in 2020. The incidence rates for males were four times those for females; however, incidence rates for females have increased faster than for males. Hanging was the most common suicide method (63%). Twenty-three percent of patients received resuscitation attempts by paramedics. Among those, the rates of return of spontaneous circulation (ROSC) sustained to hospital arrival, survival to hospital discharge, and survival to 30 days were 28.6, 8.5, and 8.0%, respectively. Over time, the rates of ROSC upon hospital arrival increased, whereas the rates of survival to discharge and 30-day survival remained stable. CONCLUSION: The incidence of prehospital-identified suicide-related OHCA in Queensland has increased over time. The prognosis of suicide-related OHCA is poor. Prevention measures should focus on early identification and treatment of individuals having a high risk of suicide. Emergency medical services need to have sufficient training for telecommunicators and paramedics in suicide risk assessment and identification.

Intestinal colonization regulates systemic anti-commensal immune sensitivity and hyperreactivity.

Healthy host-microbial mutualism with our intestinal microbiota relies to a large degree on compartmentalization and careful regulation of adaptive mucosal and systemic anti-microbial immune responses. However, commensal intestinal bacteria are never exclusively or permanently restricted to the intestinal lumen and regularly reach the systemic circulation. This results in various degrees of commensal bacteremia that needs to be appropriately dealt with by the systemic immune system. While most intestinal commensal bacteria, except for pathobionts or opportunistic pathogen, have evolved to be non-pathogenic, this does not mean that they are non-immunogenic. Mucosal immune adaptation is carefully controlled and regulated to avoid an inflammatory response, but the systemic immune system usually responds differently and more vigorously to systemic bacteremia. Here we show that germ-free mice have increased systemic immune sensitivity and display anti-commensal hyperreactivity in response to the addition of a single defined T helper cell epitope to the outer membrane porin C (OmpC) of a commensal Escherichia coli strain demonstrated by increased E. coli-specific T cell-dependent IgG responses following systemic priming. This increased systemic immune sensitivity was not observed in mice colonized with a defined microbiota at birth indicating that intestinal commensal colonization also regulates systemic, and not only mucosal, anti-commensal responses. The observed increased immunogenicity of the E. coli strain with the modified OmpC protein was not due to a loss of function and associated metabolic changes as a control E. coli strain without OmpC did not display increased immunogenicity.

The novel use of urinary androgens to optimise detection of the fertile window in giant pandas.

Abstract: Giant pandas are mono-estrus seasonal breeders, with the breeding season typically occurring in the spring. Successful fertilization is followed by an embryonic diapause, of variable length, with birth in the late summer/autumn. There is a need for additional understanding of giant panda reproductive physiology, and the development of enhanced biomarkers for impending proestrus and peak fertility. We aimed to determine the utility of non-invasive androgen measurements in the detection of both proestrus and estrus. Urine from 20 cycles (-40 days to +10 days from peak estrus) from 5 female giant pandas was analyzed for estrogen, progestogens and androgens (via testosterone and DHEA assays), and hormone concentrations were corrected against urinary specific gravity. Across proestrus, estrogens increased while progestogens and androgens decreased - at the point of entry into proestrus, androgens (as detected by the testosterone assay) decreased prior to progestogens and gave 4 days advanced warning of proestrus. At the time of peak estrus, androgens (as detected by the DHEA assay) were significantly increased at the time of the decrease in estrogen metabolites from the peak, acting as an alternative confirmatory indicator of the fertile window. This novel finding allows for enlargement of the preparative window for captive breeding and facilitates panda management within breeding programmes. Androgens allow an enhanced monitoring of giant panda estrus, not only advancing the warning of impending proestrus, but also prospectively identifying peak fertility. Lay summary: Giant pandas have one chance at pregnancy per year. The 2-day fertile window timing varies by year and panda. This is monitored by measuring the level of estrogens in the urine, which increase, indicating an upcoming fertile period. After 1-2 weeks of increase, estrogens peak and fall, marking the optimal fertile time. We tested other hormones to see if we can predict the fertile window in advance, and the specific fertile time with more accuracy. In 20 breeding seasons from 5 females, we found androgens, usually thought of as male hormones, had an important role. Testosterone gives 4 days advanced warning of estrogens increasing. DHEA identified peak estrogen and the fertile time before needing to see a confirmed decrease in estrogen itself. Therefore, androgens help improve monitoring of the giant panda breeding season, giving early warning of fertility, key in facilitating captive breeding and giant panda conservation.

Leukemia inhibitory factor-receptor signalling negatively regulates gonadotrophin-stimulated testosterone production in mouse Leydig Cells.

Testicular Leydig cells (LCs) are the principal source of circulating testosterone in males. LC steroidogenesis maintains sexual function, fertility and general health, and is influenced by various paracrine factors. The leukemia inhibitory factor receptor (LIFR) is expressed in the testis and activated by different ligands, including leukemia inhibitory factor (LIF), produced by peritubular myoid cells. LIF can modulate LC testosterone production in vitro under certain circumstances, but the role of consolidated signalling through LIFR in adult LC function in vivo has not been established. We used a conditional Lifr allele in combination with adenoviral vectors expressing Cre-recombinase to generate an acute model of LC Lifr-KO in the adult mouse testis, and showed that LC Lifr is not required for short term LC survival or basal steroidogenesis. However, LIFR-signalling negatively regulates steroidogenic enzyme expression and maximal gonadotrophin-stimulated testosterone biosynthesis, expanding our understanding of the intricate regulation of LC steroidogenic function.

Survival in Patients with Paramedic-Identified ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Field identification and treatment of ST-segment elevation myocardial infarction (STEMI) by paramedics is an important component of care for these patients. There is a paucity of studies in the setting of paramedic-identified STEMI. This study investigated mortality and factors associated with mortality in a large state-wide prehospital STEMI sample. Methods: Included were adult STEMI patients identified and treated with reperfusion therapy by paramedics in the field between January 2016 and December 2018 in Queensland, Australia. 30-day and one-year all-cause mortality was compared between two prehospital reperfusion pathways: prehospital fibrinolysis versus direct referral to a hospital for primary percutaneous coronary intervention (direct percutaneous coronary intervention [PCI] referral). For prehospital fibrinolysis patients, factors associated with failed fibrinolysis were investigated. For direct PCI referral patients, factors associated with mortality were examined. Results: The 30-day mortality was 2.2% for prehospital fibrinolysis group and 1.8% for direct PCI referral group (p = 0.661). One-year mortality for the two groups was 2.7% and 3.2%, respectively (p = 0.732). Failed prehospital fibrinolysis was observed in 20.1% of patients receiving this therapy, with male gender and history of heart failure being predictors. For direct PCI referral group, low left ventricular ejection fraction (LVEF) on admission and cardiogenic shock prior to PCI were predictors of both 30-day and one-year mortality. Aboriginal and Torres Strait Islander status, and impaired kidney function on admission, were associated with one-year but not 30-day mortality. Being overweight was associated with lower 30-day mortality. Conclusions: Mortality in STEMI patients identified and treated by paramedics was low, and the prehospital fibrinolysis treatment pathway was effective with a mortality rate comparable to that of patients undergoing primary PCI. Key words: prehospital; Queensland; cardiac reperfusion; STEMI.

"We're All the Same Here"-Investigating the Rehabilitative Climate of a Re-Rolled Sexual Offender Prison: A Qualitative Longitudinal Study.

Understanding how individuals with sexual convictions experience prison and its environment is important because such experiences can affect rehabilitation outcomes. This is the first qualitative longitudinal investigation that explores the experiences of prisoners in a prison exclusively for individuals with sexual convictions over time. The purpose of this research was to explore the rehabilitative and therapeutic climate of a recently re-rolled prison (a general prison turned into a prison only for individuals who have sexually offended) at two time points (T1 at re-roll and T2 a year later). The study focuses on prisoners' perspectives of the purpose of the prison, experience of prison life, relationships in the prison, and the prison regime over time. Twenty interviews were conducted across the time points and revealed two main superordinate themes: "'Being' in a prison for individuals with sexual convictions" and "obstructions to change." This research adds to the emerging body of knowledge surrounding the importance of the wider prison environment on the rehabilitation of individuals with sexual convictions and on the benefits and risks of co-locating men who have committed sexual offenses in the same prison site. It also has implications wider than rehabilitation of those convicted of sexual offenses and has insights for the types of environment and prisoner-staff relationships that are conducive to rehabilitation.

Urinary estrogens as a non-invasive biomarker of viable pregnancy in the giant panda (Ailuropoda melanoleuca).

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development.

Cathelicidins and the Onset of Labour.

Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of neonatal mortality and morbidity. Infection and inflammation are frequent antecedents of spontaneous preterm birth. Cathelicidin, an antimicrobial host defence peptide, is induced by infection and inflammation and although expressed in the reproductive tract and fetal tissues, its role in the pathogenesis of spontaneous preterm birth is unknown. Here we demonstrate that cathelicidin expression is increased at RNA and protein level in the mouse uterus in a model of inflammation-induced labour, where ultrasound guided intrauterine injection of lipopolysaccharide (LPS) at E17 stimulates preterm delivery within 24 hours. Cathelicidin-deficient (Camp-/-) mice are less susceptible to preterm delivery than wild type mice following intrauterine injection of 1 µg of LPS, and this is accompanied by a decrease in circulating IL-6, an inflammatory mediator implicated in the onset of labour. We also show that the proportion of cathelicidin expressing cells in the myometrium is higher in samples obtained from women in labour at term than pre-labour. Together, these data suggest that cathelicidin has roles in mediating pro-inflammatory responses in a murine model of inflammation-induced labour, and in human term labour.

Confirmation of ovulation from urinary progesterone analysis: assessment of two automated assay platforms.

Urinary concentrations of the major progesterone (P4) metabolite pregnanediol-3-glucuronide (PDG) are used to confirm ovulation. We aimed to determine whether automated immunoassay of urinary P4 was as efficacious as PDG to confirm ovulation. Daily urine samples from 20 cycles in 14 healthy women in whom ovulation was dated by ultrasound, and serial weekly samples from 21 women in whom ovulation was unknown were analysed. Daily samples were assayed by two automated P4 immunoassays (Roche Cobas and Abbott Architect) and PDG ELISA. Serial samples were assayed for P4 by Architect and PDG by ELISA. In women with detailed monitoring of ovulation, median (95% CI) luteal phase increase was greatest for PDG, 427% (261-661), 278% (187-354) for P4 Architect and least for P4 Cobas, 146% (130-191), p < 0.0001. Cobas P4 also showed marked inaccuracy in serial dilution. Similar ROC AUCs were observed for individual threshold values and two-sample percent rise analyses for P4 Architect and PDG (both >0.92). In serial samples classified as (an)ovulatory by PDG, P4 Architect gave ROC AUC 0.95 (95% CI 0.89 to 1.01), with sensitivity and specificity for confirmation of ovulation of 0.90 and 0.91 at a cutoff of 1.67 µmol/mol. Automated P4 may potentially be as efficacious as PDG ELISA but research from a range of clinical settings is required.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Melatonin limits paclitaxel-induced mitochondrial dysfunction in vitro and protects against paclitaxel-induced neuropathic pain in the rat.

Chemotherapy-induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20-100 µmol/L). Mitochondrial volume was increased dose-dependently by paclitaxel (200% increase at 100 µmol/L). These effects were prevented by co-treatment with 1 µmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co-exposure to 1 µmol/L melatonin of either the breast cancer cell line MCF-7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel-induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose-dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel-induced elevated 8-isoprostane F2 α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel-induced reduction in C-fibre activity-dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.

Inflammatory Pain Reduces C Fiber Activity-Dependent Slowing in a Sex-Dependent Manner, Amplifying Nociceptive Input to the Spinal Cord.

C fibers display activity-dependent slowing (ADS), whereby repetitive stimulation (≥1 Hz) results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. However, the impact of ADS on spinal pain processing has not been explored, nor whether ADS is altered in inflammatory pain conditions. To investigate, compound action potentials were made, from dorsal roots isolated from rats with or without complete Freund's adjuvant (CFA) hindpaw inflammation, in response to electrical stimulus trains. CFA inflammation significantly reduced C fiber ADS at 1 and 2 Hz stimulation rates. Whole-cell patch-clamp recordings in the spinal cord slice preparation with attached dorsal roots also demonstrated that CFA inflammation reduced ADS in the monosynaptic C fiber input to lamina I neurokinin 1 receptor-expressing neurons (1-10 Hz stimulus trains) without altering the incidence of synaptic response failures. When analyzed by sex, it was revealed that females display a more pronounced ADS that is reduced by CFA inflammation to a level comparable with males. Cumulative ventral root potentials evoked by long and short dorsal root stimulation lengths, to maximize and minimize the impact of ADS, respectively, demonstrated that reducing ADS facilitates spinal summation, and this was also sex dependent. This finding correlated with the behavioral observation of increased noxious thermal thresholds and enhanced inflammatory thermal hypersensitivity in females. We propose that sex/inflammation-dependent regulation of C fiber ADS can, by controlling the temporal relay of nociceptive inputs, influence the spinal summation of nociceptive signals contributing to sex/inflammation-dependent differences in pain sensitivity.SIGNIFICANCE STATEMENT The intensity of a noxious stimulus is encoded by the frequency of action potentials relayed by nociceptive C fibers to the spinal cord. C fibers conduct successive action potentials at progressively slower speeds, but the impact of this activity-dependent slowing (ADS) is unknown. Here we demonstrate that ADS is more prevalent in females than males and is reduced in an inflammatory pain model in females only. We also demonstrate a progressive delay of C fiber monosynaptic transmission to the spinal cord that is similarly sex and inflammation dependent. Experimentally manipulating ADS strongly influences spinal summation consistent with sex differences in behavioral pain thresholds. This suggests that ADS provides a peripheral mechanism that can regulate spinal nociceptive processing and pain sensation.