Human muscle cells sensitivity to chikungunya virus infection relies on their glycolysis activity and differentiation stage.

Changes to our environment have led to the emergence of human pathogens such as chikungunya virus. Chikungunya virus infection is today a major public health concern. It is a debilitating chronic disease impeding patients' mobility, affecting millions of people. Disease development relies on skeletal muscle infection. The importance of skeletal muscle in chikungunya virus infection led to the hypothesis that it could serve as a viral reservoir and could participate to virus persistence. Here we questioned the interconnection between skeletal muscle cells metabolism, their differentiation stage and the infectivity of the chikungunya virus. We infected human skeletal muscle stem cells at different stages of differentiation with chikungunya virus to study the impact of their metabolism on virus production and inversely the impact of virus on cell metabolism. We observed that chikungunya virus infectivity is cell differentiation and metabolism-dependent. Chikungunya virus interferes with the cellular metabolism in quiescent undifferentiated and proliferative muscle cells. Moreover, activation of chikungunya infected quiescent muscle stem cells, induces their proliferation, increases glycolysis and amplifies virus production. Therefore, our results showed that Chikungunya virus infectivity and the antiviral response of skeletal muscle cells relies on their energetic metabolism and their differentiation stage. Then, muscle stem cells could serve as viral reservoir producing virus after their activation.

Stability of Lisdexamfetamine in Sampled Whole Blood-Implications of Sampling Tube Additives and Storage Temperature for Interpretation of Impairment.

Lisdexamfetamine (LDX) is a prodrug that is enzymatically converted into dextroamphetamine (d-AMP), a central nervous system stimulant. The stability of LDX in sampled whole blood is an important issue that may be crucial in the assessment of impaired driving caused by d-AMP. This study investigated the stability of LDX in whole blood collected in two different tubes containing a fluoride oxalate (FX) mixture and a fluoride citrate (FC) mixture. Without additives, LDX was unstable. LDX was also unstable in FX blood stored at ambient temperature or 4°C. After 3 days of storage at ambient temperature, an initial LDX concentration of 47 ± 1 ng/g (mean ± SD) was no longer detectable in the samples (n = 3). Instead, 19 ± 0.6 ng/g d-AMP was formed. The stability was improved at 4°C. After 7 days of storage at 4°C, 88 ± 5% of an initial LDX concentration of 50 ± 0.4 ng/g was recovered and 3.8 ± 0.3 ng/g d-AMP was formed. The stability of LDX was greater in FC blood than in FX blood; 79 ± 10% and 93 ± 4% of initial LDX concentrations of 48 ± 2 and 51 ± 0.5 ng/g were recovered from FC blood after 7 days of storage at ambient temperature and 4°C, respectively, and the corresponding formation of d-AMP was 5.8 ± 0.6 and 0.5 ± 0.3 ng/g, respectively. When FX and FC blood were stored at -20°C or -80°C, no detectable degradation of LDX or formation of d-AMP was observed after 3 weeks of storage.

Senicapoc treatment in COVID-19 patients with severe respiratory insufficiency-A randomized, open-label, phase II trial.

BACKGROUND: The aim of the current study was to determine if treatment with senicapoc, improves the PaO2 /FiO2 ratio in patients with COVID-19 and severe respiratory insufficiency. METHODS: Investigator-initiated, randomized, open-label, phase II trial in four intensive care units (ICU) in Denmark. We included patients aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID-19. The intervention consisted of 50 mg enteral senicapoc administered as soon as possible after randomization and again after 24 h. Patients in the control group received standard care only. The primary outcome was the PaO2 /FiO2 ratio at 72 h. RESULTS: Twenty patients were randomized to senicapoc and 26 patients to standard care. Important differences existed in patient characteristics at baseline, including more patients being on non-invasive/invasive ventilation in the control group (54% vs. 35%). The median senicapoc concentration at 72 h was 62.1 ng/ml (IQR 46.7-71.2). The primary outcome, PaO2 /FiO2 ratio at 72 h, was significantly lower in the senicapoc group (mean 19.5 kPa, SD 6.6) than in the control group (mean 24.4 kPa, SD 9.2) (mean difference -5.1 kPa [95% CI -10.2, -0.04] p = .05). The 28-day mortality in the senicapoc group was 2/20 (10%) compared with 6/26 (23%) in the control group (OR 0.36 95% CI 0.06-2.07, p = .26). CONCLUSIONS: Treatment with senicapoc resulted in a significantly lower PaO2 /FiO2 ratio at 72 h with no differences for other outcomes.

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

A validated UHPLC-MS/MS method for rapid determination of senicapoc in plasma samples.

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-µL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.

Entrapment of drugs in dental calculus - Detection validation based on test results from post-mortem investigations.

For prospective investigation of drugs and metabolites in archaeological and contemporary dental calculus, a sensitive, broadly applicable ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) was developed. The dental calculus was treated with citric acid and the dissolution extracts were cleaned using weak and strong polymeric cation-exchange sorbents. The method was validated on hydroxyapatite for the analysis of 67 drugs and metabolites. Typically, the lower limits of quantification were in the range of 0.01-0.05ng for the sample mass extracted. The general applicability of the method was tested using dental calculus material sampled from 10 corpses undergoing forensic autopsy. The calculus material was washed several times before dissolution to remove residual substances originating from saliva, gingival crevicular fluid and blood. The wash extracts and the calculus samples (cleaned calculus material) were analysed using the same instrumental conditions. The dry mass of the calculus samples ranged from 1 to 10mg. The total number of drug detections was 131 in the dental calculus samples and 117 in the whole blood samples. From the analyses of the wash extracts and calculus samples, it was proven that drug residues were trapped in the interior of the calculus material. In 82 of the drug detections, the drug concentrations were higher in the dental calculus than in the blood. Among substances detected in the dental calculus but not in the blood were cocaine, heroin, 6-MAM and THCA-A.

Effect of a critical thinking intervention on stress management among undergraduates of adult education and extramural studies programs.

OBJECTIVE: The objective of this study was to examine the effect of a critical thinking intervention (CTI) on stress management among undergraduates of adult education and extramural studies programs. METHOD: A total of 44 undergraduates were randomly sorted into experimental and waitlist control groups. We used the Perceived Stress Scale for data collection at the pre-test, post-test, and follow-up stages. We used unpaired t and paired t-tests to analyze the data collected. SPSS version 22.0 was used for the data analyses (SPSS Inc., Chicago, IL). RESULTS: It was shown that the CTI was effective in reducing the mean stress of the participants compared to the control group both in the post-test (t[42] = -22.453, P < .001) and follow-up periods (t[42] = -34.292, P < .001). There were statistically significant changes in the mean stress of participants in the experimental group from the pre-test to post-test phases (t[23] = 26.30, P = .000, r = .08], and from pre-test to follow-up(t[23] = 37.10, P = .000, r = .30). The mean stress of the participants in the experimental group from post-test to follow-up signified the sustained positive influence of the CTI on the mean stress (t[23] = 2.41, P = .000, r = .46) of the undergraduates. CONCLUSION: This study adds to the literature by showing that a CTI is a valuable strategy for stress reduction in a university environment. Given that the CTI demonstrated the ability to reduce stress among undergraduates enrolled in adult education and extramural studies programs, we hope that similar interventions will be adopted to manage and prevent stress among students in other departments and disciplines.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

Enriched environment exposure accelerates rodent driving skills.

Although rarely used, long-term behavioral training protocols provide opportunities to shape complex skills in rodent laboratory investigations that incorporate cognitive, motor, visuospatial and temporal functions to achieve desired goals. In the current study, following preliminary research establishing that rats could be taught to drive a rodent operated vehicle (ROV) in a forward direction, as well as steer in more complex navigational patterns, male rats housed in an enriched environment were exposed to the rodent driving regime. Compared to standard-housed rats, enriched-housed rats demonstrated more robust learning in driving performance and their interest in the ROV persisted through extinction trials. Dehydroepiandrosterone/corticosterone (DHEA/CORT) metabolite ratios in fecal samples increased in accordance with training in all animals, suggesting that driving training, regardless of housing group, enhanced markers of emotional resilience. These results confirm the importance of enriched environments in preparing animals to engage in complex behavioral tasks. Further, behavioral models that include trained motor skills enable researchers to assess subtle alterations in motivation and behavioral response patterns that are relevant for translational research related to neurodegenerative disease and psychiatric illness.

Ascorbic Acid Improves the Stability of Buprenorphine in Frozen Whole Blood Samples.

Buprenorphine (BUP) was not long-term stable in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures when stored at -20°C. On average, only half of the initial concentrations of BUP was recovered after 12 weeks of storage at -20°C when interrupted by 3-4 thaw/freeze cycles. Norbuprenorphine (NBUP) was less unstable; approximately 90% was recovered after 12 weeks of storage at -20°C. The instability was less at 5°C, but the formation of BUP and NBUP from their glucuronides was observed at that temperature, especially in FC-preserved blood. The substances were stable for at least 5 months when stored uninterrupted at -80°C. The instability of BUP and NBUP in FC- and FX-preserved whole blood stored at -20°C was eliminated when the samples were modified with 30 mM ascorbic acid (ASC) prior to storage. The mean recoveries were greater than 95% after a 5-month interrupted storage period at -20°C when modified with ASC.

Sensitive determination of monoamine neurotransmitters, their main metabolites and precursor amino acids in different mouse brain components by liquid chromatography-electrospray tandem mass spectrometry after selective sample clean-up.

For the assessment of diets and supplements formulated for the treatment of phenylketonuria, a highly sensitive and selective method was developed and validated for the quantification of dopamine (DA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), phenylalanine, tyrosine and tryptophan in mouse cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform cortex and bulbus olfactorius. Samples were extracted by deproteinization with acetonitrile, and the extracts were cleaned up by strong anion exchange and weak cation exchange applied sequentially. The substances were detected by rapid liquid chromatography tandem mass spectrometry. Matrix components were largely removed by the clean-up, resulting in low matrix effects. The lower limits of quantification for an extracted tissue mass of 100 mg were 0.3, 0.3, 0.2 and 2 ng/g for DA, 5-HT, 5-HIAA and DOPAC, respectively. The mean true extraction recoveries were 80-102%. The relative intra-laboratory reproducibility standard deviations were generally <11% at concentrations of 20-1000 ng/g for DA, 5-HT, 5-HIAA and DOPAC and 7% at concentrations of 5-50 µg/g for the amino acids. This method was successfully used in a phenylketonuria mice study including nearly 300 brain tissue samples and for small sample masses (for example, 2 mg of bulbus olfactorius).

Efficacy of a Mediterranean diet supplemented with fatty fish in ameliorating inflammation in paediatric asthma: a randomised controlled trial.

BACKGROUND: Childhood asthma is the most common respiratory disorder worldwide, being associated with increased morbidity and a decreased quality of life. Omega-3 fatty acids have anti-inflammatory and immunomodulating properties; however, their efficacy in asthma is controversial. The present study aimed to examine the efficacy of a Mediterranean diet supplemented with a high omega-3 'fatty' fish intake in Greek asthmatic children. METHODS: A single-centred, 6-month, parallel randomised controlled trial compared the consumption of a Mediterranean diet supplemented with two meals of 150 g of cooked fatty fish weekly (intervention) with the usual diet (control) with respect to pulmonary function in children (aged 5-12 years) with mild asthma. Pulmonary function was assessed using spirometry and bronchial inflammation by fractional exhaled nitric oxide analysis. RESULTS: Sixty-four children (52% male, 48% female) successfully completed the trial. Fatty fish intake increased in the intervention group from 17 g day-1 at baseline to 46 g day-1 at 6 months (P < 0.001). In the unadjusted analysis, the effect of the intervention was of borderline significance (P = 0.06, ß = -11.93; 95% confidence interval = -24.32 to 0.46). However, after adjusting for age, sex, body mass index and regular physical activity, a significant effect was observed (P = 0.04, ß = -14.15 ppb; 95% confidence interval = -27.39 to -0.91). No difference was observed for spirometry, asthma control and quality of life scores. CONCLUSIONS: A Mediterranean diet supplemented with two fatty fish meals per week might be a potential strategy for reducing airway inflammation in childhood asthma. Future robust clinical trials are warranted to replicate and corroborate these findings.

Contingency Training Alters Neurobiological Components of Emotional Resilience in Male and Female Rats.

Prior research with a rat model of behavioral therapy [i.e., effort-based reward (EBR) contingency training] suggests that strengthened associations between physical effort and desired outcomes enhance neurobiological indices of resilience. In the current study, male and female Long-Evans rats were exposed to either six weeks of EBR training or noncontingent training prior to 10 days of exposure to chronic unpredictable stress (CUS). Subsequently, all animals were exposed to a problem-solving task and then trained in a spatial learning/foraging task, the Dry Land Maze (DLM). Following habituation training and test trials, rats were assessed in a probe trial that generated a prediction error (cognitive uncertainty). Results indicated that, during CUS exposure, contingency-training enhanced dehydroepiandrosterone/corticosterone ratios (consistent with healthier stress responses), especially in male rats. Additionally, contingency training increased exploratory behaviors in the probe trial as well as differentially influenced on-task problem-solving performance in males and females. Following the probe trial, brains were exposed to histological analyses to determine the effects of sex and contingency training on various neurobiological markers. Contingency training decreased BDNF-immunoreactivity (ir) in the hippocampus CA1 and lateral habenula, implicating differential neuroplasticity responses in the training groups. Further, coordinated fos-ir activation in areas associated with emotional resilience (i.e., motivation-regulation) was observed in contingent-trained animals. In sum, the current findings confirm that behavioral training is associated with neurobiological markers of emotional resilience; however, further assessments are necessary to more accurately determine the therapeutic potential for the EBR contingency training model.

Outdoor pollen is a trigger of child and adolescent asthma emergency department presentations: A systematic review and meta-analysis.

BACKGROUND: In the context of increased asthma exacerbations associated with climatic changes such as thunderstorm asthma, interest in establishing the link between pollen exposure and asthma hospital admissions has intensified. Here, we systematically reviewed and performed a meta-analysis of studies on pollen and emergency department (ED) attendance. METHODS: A search for studies with appropriate search strategy in MEDLINE, EMBASE, Web of Science and CINAHL was conducted. Each study was assessed for quality and risk of bias. The available evidence was summarized both qualitatively and meta-analysed using random-effects models when moderate heterogeneity was observed. RESULTS: Fourteen studies were included. The pollen taxa investigated differed between studies, allowing meta-analysis only of the effect of grass pollen. A statistically significant increase in the percentage change in the mean number of asthma ED presentations (MPC) (pooled results from 3 studies) was observed for an increase in 10 grass pollen grains per cubic metre of exposure 1.88% (95% CI = 0.94%, 2.82%). Time series studies showed positive correlations between pollen concentrations and ED presentations. Age-stratified studies found strongest associations in children aged 5-17 years old. CONCLUSION: Exposure to ambient grass pollen is an important trigger for childhood asthma exacerbations requiring ED attendance. As pollen exposure is increasingly a problem especially in relation to thunderstorm asthma, studies with uniform measures of pollen and similar analytical methods are necessary to fully understand its impact on human health.

The effect of antioxidants on the long-term stability of THC and related cannabinoids in sampled whole blood.

The stability of cannabinoids is complex and crucial for the assessment of impaired driving caused by cannabis. Therefore, the effect of antioxidants on the long-term stability of Δ9 -tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-Δ9 -tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures was investigated at different temperatures. The measured concentrations of the cannabinoids in authentic whole blood preserved solely with FC or FX mixtures decreased significantly during prolonged storage at -20°C. On average, less than 5% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The rate of decrease was greatest in FC-preserved blood. The repeated thawing/freezing of the samples accelerated the instability progression. At 5°C approximately 60% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage. No significant decrease was observed in samples stored at -80°C during the test period of 5 months. The instability at -20°C was to a great extend avoided by adding 30 mM ascorbic acid (ASC) to the samples before storage. Samples preserved with a combination of the FX mixture and ASC showed no significant decrease in the recovered concentrations during a 5-month storage period interrupted by 6 thawing/freezing cycles. Samples preserved with a combination of the FC mixture and ASC showed almost similar improvements in cannabinoid stability. Other reducing agents such as sodium metabisulfite and glutathione also improved the stability in FX-preserved blood stored at -20°C.

Hormone-secreting adrenal tumours cause severe hypertension and high rates of poor pregnancy outcome; a UK Obstetric Surveillance System study with case control comparisons.

OBJECTIVE: To examine the management and outcomes of adrenal tumours in pregnancy. DESIGN: A national observational, cohort study over 4 years using the UK Obstetric Surveillance System (UKOSS). SETTING: Consultant-led obstetric units. PATIENTS: Women with phaeochromocytoma, primary aldosteronism or Cushing's syndrome diagnosed before or during pregnancy. METHODS: Clinical features of UKOSS cases were compared with those of women with adrenal tumours reported from 1985-2015. Nested case-control comparisons involving the UKOSS cases as well as those identified in the literature were performed for pregnancy outcome data using UKOSS controls with uncomplicated singleton (n = 2250) pregnancy and data from the Office of National Statistics (ONS). MAIN OUTCOME MEASURES: Incidence, management and frequency of adverse maternal and offspring outcomes of adrenal tumours in pregnancy. RESULTS: Fifteen pregnant women met the inclusion criteria: ten phaeochromocytoma, three primary aldosteronism and two Cushing's syndrome. All of the tumours had an incidence rate <2 per 100 000 pregnancies. Clinical symptoms were similar to those in non-pregnant women due to the hormones released. All women had severe hypertension, and in those diagnosed in pregnancy prior to conception. There was a significantly increased risk of adverse pregnancy outcomes in affected women, with increased rates of stillbirth, preterm labour and operative delivery. CONCLUSIONS: Adrenal tumours are associated with increased risks for pregnant women and their babies. Data on these tumours to inform practice are limited and international collaborative efforts are likely to be needed. TWEETABLE ABSTRACT: Study of hormone-secreting adrenal tumours in pregnancy linked with high BP and high rates of fetal morbidity.

cGAS is activated by DNA in a length-dependent manner.

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.