LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Net water uptake within the ischemic penumbra predicts the presence of the midline shift in patients with acute ischemic stroke.

Objective: The study aimed to explore the association between midline shift (MLS) and net water uptake (NWU) within the ischemic penumbra in acute ischemic stroke patients. Methods: This was a retrospective cohort study that examined patients with anterior circulation stroke. Net water uptake within the acute ischemic core and penumbra was calculated using data from admission multimodal CT scans. The primary outcome was severe cerebral edema measured by the presence of MLS on 24 to 48 h follow-up CT scans. The presence of a significant MLS was defined by a deviation of the septum pellucidum from the midline on follow-up CT scans of at least 3 mm or greater due to the mass effect of ischemic edema. The net water uptake was compared between patients with and without MLS, followed by logistic regression analyses and receiver operating characteristics (ROCs) to assess the predictive power of net water uptake in MLS. Results: A total of 133 patients were analyzed: 50 patients (37.6%) with MLS and 83 patients (62.4%) without. Compared to patients without MLS, patients with MLS had higher net water uptake within the core [6.8 (3.2-10.4) vs. 4.9 (2.2-8.1), P = 0.048] and higher net water uptake within the ischemic penumbra [2.9 (1.8-4.3) vs. 0.2 (-2.5-2.7), P < 0.001]. Penumbral net water uptake had higher predictive performance than net water uptake of the core in MLS [area under the curve: 0.708 vs. 0.603, p < 0.001]. Moreover, the penumbral net water uptake predicted MLS in the multivariate regression model, adjusting for age, sex, admission National Institutes of Health Stroke Scale (NIHSS), diabetes mellitus, atrial fibrillation, ischemic core volume, and poor collateral vessel status (OR = 1.165; 95% CI = 1.002-1.356; P = 0.047). No significant prediction was found for the net water uptake of the core in the multivariate regression model. Conclusion: Net water uptake measured acutely within the ischemic penumbra could predict severe cerebral edema at 24-48 h.

Pharmacokinetic Drug-Drug Interaction With Coadministration of Cannabidiol and Everolimus in a Phase 1 Healthy Volunteer Trial.

When highly purified cannabidiol (CBD; Epidiolex) and the mammalian target of rapamycin inhibitor everolimus are used concomitantly in the treatment of tuberous sclerosis complex, there is evidence of a pharmacokinetic (PK) interaction, leading to increased everolimus systemic exposure. We evaluated the effect of steady-state CBD exposure following multiple clinically relevant CBD doses on everolimus PK in healthy adult participants in a single-center, fixed-sequence, open-label, phase 1 study. All participants received oral everolimus 5 mg on day 1, followed by a 7-day washout. On days 9-17, participants received CBD (100 mg/mL oral solution) at 12.5 mg/kg in the morning and evening. On the morning of day 13, participants also received a single dose of oral everolimus 5 mg. Medications were taken 30 or 45 minutes (morning or evening dose) after starting a standardized meal. Maximum concentration and area under the concentration-time curve (AUC) from time of dosing to the last measurable concentration and extrapolated to infinity, of everolimus in whole blood were estimated using noncompartmental analysis, with geometric mean ratios and 90% confidence intervals for the ratios of everolimus dosed with CBD to everolimus dosed alone. A single dose of everolimus 5 mg was well tolerated when administered with multiple doses of CBD. Log-transformed everolimus maximum concentration, AUC from time of dosing to the last measurable concentration, and AUC extrapolated to infinity values increased by ≈2.5-fold, and everolimus half-life remained largely unchanged in the presence of steady-state CBD relative to everolimus dosed alone. Everolimus blood concentration monitoring should be strongly advised with appropriate dose reduction when coadministered with CBD.

Metabolomics analysis reveals metabolite changes during freeze-drying and oven-drying of Angelica dahurica.

Angelica dahurica (Angelica dahurica Fisch. ex Hoffm.) is widely used as a traditional Chinese medicine and the secondary metabolites have significant pharmacological activities. Drying has been shown to be a key factor affecting the coumarin content of Angelica dahurica. However, the underlying mechanism of metabolism is unclear. This study sought to determine the key differential metabolites and metabolic pathways related to this phenomenon. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted metabolomics analysis was performed on Angelica dahurica that were freeze-drying (- 80 °C/9 h) and oven-drying (60 °C/10 h). Furthermore, the common metabolic pathways of paired comparison groups were performed based on KEEG enrichment analysis. The results showed that 193 metabolites were identified as key differential metabolites, most of which were upregulated under oven drying. It also displayed that many significant contents of PAL pathways were changed. This study revealed the large-scale recombination events of metabolites in Angelica dahurica. First, we identified additional active secondary metabolites apart from coumarins, and volatile oil were significantly accumulated in Angelica dahurica. We further explored the specific metabolite changes and mechanism of the phenomenon of coumarin upregulation caused by temperature rise. These results provide a theoretical reference for future research on the composition and processing method of Angelica dahurica.

Effect of short-term mindfulness-based stress reduction on sleep quality in male patients with alcohol use disorder.

Background: Sleep disturbance is one of the most prominent complaints of patients with alcohol use disorder (AUD), with more than 70% of patients with AUD reporting an inability to resolve sleep problems during abstinence. Mindfulness-based stress reduction (MBSR) has been shown to improve sleep quality and as an alternative therapy to hypnotics for sleep disorders. Objective: The aim of the present study was to evaluate the effect of short-term MBSR on sleep quality in male patients with AUD after withdrawal. Methods: A total of 91 male patients with AUD after 2 weeks of routine withdrawal therapy were randomly divided into two groups using a coin toss: the treatment group (n = 50) and the control group (n = 41). The control group was received supportive therapy, and the intervention group added with MBSR for 2 weeks on the basis of supportive therapy. Objective sleep quality was measured at baseline and 2 weeks after treatment using the cardiopulmonary coupling (CPC). Indicators related to sleep quality include total sleep time, stable sleep time, unstable sleep time, rapid eye movement (REM) sleep time, wake-up time, stable sleep latency, sleep efficiency, and apnea index. These indicators were compared by an analysis of covariance (ANCOVA) between the two groups, controlling for individual differences in the respective measures at baseline. Results: The results showed that there were no significant differences in the age [t (89) = -0.541, P = 0.590), BMI [t (89) = -0.925, P = 0.357], educational status [t (89) = 1.802, P = 0.076], years of drinking [t (89) = -0.472, P = 0.638), daily intake [t (89) = 0.892, P = 0.376], types of alcohol [χ2 (1) = 0.071, P = 0.789], scores of CIWA-AR [t (89) = 0.595, P = 0.554], scores of SDS [t (89) = -1.151, P = 0.253), or scores of SAS [t (89) = -1.209, P = 0.230] between the two groups. Moreover, compared with the control group, the total sleep time [F (1.88) = 4.788, P = 0.031) and stable sleep time [F (1.88) = 6.975, P = 0.010] were significantly increased in the treatment group. Furthermore, the average apnea index in the patients who received MBSR was significantly decreased than in the control group [F (1.88) = 5.284, P = 0.024]. Conclusion: These results suggest that short-term MBSR could improve sleep quality and may serve as an alternative treatment to hypnotics for sleep disturbance in patients with AUD after withdrawal.

EGFR promotes the apoptosis of CD4+ T lymphocytes through TBK1/Glut1 induced Warburg effect in sepsis.

INTRODUCTION: Sepsis-induced apoptosis leads to lymphopenia including the decrease of CD4+ T cells thus favoring immunosuppression. OBJECTIVES: Although epidermal growth factor receptor (EGFR) inhibitors significantly improve the survival rate of septic mice, the effect of EGFR on the function and metabolism of CD4+ T cells in sepsis remained unknown. METHODS: CD4+ T cells from septic mice and patients were assessed for apoptosis, activation, Warburg metabolism and glucose transporter 1 (Glut1) expression with or without the interference of EGFR activation. RESULTS: EGFR facilitates CD4+ T cell activation and apoptosis through Glut1, which is a key enzyme that controls glycolysis in T cells. EGFR, TANK binding kinase 1 (TBK1) and Glut1 form a complex to facilitate Glut1 transportation from cytoplasm to cell surface. Both the levels of membrane expression of EGFR and Glut1 and the activation levels of CD4+ T cells were significantly higher in patients with sepsis as compared with healthy subjects. CONCLUSION: Our data demonstrated that through its downstream TBK1/Exo84/RalA protein system, EGFR regulates Glut1 transporting to the cell surface, which is a key step for inducing the Warburg effect and the subsequent cellular activation and apoptosis of CD4+ T lymphocytes and may eventually affect the immune functional status, causing immune cell exhaustion in sepsis.

EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis.

EGFR phosphorylation is required for TLR4-mediated macrophage activation during sepsis. However, whether and how intracellular EGFR is transported during endotoxemia have largely been unknown. Here, we show that LPS promotes high levels cell surface expression of EGFR in macrophages through two different transport mechanisms. On one hand, Rab10 is required for EEA1-mediated the membrane translocation of EGFR from the Golgi. On the other hand, EGFR phosphorylation prevents its endocytosis in a kinase activity-dependent manner. Erlotinib, an EGFR tyrosine kinase inhibitor, significantly reduced membrane EGFR expression in LPS-activated macrophage. Mechanistically, upon LPS induced TLR4/EGFR phosphorylation, MAPK14 phosphorylated Rab7a at S72 impaired membrane receptor late endocytosis, which maintains EGFR membrane localization though blocking its lysosomal degradation. Meanwhile, Rab5a is also involved in the early endocytosis of EGFR. Subsequently, inhibition of EGFR phosphorylation switches M1 phenotype to M2 phenotype and alleviates sepsis-induced acute lung injury. Mechanistic study demonstrated that Erlotinib suppressed glycolysis-dependent M1 polarization via PKM2/HIF-1ɑ pathway and promoted M2 polarization through up-regulating PPARγ induced glutamine metabolism. Collectively, our data elucidated a more in-depth mechanism of macrophages activation, and provided stronger evidence supporting EGFR as a potential therapeutic target for the treatment of sepsis.

Colon-targeted oral nanoparticles based on ROS-scavenging hydroxyethyl starch-curcumin conjugates for efficient inflammatory bowel disease therapy.

Co-delivery of anti-inflammatory drugs and reactive oxygen species (ROS) scavengers by stimuli-responsive oral nanoparticles is deemed to be a favorable strategy for inflammatory bowel disease (IBD) therapy. In this study, using micelles formed by CUR conjugated hydroxyethyl starch (HES) as vehicles, dexamethasone (DEX)-loaded HES-CUR nanoparticles (DHC NPs) with desirable size, negative surface charge, good stability in the harsh gastric environment, and excellent ROS scavenging activity are developed as a colon-targeted oral formulation for treating IBD. Due to the degradation of HES in response to α-amylase overexpressed in the inflamed colon, the DHC NPs release drugs in an α-amylase-responsive manner. Meanwhile, the DHC NPs can be effectively internalized by macrophages and show excellent cytocompatibility with macrophages since they are composed of food-derived compounds. Importantly, in vivo studies reveal that the DHC NPs are capable of targeting the inflamed colon induced by dextran sulfate sodium (DSS), and the targeted and combination therapy enhances the efficacy of free DEX and significantly relieves the impairment caused by DSS-induced ulcerative colitis. Incorporating the merits of targeted drug delivery and combined therapy with an anti-inflammatory drug and ROS scavenger, the DHC NPs are promising for developing novel oral formulations for IBD therapy.

Development of a lower-sodium oxybate formulation for the treatment of patients with narcolepsy and idiopathic hypersomnia.

INTRODUCTION: Sodium oxybate (SXB) is a standard of care for cataplexy, excessive daytime sleepiness, and disrupted nighttime sleep in narcolepsy. At recommended dosages in adults (6-9 g/night), SXB increases daily dietary intake of sodium by 1100-1640 mg. Because excess sodium intake is associated with increased blood pressure and cardiovascular risk, an oxybate formulation containing 92% less sodium than SXB (lower-sodium oxybate; LXB) was developed to provide an alternative oxybate treatment option. In 2020, LXB was approved for treatment of cataplexy or excessive daytime sleepiness in patients 7 years of age and older with narcolepsy, and in 2021, for treatment of idiopathic hypersomnia in adults. AREAS COVERED: Development of LXB from initial concept to regulatory approval is described, including formulation development and preclinical and clinical studies. Pharmacokinetic parameters and bioequivalence evaluations from phase 1 clinical trials are detailed. Efficacy and safety results from phase 3 clinical trials of LXB in patients with narcolepsy or idiopathic hypersomnia are presented and discussed. EXPERT OPINION: Reducing sodium from high sodium‒containing medications is an important step to offset cardiovascular risks associated with high sodium consumption. The development of LXB exemplifies the importance of a collaborative approach to drug development, with patient needs paramount. PLAIN LANGUAGE SUMMARY: Sodium oxybate (Xyrem®) is a medication for people with narcolepsy aged 7 years and older. Xyrem treats symptoms of excessive daytime sleepiness (EDS) or cataplexy (attacks of muscle weakness caused by emotion) in narcolepsy. At the recommended dosages in adults, Xyrem adds a large amount of sodium to daily dietary intake. Too much sodium in the diet is associated with increased blood pressure and risks of damage to the heart and blood vessels. Researchers used calcium, magnesium, and potassium ions in addition to a small amount of sodium to make a new oxybate medication, called Xywav®, that has 92% less sodium than Xyrem. Xywav and Xyrem were similar in laboratory and animal studies. In people, the body absorbs and processes Xywav slightly differently than Xyrem, but Xywav treatment has been shown to work the same to reduce symptoms of cataplexy and EDS in people with narcolepsy and is approved by the US Food and Drug Administration. Another neurological disorder with EDS is called idiopathic hypersomnia. Based on a clinical study, Xywav also reduced EDS and other symptoms in people with idiopathic hypersomnia. Side effects with Xywav are similar to those seen in previous studies with Xyrem.

A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus.

BACKGROUND: The determinate growth habits is beneficial for plant architecture modification and the development of crops cultivars suited to mechanized production systems. Which play an important role in the genetic improvement of crops. In Brassica napus, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross, but there are few reports on it. We fine mapped a determinate inflorescence locus, and evaluated the effect of the determinate growth habit on agronomic traits. RESULTS: In this study, we assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity, enhancing lodging resistance, increasing plant branches and maintaining productivity. Genetic analysis in the determinate (4769) and indeterminate (2982) genotypes revealed that two independently inherited recessive genes (Bnsdt1, Bnsdt2) are responsible for this determinate growth trait. Bnsdt2 was subsequently mapped in BC2 and BC3 populations derived from the combination 2982 × 4769. Bnsdt2 could be delimited to an approximately 122.9 kb region between 68,586.2 kb and 68,709.1 kb on C09. BLAST analysis of these candidate intervals showed that chrC09g006434 (BnaC09.TFL1) is homologous to TFL1 of A. thaliana. Sequence analysis of two alleles identified two non-synonymous SNPs (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, qRT-PCR revealed that BnaC09.TFL1 expression in shoot apexes was significantly higher in NIL-4769 than in 4769, suggesting its essential role in sustaining the indeterminate growth habit. CONCLUSIONS: In this study, the novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) that may be responsible for inflorescence traits were found.

Erlotinib protests against LPS-induced parthanatos through inhibiting macrophage surface TLR4 expression.

Sepsis is a life-threatening cascading systemic inflammatory response syndrome on account of serve infection. In inflamed tissues, activated macrophages generate large amounts of inflammatory cytokines reactive species, and are exposed to the damaging effects of reactive species. However, comparing with necroptosis and pyroptosis, so far, there are few studies focusing on the overproduction-related cell death, such as parthanatos in macrophage during sepsis. In LPS-treated macrophage, we observed PARP-1 activation, PAR formation and AIF translocation. All these phenomena could be inhibited by both erlotinib and 3-AB, indicating the presence of parthanatos in endotoxemia. We further found that LPS induced the increase of cell surface TLR4 expression responsible for the production of ROS and subsequent parthanatos in endotoxemia. All these results shed a new light on how TLR4 regulating the activation of PARP-1 by LPS in macrophage.

Pharmacokinetics, bioavailability, and bioequivalence of lower-sodium oxybate in healthy participants in two open-label, randomized, crossover studies.

American Academy of Sleep Medicine practice parameters designate sodium oxybate (SXB) as a standard of care for cataplexy, excessive daytime sleepiness (EDS), and disrupted night-time sleep in narcolepsy. Recently, a lower-sodium oxybate (LXB) with 92% less sodium than SXB was approved in the United States for the treatment of cataplexy or EDS in patients 7 years of age and older with narcolepsy. Two phase I, open-label, randomized, single-dose crossover pharmacokinetic studies in healthy adults were conducted. Single 4.5-g oral doses of LXB and SXB were administered in a fasted or fed state. In the fasted state at equivalent oxybate doses, LXB, compared with SXB, had a lower maximum plasma concentration (Cmax ; study 1 [total aqueous volume, 240 ml]: 101.8 vs. 135.7 µg/ml; study 2 [60 ml]: 94.6 vs. 123.0 µg/ml), delayed time to Cmax (Tmax ; study 1: 0.75 vs. 0.5 h; study 2: 1.0 vs. 0.5 h), but similar area under the curve (AUC; study 1: AUC0-t , 235.4 vs. 263.9 µg∙h/ml; AUC0-∞ , 236.5 vs. 265.2 µg∙h/ml; study 2: AUC0-t , 241.5 vs. 254.7 µg∙h/ml; AUC0-∞ , 243.1 vs. 256.3 µg∙h/ml). Bioequivalence criteria were met for AUC but not Cmax (both studies). Cmax and AUC were lower under fed than fasted conditions (LXB and SXB); differences between fed versus fasted were smaller for LXB than SXB. These pharmacokinetic differences between LXB and SXB are likely due to the lower sodium content in LXB. Pooled analyses demonstrated that a higher Cmax is associated with a higher incidence of nausea and vomiting.

White Matter Hypoperfusion Associated with Leukoaraiosis Predicts Intracranial Hemorrhage after Intravenous Thrombolysis.

OBJECTIVES: White matter hyperintensity is common in patients receiving intravenous thrombolysis. Some studies have expressed concern about the increased risk of hemorrhagic transformation and poor prognosis for those patients with pre-existing leukoaraiosis. The purpose of this study was to evaluate hypoperfusion associated with leukoaraiosis before thrombolysis using CT perfusion and to explore whether chronic white matter hypoperfusion increases risks of intracranial hemorrhage and poor clinical prognosis. MATERIALS AND METHODS: We collected 175 patients underwent intravenous thrombolysis with complete CT perfusion data and follow-up MRI between June 2017 and January 2020. We measured cerebral blood flow, cerebral blood volume, mean transit time and transit time to the peak at both periventricular and subcortical layers in the cerebral hemisphere contralateral to the stroke. The differences of white matter perfusion were compared between groups with different leukoaraiosis severity. Univariate analysis was used to compare in incidence of hemorrhagic transformation and poor prognosis between the hypoperfusion and normal perfusion groups. Further, we examined association between white matter hypoperfusion and intracranial hemorrhage after thrombolysis using logistic regression. RESULTS: The length of periventricular transit time to the peak was independently associated with a higher risk of intracranial hemorrhage after thrombolysis (OR=4.740, 95%CI=1.624-13.837, P=0.004). The best predictive value was 4.012. But there was no significant difference in poor prognosis at 3 months between hypoperfusion (periventricular transit time to the peak≥4.012 s) and normal perfusion (periventricular transit time to the peak<4.012 s) group. CONCLUSIONS: Image presentations of white matter hypoperfusion reflected the severity of leukoaraiosis. White matter hypoperfusion was independently associated with intracranial hemorrhage after intravenous thrombolysis. However, hypoperfusion would not increase the risk of poor prognosis.

Transcriptome analysis of flavonoid biosynthesis in safflower flowers grown under different light intensities.

BACKGROUND: Safflower (Carthamus tinctorius L.) is a domesticated species with a long history of cultivation and widespread distribution across the globe, and light plays an important role in controlling its distribution boundary. Flowers from safflower have been widely used in traditional Chinese medicine because of their ability to improve cerebral blood flow. Flavonoids are the main active compounds in safflower and have many pharmacological effects. In this study, we aimed to explore the relationship between different light intensities and flavonoid biosynthesis in safflower flowers cultivated in greenhouse. METHODS: The transcriptome of safflower flowers grown under different light intensities were sequenced through BGISEQ-500 platform. After assembled and filtered, Unigenes were annotated by aligning with seven functional databases. Differential expression analysis of two samples was performed with the DEseq2 package. Differentially expressed genes (DEGs) related with flavonoids biosynthesis were analyzed by Real-time PCR (RT-PCR). Flavonoids accumulation in flowers were determined by high performance liquid chromatography and spectrophotometer. RESULTS: Transcriptome analysis of safflower flowers cultivated under different light intensities was performed. A total of 99.16 Gb data were obtained, and 78,179 Unigenes were annotated. Among the DEGs, 13 genes were related to flavonoid biosynthesis. The differential expressions of seven key genes were confirmed by RT-PCR. In addition, the levels of some flavonoids were measured in safflower flowers grown under different light intensities. CtHCT3 gene expression showed a significantly negative correlation with kaempferol content in safflower grown under different light intensities. CONCLUSION: Our results strongly suggested that the reduction in light intensity in a suitable range promoted flavonoid biosynthesis in safflower flowers. We suggest that the expressions of HCT genes played an important role in flavonoid accumulation in safflower flowers. Our study lays a foundation for further research on the effects of light on flavonoid biosynthesis in safflower.

Population and Noncompartmental Pharmacokinetics of Sodium Oxybate Support Weight-Based Dosing in Children and Adolescents With Narcolepsy With Cataplexy.

The pharmacokinetics (PKs) of sodium oxybate (SXB) was evaluated in a subset of participants from a study of SXB treatment in children (aged 7-11 years; n = 11) and adolescents (aged 12-17 years; n = 18) with narcolepsy with cataplexy. PK evaluation was conducted over 2 nights during the period when participants received a stable nightly SXB dose. The SXB dose on night 1 was half of night 2 and was administered in two equally divided doses: dose 1 was administered > 2 hours after the evening meal, and dose 2 was administered ≥ 4 hours after dose 1. Noncompartmental PK analysis demonstrated higher plasma concentrations post-dose 2 vs. post-dose 1, higher than dose-proportional increases in area under the concentration-time curve from 0 to 4 hours (AUC0-4h ) after dose 1, indicating nonlinear clearance, and better correlation between exposure and mg/kg than exposure and gram dose. To confirm the noncompartmental findings, identify factors affecting SXB PK, and compare with prior results in adults, a population PK (PopPK) model was established combining PK data from the current study with prior data from adults (132 healthy volunteers and 13 with narcolepsy). A two-compartment PopPK model with first-order absorption and nonlinear clearance from the central compartment described the data well. PopPK identified weight as the main intrinsic factor and food as the main extrinsic factor affecting SXB PK, and predicts similar PK profiles on a mg/kg basis across ages. These results, along with previously reported efficacy and safety outcomes, support weight-based SXB dose initiation in pediatric patients.

Neutrophil-to-Lymphocyte Ratio and Platelet-to-Lymphocyte Ratio as Potential Predictors of Prognosis in Acute Ischemic Stroke.

Objective: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been emerging as the novel inflammatory biomarkers for determining the prognosis of various diseases. This study aimed to investigate the individual and joint effects of NLR and PLR on functional outcomes of acute ischemic stroke (AIS). Methods: Our study involved 448 eligible patients with first-ever AIS. Clinical and laboratory data were collected on admission within 72 h from stroke onset. Unfavorable functional outcome was defined as a modified Rankin Scale score of 3-6 at 3 months after AIS. Cox proportional hazard model and spline regression models was used to estimate the effect of NLR and PLR on risk of adverse outcomes after the last patient who completed a 3-months follow-up was enrolled. Results: After adjusting confounders, NLR were significantly associated with the unfavorable functional outcomes (P-trend < 0.001). So were PLR (P-trend < 0.001). NLR was discovered to have higher predictive value than PLR (AUC = 0.776, 95%CI = 0.727-0.825, P < 0.001; AUC = 0.697, 95%CI = 0.641-0.753, P < 0.001). The optimal cutoff values for NLR and PLR was 3.51 and 141.52, respectively. Stratified analysis performed by cox proportional hazard model showed that high level of NLR and PLR (NLR ≥ 3.51, PLR ≥ 141.52) presented the highest risk of unfavorable functional outcomes (adjusted HR, 3.77; 95% CI: 2.38-5.95; P < 0.001). Followed by single high level of NLR (adjusted HR, 2.32; 95% CI: 1.10-4.87; P = 0.027). Single high level of PLR (NLR < 3.51, PLR ≥ 141.52) also showed higher risk than low level of the combination, but it did not reach statistical significance (adjusted HR, 1.42; 95% CI: 0.75-2.70; P = 0.285). No obvious additive [relative excess risk due to interaction (RERI) not significant] or multiplicative (adjusted HR, 0.71; 95%CI: 0.46-1.09; P = 0.114) interaction was found between the effects of NLR and PLR on the risk of unfavorable functional outcomes. Conclusion: This study demonstrated that both NLR and PLR were independent predictors of 3-months functional outcomes of AIS. They may help to identify high-risk patients more forcefully when combined together.

Variation in the microbial community contributes to the improvement of the main active compounds of Magnolia officinalis Rehd. et Wils in the process of sweating.

BACKGROUND: Magnolia officinalis Rehd. et Wils, commonly called Houpo, has been used for thousands of years in China as a traditional herbal medicine. The primary processing of Houpo requires sweating treatment, which is a special drying process and is considered to be an essential embodiment of high quality and genuine medicinal materials. The sweating of Houpo leads to peculiar changes in the microbial community structure and the content of main active substances (magnolol, honokiol, syringin and magnoflorine). Variation in the microbial community was considered the cause of the change in content of active substances of Houpo, although the microbial taxa responsible for the improvement of content remain unidentified. METHODS: In this study, we used MiSeq high-throughput sequencing methods for partial bacterial 16S rRNA and 18S rRNA gene sequences to compare the bacterial and fungal community structures at different timepoints in the process of sweating. The content of the main active substances (magnolol, honokiol, syringin and magnoflorine) were determined by high-performance liquid chromatography analysis to evaluate the effects of sweating. UPLC-Q-Extractive Orbitrap mass spectrometry (UPLC-QE Orbitrap MS) was used to detection of differential metabolites of unsweated Houpo before and after co-culture with core bacterial solutions. RESULTS: In this study, the total contents of magnolol (MG) and honokiol (HK) were significantly increased at 4 dp (dp for day PM sample), up to 3.75%, and the contents of syringin (SG) and magnoflorine (MF) were as high as 0.12% and 0.06%, respectively. Bacterial abundance and diversity were higher in the early stage (0 day-2 da; da for day AM sample) than in the later stage (4-5 dp), while fungal abundance was more obvious in the later stage than in the early stage. Positive correlation coefficients revealed that the relative abundance of Enterobacter (P < 0.05), Klebsiella (P < 0.05), Weissella (P < 0.05), Bacillus (P < 0.05) and Candida (P < 0.05) would be conducive to improving the quality of Houpo. Negative correlation coefficients revealed that the relative abundance of Actinomycetospora, Singulisphaera, Mucilaginibacter, Deinococcus, Gemmatirosa, Methylobacterium, Sphingomonas, Hymenobacter, Halomonas and Capnobotryella could be a potential antagonist for the decrease in the quality of Houpo. After co-culture of single core strain and unsweated Houpo, there was no significant difference in the four main active components, but there were other metabolites with significant difference. CONCLUSIONS: Our findings reveal that sweating increased the content of the main active compounds, promoted the relative abundance of potentially beneficial microbes, decreased the abundance of potentially harmful microbes, the core functional genera group together, forming a core microbiome, these genera are dominant across the different stages of the sweating process and contribute to the quality development of the characteristics of Houpo. Meanwhile, this study presented a clear scope for potential beneficial microbes that improve the quality of Houpo.

A comparison of contrast transthoracic echocardiography and contrast transcranial Doppler in cryptogenic stroke patients with patent foramen ovale.

OBJECTIVE: In recent years, increasing attention has been paid to cryptogenic stroke (CS) caused by the patent foramen ovale (PFO). This study aims to compare contrast transthoracic echocardiography (cTTE) and contrast transcranial Doppler (cTCD) to determine whether cTTE is more suitable and reliable than cTCD for clinical use. METHODS: From March 2017 to May 2018, patients who suffered from migraines, stroke, hypomnesis, or asymptomatic stroke found casually were included in our study. Patients with CS were semirandomly divided into two groups (cTTE and cTCD) according to the date of the outpatient visit. Patients with either of the examination above found positive were selected to finish transesophageal echocardiography (TEE). RESULTS: In our study, the sensitivities of group cTTE positive (group cTTE+) and group cTCD positive (group cTCD+) did not have any statistical difference (89% vs. 80%, p = 0.236). Focusing on group cTCD+, we discovered that the semiquantitative shunt grading was not correlated with whether a PFO was present or not (p = 0.194). However, once the PFO has been diagnosed, the shunt grading was shown to be related to the width of the gaps (p = 0.032, pdeviation  = 0.03). CONCLUSION: Both cTTE and the cTCD can be used for preliminary PFO findings. The semiquantitative shunt grading of cTCD and cTTE can suggest the size of the PFO and the next course of treatment. The cTTE may be more significant to a safe PFO (a PFO does not have right-to-left shunts, RLSs). Combining cTTE and TEE could help diagnose PFO and assess CS risk.

Comparative Mapping Combined With Map-Based Cloning of the Brassica juncea Genome Reveals a Candidate Gene for Multilocular Rapeseed.

Multilocular traits exist in a variety of plants and exert important effects on plant yield. Previous genetic studies have shown that multilocular trait of the Brassica juncea cultivar Duoshi is controlled by two recessive genes, Bjln1 and Bjln2. In previous studies, the Bjln1 gene is located on chromosome A07, and the Bjln1 candidate gene is BjuA07.CLV1. In this study, a BC4 mapping population for the Bjln2 gene was generated. This population was used to construct genetic linkage maps of the Bjln2 gene using amplified fragment length polymorphism (AFLP), intron length polymorphism (IP) and simple sequence repeat (SSR) methodology. The results showed that the Bjln2 gene was restricted to a 0.63 cM interval. BLAST alignment with B. juncea revealed the Bjln2 gene was located within a 11.81-16.65 Mb region on chromosome B07. Moreover, the candidate gene BjuB07.CLV1 (equivalent to Bjln2) was cloned by comparing mapping and map-based cloning, and BjuB07.CLV1 gene was shown to have the ability to restore the bilocular traits in a genetic complementation experiment. The sequencing revealed that a 4961 bp insertion interrupted the coding sequence of the BjuB07.CLV1 gene, resulting in an increase in locule number. Expression analysis revealed that BjuB07.CLV1 was expressed in all tissues and the expression level in bilocular plants was significantly higher than that in multilocular plants. In addition, markers closely linked to the Bjln2 gene were developed and used for molecular marker-assisted breeding of multilocular traits.

Mutations in the CDS and promoter of BjuA07.CLV1 cause a multilocular trait in Brassica juncea.

Multilocular trait has recently attracted considerable attention for its potential to increase yield. Our previous studies indicated that two genes (Bjln1 and Bjln2) are responsible for multilocular siliques in Brassica juncea and the Bjln1 gene has been delimited to a 208-kb region. In present study, the Bjln1 gene was successfully isolated using the map-based cloning method. Complementation test indicated that the BjuA07.CLV1 (equivalent to BjLn1) could rescue the multilocular phenotype and generate bilocular siliques. Two amino acids changes at positions 28 and 63 in BjuA07.clv1 as well as a 702-bp deletion in its promoter have been proved to affect the carpel numbers. Microscopic analyses suggested that BjuA07.CLV1 is involved in the maintenance of shoot and floral meristem size. The expression level of BjuA07.clv1 was significantly reduced in the SAM. Furthermore, WUS, CLV2, CLV3, RPK2 and POL, key genes in the CLV/WUS signal pathway, showed lower expression level in the multilocular plants. These data suggest that the mutations in the CDS and promoter of BjuA07.clv1 reduced its function and expression level, which disturbed CLV/WUS signal pathway, thereby leading to the enlargement of the shoot and floral meristem and resulting in the multilocular siliques.