Corticotropin-stimulated steroid profiles to predict shock development and mortality in sepsis: From the HYPRESS study.

RATIONALE: Steroid profiles in combination with a corticotropin stimulation test provide information about steroidogenesis and its functional reserves in critically ill patients. OBJECTIVES: We investigated whether steroid profiles before and after corticotropin stimulation can predict the risk of in-hospital death in sepsis. METHODS: An exploratory data analysis of a double blind, randomized trial in sepsis (HYPRESS [HYdrocortisone for PRevention of Septic Shock]) was performed. The trial included adult patients with sepsis who were not in shock and were randomly assigned to placebo or hydrocortisone treatment. Corticotropin tests were performed in patients prior to randomization and in healthy subjects. Cortisol and precursors of glucocorticoids (17-OH-progesterone, 11-desoxycortisol) and mineralocorticoids (11-desoxycorticosterone, corticosterone) were analyzed using the multi-analyte stable isotope dilution method (LC-MS/MS). Measurement results from healthy subjects were used to determine reference ranges, and those from placebo patients to predict in-hospital mortality. MEASUREMENTS AND MAIN RESULTS: Corticotropin tests from 180 patients and 20 volunteers were included. Compared to healthy subjects, patients with sepsis had elevated levels of 11-desoxycorticosterone and 11-desoxycortisol, consistent with activation of both glucocorticoid and mineralocorticoid pathways. After stimulation with corticotropin, the cortisol response was subnormal in 12% and the corticosterone response in 50% of sepsis patients. In placebo patients (n = 90), a corticotropin-stimulated cortisol-to-corticosterone ratio > 32.2 predicted in-hospital mortality (AUC 0.8 CI 0.70-0.88; sensitivity 83%; and specificity 78%). This ratio also predicted risk of shock development and 90-day mortality. CONCLUSIONS: In this exploratory analysis, we found that in sepsis mineralocorticoid steroidogenesis was more frequently impaired than glucocorticoid steroidogenesis. The corticotropin-stimulated cortisol-to-corticosterone ratio predicts the risk of in-hospital death. Trial registration Clinical trial registered with www. CLINICALTRIALS: gov Identifier: NCT00670254. Registered 1 May 2008, https://clinicaltrials.gov/ct2/show/NCT00670254 .

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

AIMS: Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. METHODS AND RESULTS: A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. CONCLUSION: This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

The impact of low back pain on health-related quality of life in old age: results from a survey of a large sample of Swiss elders living in the community.

PURPOSE: The present study aims at investigating the effects of low back pain (LBP), i.e., type of symptoms, activity limitations, frequency, duration, and severity on health-related quality of life (HRQoL) in a sample of 707 community-dwelling men and women aged ≥ 65 years living in Switzerland. METHODS: The study is part of a larger survey conducted in Switzerland on a sample of older adults selected randomly from population records, stratified by age and sex. The Standardized Back Pain Definition was used to investigate LBP, and HRQoL was assessed by means of the EQ-5D, including Health Utility Index (HUI) measures. RESULTS: For more than half of the sufferers, pain was chronic, occurred most days or every day and induced activity limitations. One-third of the sufferers reported sciatica symptoms. Individuals reporting every day pain, severe pain and more than 3 years since the last episode without pain lost nearly 10 points of HRQoL. Amongst the dimension of HRQoL, Mobility was the most affected by LBP. CONCLUSIONS: These results provide further insight into the impact of qualitative aspects of LBP and in particular the importance of radiating leg pain and pain frequency and duration. While LBP-related activity limitations had little impact on both self-rated overall health and HUI, radiating leg pain and pain frequency and duration were associated with significantly decreased scores on both dimensions.

MicroRNA-146a controls Th1-cell differentiation of human CD4+ T lymphocytes by targeting PRKCε.

T-cell functions must be tightly controlled to keep the balance between vital proinflammatory activity and detrimental overactivation. MicroRNA-146a (miR-146a) has been identified as a key negative regulator of T-cell responses in mice. Its role in human T cells and its relevance to human inflammatory disease, however, remains poorly defined. In this study, we have characterized miR-146a-driven pathways in primary human T cells. Our results identify miR-146a as a critical gatekeeper of Th1-cell differentiation processes acting via molecular mechanisms not uncovered so far. MiR-146a targets protein kinase C epsilon (PRKCε), which is part of a functional complex consisting of PRKCε and signal transducer and activator of transcription 4 (STAT4). Within this complex, PRKCε phosphorylates STAT4, which in turn is capable of promoting Th1-cell differentiation processes in human CD4(+) T lymphocytes. In addition, we observed that T cells of sepsis patients had reduced levels of miR-146a and an increased PRKCε expression in the initial hyperinflammatory phase of the disease. Collectively, our results identify miR-146a as a potent inhibitor of Th1-cell differentiation in human T cells and suggest that dysregulation of miR-146a contributes to the pathogenesis of sepsis.

miR-124a and miR-155 enhance differentiation of regulatory T cells in patients with neuropathic pain.

BACKGROUND: Accumulating evidence indicates that neuropathic pain is a neuro-immune disorder with enhanced activation of the immune system. Recent data provided proof that neuropathic pain patients exhibit increased numbers of immunosuppressive regulatory T cells (Tregs), which may represent an endogenous attempt to limit inflammation and to reduce pain levels. We here investigate the molecular mechanisms underlying these alterations. METHODS: Our experimental approach includes functional analyses of primary human T cells, 3'-UTR reporter assays, and expression analyses of neuropathic pain patients' samples. RESULTS: We demonstrate that microRNAs (miRNAs) are involved in the differentiation of Tregs in neuropathic pain. We identify miR-124a and miR-155 as direct repressors of the histone deacetylase sirtuin1 (SIRT1) in primary human CD4(+) cells. Targeting of SIRT1 by either specific siRNA or by these two miRNAs results in an increase of Foxp3 expression and, consecutively, of anti-inflammatory Tregs (siRNA: 1.7 ± 0.4; miR-124a: 1.5 ± 0.4; miR-155: 1.6 ± 0.4; p < 0.01). As compared to healthy volunteers, neuropathic pain patients exhibited an increased expression of miR-124a (2.5 ± 0.7, p < 0.05) and miR-155 (1.3 ± 0.3; p < 0.05) as well as a reduced expression of SIRT1 (0.5 ± 0.2; p < 0.01). Moreover, the expression of these two miRNAs was inversely correlated with SIRT1 transcript levels. CONCLUSIONS: Our findings suggest that in neuropathic pain, enhanced targeting of SIRT1 by miR-124a and miR-155 induces a bias of CD4(+) T cell differentiation towards Tregs, thereby limiting pain-evoking inflammation. Deciphering miRNA-target interactions that influence inflammatory pathways in neuropathic pain may contribute to the discovery of new roads towards pain amelioration. TRIAL REGISTRATION: German Clinical Trial Register DRKS00005954.

Differential expression of P2X7 receptor and IL-1ß in nociceptive and neuropathic pain.

BACKGROUND: Despite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1ß in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy. METHODS: P2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1ß serum levels were measured with a multiplex cytokine assay. RESULTS: Compared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 ± 6.2, healthy volunteers: 17.0 ± 5.4; p = 0.002) and lymphocytes (NeP: 21.8 ± 6.5, healthy volunteers: 15.6 ± 5.2; p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1ß serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04). CONCLUSIONS: A significant upregulation of P2X7R and increased IL-1ß release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain. German Clinical Trial Register (DRKS): Registration Trial DRKS00005954.

Antimicrobial susceptibility monitoring of dermatological bacterial pathogens isolated from diseased dogs and cats across Europe (ComPath results).

AIMS: The ComPath project is a pan-European programme dedicated to the monitoring of antimicrobial susceptibility of pathogens from diseased dogs and cats using standardized methods and centralized minimum inhibitory concentration (MIC) determination. Here, the susceptibility of major pathogens is reported from antimicrobial nontreated animals with acute clinical signs of skin, wound or ear infections in 2008-2010. METHODS AND RESULTS: MICs were determined by agar dilution for commonly used antibiotics and interpreted using CLSI breakpoints, if available. Of the 1408 strains recovered, the main canine species was Staphylococcus pseudintermedius, followed by Pseudomonas and Streptococcus. In cats, Pasteurella multocida and Staph. pseudintermedius were most prevalent. For Staph. pseudintermedius, resistance was 18·4-25·2% for penicillin, clindamycin and chloramphenicol, but below 11% for ampicillin, amoxi/clav and fluoroquinolones. For Staphylococcus aureus, beta-lactam resistance was high (26·7-62·1%) but low (0·0-4·4%) for other antibiotics. 6·3% of Staph. pseudintermedius and 5·4% of Staph. aureus were confirmed mecA-positive. Gentamicin and fluoroquinolones exhibited moderate activity against Pseudomonas aeruginosa. For streptococci, resistance was absent/very low for penicillin, ampicillin, chloramphenicol and fluoroquinolones. For Escherichia coli, resistance was low to fluoroquinolones, chloramphenicol and gentamicin. No resistance was observed in Past. multocida. CONCLUSIONS: Overall, antimicrobial resistance was low in skin and soft tissue infections in dogs and cats. The results show the need for ongoing monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are a reference baseline for future surveillance. The paucity of clinical breakpoints underlines the need to set breakpoints for relevant antibiotics.

Low back pain and health-related quality of life in community-dwelling older adults.

PURPOSE: Investigation of self-reported of low back pain (LBP) over the last month and associated health-related quality of life (HRQoL) in a sample of a community-dwelling population aged ≥65. METHODS: Cross-sectional study including older adults selected randomly from population records. Data were collected within a sample stratified by age and sex. Physical and psychological healths were investigated using a standardized definition of LBP and the EuroQoL-5D for HRQoL. Analyses were first conducted on the entire sample (N = 3042) and subsequently considering the subsample who reported LBP and a paired sample drawn from the pool of LBP-free respondents. RESULTS: 889 (29 %) respondents reported LBP within the past month, present 'most days' or 'every day' in 52 % and limiting activities in the same proportion. Average pain score was 4.6 (SD 2.2; 0-10 scale). Age was associated with pain frequency and duration, with younger groups more often reporting pain 'some days' and 'dating back <3 months'. Results of regression analyses showed that individuals suffering from LBP had significantly more problems than LBP non-sufferers on all EQ-5D subscales, except self-care: pain/discomfort (OR 5.33; 95 % CI [4.19-6.79]), mobility (OR 2.66; 95 % CI [2.04-3.46]), usual activities (OR 1.92; 95 % CI [1.42-2.60]), anxiety/depression (OR 1.59; 95 % CI [1.23-2.04]) and self-care (OR 1.29; 95 % CI [0.84-1.98]). CONCLUSION: LBP appears to be a more permanent condition in the older groups. LBP may be a part of the definition of a subgroup of elderly at risk of becoming frail in relation with higher levels of functional limitations, psychological difficulties and social restrictions, hence globally impaired HRQoL.

Patterns of early resuscitation associated with mortality after penetrating injuries.

BACKGROUND: Penetrating injuries are rare in European populations so their management represents a particular challenge. The aim was to assess early therapeutic aspects that are associated with favourable outcomes in patients with penetrating trauma. METHODS: Patients with penetrating injuries documented from 2009 to 2013 in the TraumaRegister DGU® were analysed. Patients with a primary admission and an Injury Severity Score (ISS) of at least 9 were included. The Revised Injury Severity Classification (RISC) II score was used for mortality prediction, and a standardized mortality ratio (SMR) calculated per hospital. Hospitals with favourable outcome (SMR below 1) were compared with those with poor outcome (SMR 1 or more). RESULTS: A total of 50 centres had favourable outcome (1242 patients; observed mortality rate 15.7 per cent) and 34 centres had poor outcome (918 patients; observed mortality rate 24.4 per cent). Predicted mortality rates according to RISC-II were 20.4 and 20.5 per cent respectively. Mean(s.d.) ISS values were 22(14) versus 21(14) (P = 0.121). Patients in the favourable outcome group had a significantly shorter time before admission to hospital and a lower intubation rate. They received smaller quantities of intravenous fluids on admission to the emergency room, but larger amounts of fresh frozen plasma, and were more likely to receive haemostatic agents. A higher proportion of patients in the favourable outcome group were treated in a level I trauma centre. Independent risk factors for hospital death following penetrating trauma identified by multivariable analysis included gunshot injury mechanism and treatment in non-level I centres. CONCLUSION: Among penetrating traumas, gunshot injuries pose an independent risk of death. Treatment of penetrating trauma in a level I trauma centre was significantly and independently associated with lower hospital mortality.

[Review of the Literature for Interpretation of Endobronchial Wound Healing after Tracheobronchial Sleeve Resection]. / Literaturbasierte Empfehlung zur bronchoskopischen Interpretation einer Anastomose nach Manschettenresektion.

Sleeve resection comprises 3.1 % to 27.7 % of all anatomic lung resections performed in Germany. Anastomotic insufficiency is a feared complication that should be avoided. When anastomotic insufficiency does lead to secondary pneumonectomy, postoperative morbidity and mortality is high (30 % to 80 %). It is therefore very important to standardize the technique of sleeve resection as well as postoperative care. The time-point of postoperative follow-up and the interpretation of endobronchial healing have not yet been defined. In this paper anastomotic healing is described and interpreted with the help of a 5-step classification that allows bronchoscopic evaluation and classification of the anastomosis. The aim is to provide a standardized algorithm for postoperative care after sleeve resection. The basis of this classification and postoperative care measures derived from it are described and illustrated with the help of clinical examples.

[Reduction of Tracheotomy Associated Tracheal Stenosis by Surgical Closure of the Tracheostomy]. / Reduktion Tracheostoma-assoziierter Trachealstenosen durch chirurgischen Stomaverschluss.

BACKGROUND: The most common long-term complication of tracheotomy is the benign stenosis of the trachea, which is described for up to 20% of the cases. Typically, the stenosis occurs after decannulation in the context of secondary wound healing. This study examined whether the closure of the tracheostomy by surgical procedure reduces stenosis. METHOD: With the help of our clinical database a retrospective analysis of 401 surgical tracheotomies was performed. Variables that were recorded were the indication for tracheotomy, the clinical course and complications occurred. RESULTS: 155 patients were successfully decannulated. In 92 of these patients the tracheostomy was closed by a surgical procedure, in 63 cases the closure occurred spontaneously by wound healing. After decannulation 3% (n=3) of the surgically closed and 22% (n=14) of the spontaneously closed tracheostomies developed a symptomatic tracheal stenosis (p<0.001). CONCLUSION: Secondary wound healing of the tracheostomy often leads to symptomatic tracheal stenosis. The incidence of symptomatic tracheal stenosis was significantly reduced applying closure of the tracheostomy by surgical procedure.

Contrasting adaptive strategies to terminal drought-stress gradients in Mediterranean legumes: phenology, productivity, and water relations in wild and domesticated Lupinus luteus L.

Our understanding of within-species annual plant adaptation to rainfall gradients is fragmented. Broad-scale ecological applications of Grime's C-S-R triangle are often superficial, while detailed drought physiology tends to be narrow, focusing on elite cultivars. The former lack the detail to explain how plants respond, while the latter provide little context to investigate trade-offs among traits, to explain where/why these might be adaptive. Ecophysiology, combining the breadth of the former with the detail of the latter, can resolve this disconnect and is applied here to describe adaptive strategies in the Mediterranean legume Lupinus luteus. Wild and domesticated material from low- and high-rainfall environments was evaluated under contrasting terminal drought. These opposing environments have selected for contrasting, integrated, adaptive strategies. Long-season, high-rainfall habitats select for competitive (C) traits: delayed phenology, high above- and below-ground biomass, productivity, and fecundity, leading to high water-use and early stress onset. Terminal drought-prone environments select for the opposite: ruderal (R) traits that facilitate drought escape/avoidance but limit reproductive potential. Surprisingly, high-rainfall ecotypes generate lower critical leaf water potentials under water deficit, maintaining higher relative water content than the latter. Given that L. luteus evolved in sandy, low-water-holding capacity soils, this represents a bet-hedging response to intermittent self-imposed water-deficits associated with a strongly C-selected adaptive strategy that is therefore redundant in R-selected low-rainfall ecotypes. Domesticated L. luteus is even more R-selected, reflecting ongoing selection for early maturity. Introgression of appropriate C-selected adaptive traits from wild germplasm may widen the crop production range.

Toroidal-spiral particles for codelivery of anti-VEGFR-2 antibody and irinotecan: a potential implant to hinder recurrence of glioblastoma multiforme.

Heterogeneous toroidal-spiral particles (TSPs) were generated by polymer droplet sedimentation, interaction, and cross-linking. TSPs provide a platform for encapsulation and release of multiple compounds of different sizes and physicochemical properties. As a model system, we demonstrate the encapsulation and independently controlled release of an anti-VEGFR-2 antibody and irinotecan for the treatment of glioblastoma multiforme. The anti-VEGFR-2 antibody was released from the TS channels and its binding to HUVECs was confirmed by confocal microscopy and flow cytometry, suggesting active antibody encapsulation and release. Irinotecan, a small molecule drug, was released from the dense polymer matrix of poly(ethylene glycol) diacrylate (MW ~ 700 g/mol; PEGDA 700). Released irinotecan inhibited the proliferation of U251 malignant glioma cells. Since the therapeutic compounds are released through different pathways, specifically diffusion through the polymer matrix versus TS channels, the release rate can be controlled independently through the design of the structure and material of particle components.

Association between intraoperative hypotension and postoperative nausea and vomiting: a retrospective cohort study.

OBJECTIVE: Postoperative nausea and vomiting (PONV) occurs in up to 30% of patients and its pathophysiology and mechanisms have not been completely described. Hypotension and a decrease in cardiac output are suspected to induce nausea. The hypothesis that intraoperative hypotension might influence the incidence of PONV was investigated. MATERIAL AND METHODS: The study was conducted as a retrospective large single center cohort study. The incidence of PONV was investigated until discharge from post anesthesia care unit (PACU). Surgical patients with general anesthesia during a 2-year period between 2018 and 2019 at a university hospital in Germany were included. Groups were defined based on the lowest documented mean arterial pressure (MAP) with group H50: MAP <50mmHg; group H60: MAP <60mmHg; group H70: MAP <70mmHg, and group H0: no MAP <70mmHg. Decreases of MAP in the different groups were related to PONV. Propensity-score matching was carried out to control for overlapping risk factors. RESULTS: In the 2-year period 18.674 patients fit the inclusion criteria. The overall incidence of PONV was 11%. Patients with hypotension had a significantly increased incidence of PONV (H0 vs. H50: 11.0% vs.17.4%, Risk Ratio (RR): 1.285 (99%CI: 1.102-1.498), p < 0.001; H0 vs. H60: 10.4% vs. 13.5%, RR: 1.1852 (99%CI: 1.0665-1.3172), p < 0.001; H0 vs. H70: 9.4% vs. 11.2%, RR: 1.1236 (99%CI: 1.013 - 1.2454); p = 0.0027). CONCLUSION: The study demonstrates an association between intraoperative hypotension and early PONV. A more severe decrease of MAP had a pronounced effect.

In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing.

Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3'-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3'-UTR and miRNA targeting as new mechanisms of MGMT silencing.

Minimally invasive mitral valve reconstruction on the fibrillating heart for high-risk patients.

BACKGROUND AND AIM OF THE STUDY: Mitral valve surgery after previous cardiac surgery is technically demanding and risky. In patients after coronary artery bypass grafting (CABG), mitral valve surgery is associated with a high risk of injury to the bypass graft with concomitant myocardial ischemia. An aortic valve prosthesis usually severely impairs access to the mitral valve, so that these patients are often denied surgery. Furthermore, patients with porcelain aorta may be inoperable. METHODS: A series of 10 patients undergoing minimally invasive mitral valve repair via a right-sided anterolateral minithoracotomy without aortic cross-clamping on the fibrillating heart was investigated. Four patients had an aortic valve prosthesis in situ, six patients had undergone previous CABG, and two patients presented with porcelain aorta. RESULTS: Reconstruction was possible in nine patients. Cannulation was performed femorally in three patients, and via the axillary artery in seven patients. No fatalities were observed. One patient required rethoracotomy for bleeding and subsequently developed a right-sided pneumonia, and a second patient experienced lower-limb ischemia. The postoperative course of the other eight patients was uneventful. No patient presented with significant residual mitral insufficiency at control echocardiography. CONCLUSION: Minimally invasive mitral valve reconstruction via a right-sided minithoracotomy represents an attractive surgical option in a high-risk reoperative setting.

Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

Development and validation of a sleep questionnaire, SNoRE 3.0, to evaluate sleep in companion dogs.

Disturbances in the sleep-wake cycle are a debilitating, yet rather common condition not only in humans, but also in family dogs. While there is an emerging need for easy-to-use tools to document sleep alterations (in order to ultimately treat and/or prevent them), the veterinary tools which yield objective data (e.g. polysomnography, activity monitors) are both labor intensive and expensive. In this study, we developed a modified version of a previously used sleep questionnaire (SNoRE) and determined criterion validity in companion dogs against polysomnography and physical activity monitors (PAMs). Since a negative correlation between sleep time and cognitive performance in senior dogs has been demonstrated, we evaluated the correlation between the SNoRE scores and the Canine Dementia Scale (CADES, which includes a factor concerning sleep). There was a significant correlation between SNoRE 3.0 questionnaire scores and polysomnography data (latency to NREM sleep, ρ = 0.507, p < 0.001) as well as PAMs' data (activity between 1:00 and 3:00 AM, p < 0.05). There was a moderate positive correlation between the SNoRE 3.0 scores and the CADES scores (ρ = 0.625, p < 0.001). Additionally, the questionnaire structure was validated by a confirmatory factor analysis, and it also showed an adequate test-retest reliability. In conclusion the present paper describes a valid and reliable questionnaire tool, that can be used as a cost-effective way to monitor dog sleep in clinical settings.

Retro-miRs: novel and functional miRNAs originating from mRNA retrotransposition.

BACKGROUND: Reverse-transcribed gene copies (retrocopies) have emerged as major sources of evolutionary novelty. MicroRNAs (miRNAs) are small and highly conserved RNA molecules that serve as key post-transcriptional regulators of gene expression. The origin and subsequent evolution of miRNAs have been addressed but not fully elucidated. RESULTS: In this study, we performed a comprehensive investigation of miRNA origination through retroduplicated mRNA sequences (retro-miRs). We identified 17 retro-miRs that emerged from the mRNA retrocopies. Four of these retro-miRs had de novo origins within retrocopied sequences, while 13 retro-miRNAs were located within exon regions and duplicated along with their host mRNAs. We found that retro-miRs were primate-specific, including five retro-miRs conserved among all primates and two human-specific retro-miRs. All retro-miRs were expressed, with predicted and experimentally validated target genes except miR-10527. Notably, the target genes of retro-miRs are involved in key biological processes such as metabolic processes, cell signaling, and regulation of neurotransmitters in the central nervous system. Additionally, we found that these retro-miRs play a potential oncogenic role in cancer by targeting key cancer genes and are overexpressed in several cancer types, including liver hepatocellular carcinoma and stomach adenocarcinoma. CONCLUSIONS: Our findings demonstrated that mRNA retrotransposition is a key mechanism for the generation of novel miRNAs (retro-miRs) in primates. These retro-miRs are expressed, conserved, have target genes with important cellular functions, and play important roles in cancer.