Interferon-γ induces tumor resistance to anti-PD-1 immunotherapy by promoting YAP phase separation.

Interferon-γ (IFN-γ)-mediated adaptive resistance is one major barrier to improving immunotherapy in solid tumors. However, the mechanisms are not completely understood. Here, we report that IFN-γ promotes nuclear translocation and phase separation of YAP after anti-PD-1 therapy in tumor cells. Hydrophobic interactions of the YAP coiled-coil domain mediate droplet initiation, and weak interactions of the intrinsically disordered region in the C terminus promote droplet formation. YAP partitions with the transcription factor TEAD4, the histone acetyltransferase EP300, and Mediator1 and forms transcriptional hubs for maximizing target gene transcriptions, independent of the canonical STAT1-IRF1 transcription program. Disruption of YAP phase separation reduced tumor growth, enhanced immune response, and sensitized tumor cells to anti-PD-1 therapy. YAP activity is negatively correlated with patient outcome. Our study indicates that YAP mediates the IFN-γ pro-tumor effect through its nuclear phase separation and suggests that YAP can be used as a predictive biomarker and target of anti-PD-1 combination therapy.

LATS2 condensates organize signalosomes for Hippo pathway signal transduction.

Biomolecular condensates have been proposed to mediate cellular signaling transduction. However, the mechanism and functional consequences of signal condensates are not well understood. Here we report that LATS2, the core kinase of the Hippo pathway, responds to F-actin cytoskeleton reduction and forms condensates. The proline-rich motif (PRM) of LATS2 mediates its condensation. LATS2 partitions with the main components of the Hippo pathway to assemble a signalosome for LATS2 activation and for its stability by physically compartmentalizing from E3 ligase FBXL16 complex-dependent degradation, which in turn mediates yes-associated protein (YAP)-transcriptional coactivator with PDZ-binding motif (TAZ) recruitment and inactivation. This oncogenic FBXL16 complex blocks LATS2 condensation by binding to the PRM region to promote its degradation. Disruption of LATS2 condensation leads to tumor progression. Thus, our study uncovers that the signalosomes assembled by LATS2 condensation provide a compartmentalized and reversible platform for Hippo signaling transduction and protein stability, which have potential implications in cancer diagnosis and therapeutics.

Characterizing the metabolic divide: distinctive metabolites differentiating CAD-T2DM from CAD patients.

OBJECTIVE: To delineate the metabolomic differences in plasma samples between patients with coronary artery disease (CAD) and those with concomitant CAD and type 2 diabetes mellitus (T2DM), and to pinpoint distinctive metabolites indicative of T2DM risk. METHOD: Plasma samples from CAD and CAD-T2DM patients across three centers underwent comprehensive metabolomic and lipidomic analyses. Multivariate logistic regression was employed to discern the relationship between the identified metabolites and T2DM risk. Characteristic metabolites' metabolic impacts were further probed through hepatocyte cellular experiments. Subsequent transcriptomic analyses elucidated the potential target sites explaining the metabolic actions of these metabolites. RESULTS: Metabolomic analysis revealed 192 and 95 significantly altered profiles in the discovery (FDR < 0.05) and validation (P < 0.05) cohorts, respectively, that were associated with T2DM risk in univariate logistic regression. Further multivariate regression analyses identified 22 characteristic metabolites consistently associated with T2DM risk in both cohorts. Notably, pipecolinic acid and L-pipecolic acid, lysine derivatives, exhibited negative association with CAD-T2DM and influenced cellular glucose metabolism in hepatocytes. Transcriptomic insights shed light on potential metabolic action sites of these metabolites. CONCLUSIONS: This research underscores the metabolic disparities between CAD and CAD-T2DM patients, spotlighting the protective attributes of pipecolinic acid and L-pipecolic acid. The comprehensive metabolomic and transcriptomic findings provide novel insights into the mechanism research, prophylaxis and treatment of comorbidity of CAD and T2DM.

A Randomized Phase 2 KINETIC Trial Evaluating SAGE-324/BIIB124 in Individuals with Essential Tremor.

BACKGROUND: SAGE-324/BIIB124 is an investigational positive allosteric modulator of GABAA receptors. OBJECTIVE: KINETIC (NCT04305275), a double-blind, randomized, placebo-controlled, phase 2 study, evaluated SAGE-324/BIIB124 in individuals with essential tremor (ET). METHODS: Individuals aged 18 to 80 years were randomly assigned 1:1 to orally receive 60 mg of SAGE-324/BIIB124 or placebo once daily for 28 days. The primary endpoint was change from baseline in The Essential Tremor Rating Assessment Scale-Performance Subscale (TETRAS-PS) Item 4 (upper-limb tremor) at day 29 with SAGE-324/BIIB124 versus placebo. RESULTS: Between May 2020 and February 2021, 69 U.S. participants were randomly assigned to receive SAGE-324/BIIB124 (n = 34) or placebo (n = 35). There was a significant reduction from baseline in TETRAS-PS Item 4 at day 29 with SAGE-324/BIIB124 versus placebo (least squares mean [standard error]: -2.31 [0.401] vs. -1.24 [0.349], P = 0.0491). The most common treatment-emergent adverse events included somnolence, dizziness, fatigue, and balance disorder. CONCLUSION: These results support further development of SAGE-324/BIIB124 for potential ET treatment. © 2024 Sage Therapeutics, Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Analysis of intracellular communication reveals consistent gene changes associated with early-stage acne skin.

A comprehensive understanding of the intricate cellular and molecular changes governing the complex interactions between cells within acne lesions is currently lacking. Herein, we analyzed early papules from six subjects with active acne vulgaris, utilizing single-cell and high-resolution spatial RNA sequencing. We observed significant changes in signaling pathways across seven different cell types when comparing lesional skin samples (LSS) to healthy skin samples (HSS). Using CellChat, we constructed an atlas of signaling pathways for the HSS, identifying key signal distributions and cell-specific genes within individual clusters. Further, our comparative analysis revealed changes in 49 signaling pathways across all cell clusters in the LSS- 4 exhibited decreased activity, whereas 45 were upregulated, suggesting that acne significantly alters cellular dynamics. We identified ten molecules, including GRN, IL-13RA1 and SDC1 that were consistently altered in all donors. Subsequently, we focused on the function of GRN and IL-13RA1 in TREM2 macrophages and keratinocytes as these cells participate in inflammation and hyperkeratinization in the early stages of acne development. We evaluated their function in TREM2 macrophages and the HaCaT cell line. We found that GRN increased the expression of proinflammatory cytokines and chemokines, including IL-18, CCL5, and CXCL2 in TREM2 macrophages. Additionally, the activation of IL-13RA1 by IL-13 in HaCaT cells promoted the dysregulation of genes associated with hyperkeratinization, including KRT17, KRT16, and FLG. These findings suggest that modulating the GRN-SORT1 and IL-13-IL-13RA1 signaling pathways could be a promising approach for developing new acne treatments.

Comparison of EUS-FNA and EUS-FNB for diagnosis of solid pancreatic mass lesions: a meta-analysis of prospective studies.

Objective: To quantitatively compare the diagnostic value of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) in solid pancreatic mass lesions using a systematic evaluation method.Methods: A systematic literature search was conducted on public databases to include studies comparing the diagnostic value of EUS-FNA and EUS-FNB in solid pancreatic mass lesions. The combined effect size was estimated using mean difference (MD) and risk difference (RD) respectively, and the corresponding 95% confidence interval (CI) was calculated.Results: The 12 articles (7 RCTs and 5 cohort studies) met the inclusion criteria of this study. The meta-analysis showed that compared with EUS-FNB, EUS-FNA had lower diagnostic accuracy (RD: -0.08, 95% CI: -0.15, -0.01) and specimen adequacy (RD: -0.08, 95% CI: -0.15, -0.02), while higher required number of needle passes (MD: 0.42, 95% CI: 0.12, 0.73). However, EUS-FNB and EUS-FNA presented similar overall complications (RD: 0.00, 95% CI: -0.01, 0.02) and technical failures (RD: -0.01, 95% CI: -0.02, 0.00), without statistically significant differences.Conclusions: Compared with EUS-FNA, EUS-FNB seems to be a better choice for diagnosing suspected pancreatic lesions.

Early oral hydration on demand in postanesthesia care unit effectively relieves postoperative thirst in patients after gynecological laparoscopy: a prospective randomized controlled trial.

BACKGROUND: Postoperative thirst is one of the most intense, common and easily ignored subjective discomforts in patients after gynecological surgery. This study aimed to investigate whether early oral hydration on demand in the postanesthesia care unit (PACU) after gynecological laparoscopy under general anesthesia can appease postoperative thirst and increase patient comfort. METHODS: Participants were randomized into the intervention and control groups. Patients in the intervention group were allowed to achieve early oral hydration on demand in the PACU if they were evaluated as fully conscious, with stable vital signs, grade 5 muscle strength, and well-recovered cough and swallowing reflex. However, the total amount of water intake throughout the entire study should not exceed 0.5mL/kg. During the study, the frequency of water intake, the total amount of water intake and adverse events were accurately recorded. The control group was managed according to the routine procedures and began to drink water 2 h after anesthesia. The intensity of thirst and subjective comfort in patients were assessed using the visual analog scale (VAS) when they entered and left the PACU. RESULTS: No statistically significant differences were identified in age, height, weight, body mass index, pre-operative fasting time, duration of surgery, intraoperative fluid intake, intraoperative blood loss, intraoperative urine volume, and thirst intensity and subjective comfort scores between the groups before intervention (P > 0.05). After intervention, the VAS score for thirst intensity in the intervention group significantly decreased (P < 0.05), and the VAS score for subjective comfort in the intervention group significantly increased (P < 0.05). No adverse events were detected in both groups during the entire study. CONCLUSION: Early oral hydration on demand in the PACU can safely and effectively relieve postoperative thirst in patients, and improve patient comfort after gynecological laparoscopy. TRIAL REGISTRATION: This single-center, prospective, randomized controlled trial was registered at the Chinese Clinical Trial Center on April 27, 2023. The registration number of this study is ChiCTR2300070985.

Integrated Transcriptome and Metabolome Analyses Reveal Bamboo Culm Color Formation Mechanisms Involved in Anthocyanin Biosynthetic in Phyllostachys nigra.

Phyllostachys nigra has green young culms (S1) and purple black mature culms (S4). Anthocyanins are the principal pigment responsible for color presentation in ornamental plants. We employ a multi-omics approach to investigate the regulatory mechanisms of anthocyanins in Ph. nigra. Firstly, we found that the pigments of the culm of Ph. nigra accumulated only in one to four layers of cells below the epidermis. The levels of total anthocyanins and total flavonoids gradually increased during the process of bamboo culm color formation. Metabolomics analysis indicated that the predominant pigment metabolites observed were petunidin 3-O-glucoside and malvidin O-hexoside, exhibiting a significant increase of up to 9.36-fold and 13.23-fold, respectively, during pigmentation of Ph. nigra culm. Transcriptomics sequencing has revealed that genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and starch and sucrose metabolism pathways were significantly enriched, leading to color formation. A total of 62 differentially expressed structural genes associated with anthocyanin synthesis were identified. Notably, PnANS2, PnUFGT2, PnCHI2, and PnCHS1 showed significant correlations with anthocyanin metabolites. Additionally, certain transcription factors such as PnMYB6 and PnMYB1 showed significant positive or negative correlations with anthocyanins. With the accumulation of sucrose, the expression of PnMYB6 is enhanced, which in turn triggers the expression of anthocyanin biosynthesis genes. Based on these findings, we propose that these key genes primarily regulate the anthocyanin synthesis pathway in the culm and contribute to the accumulation of anthocyanin, ultimately resulting in the purple-black coloration of Ph. nigra.

Pharmacokinetics and pharmacodynamics of sacubitril/valsartan in peritoneal dialysis patients.

BACKGROUND: There is little information on the pharmacokinetics and pharmacodynamics of sacubitril/valsartan (SV) in patients undergoing peritoneal dialysis (PD) complicated with hypertension or heart failure (HF). This study was designed to evaluate the pharmacokinetics and pharmacodynamics of SV in PD patients with complications of hypertension or HF. METHODS: This was an open-label and cross-sectional study investigating PD patients diagnosed with hypertension or New York Heart Association Class II-IV HF. The concentrations of valsartan, sacubitril and sacubitrilat (LBQ657) were measured by ultra-performance liquid chromatography tandem mass spectrometry in plasma, urine and peritoneal dialysate samples. Pharmacodynamics were evaluated by comparing changes in mean sitting systolic blood pressure (msSBP), mean sitting diastolic blood pressure (msDBP), mean sitting heart rate, N-terminal-pro B-type natriuretic peptide (NT-proBNP) and left ventricular ejection fraction (LVEF). RESULTS: Forty patients with PD were enrolled including 27 (67.5%) patients with hypertension, 4 (10%) patients with HF and 9 (22.5%) patients with both hypertension and HF. This study included three treatment cohorts: 50 mg twice daily (BID), 100 mg once daily and 100 mg BID. The plasma maximum drug concentrations in the 100 mg BID group were 1995 ± 1499 ng/mL for valsartan, 171 ± 148 ng/mL for sacubitril and 13 686 ± 7418 ng/mL for LBQ657. The 24-h recovery rate of LBQ657 was 3.77% in urine and 2.23% in peritoneal dialysate. After taking SV, msSBP and msDBP decreased by 19.25 ± 10.32 mmHg and 10.10 ± 8.00 mmHg from baseline, respectively. NT-proBNP decreased by 1436.50 (0.00-18 198.00) from baseline, while LVEF increased by 5.00 (-0.25 to 9.25) from baseline after SV treatment. CONCLUSIONS: PD and residual renal function contributed only to a minor degree to the elimination of LBQ657. Additionally, a dose of 100 mg BID SV is safe and effective in patients with PD with complications of hypertension or HF.

Key emergency response technologies for abrupt air pollution accidents in China.

Abrupt air pollution accidents can endanger people's health and destroy the local ecological environment. The appropriate emergency response can minimize the harmful effects of accidents and protect people's lives and property. This paper provides an overview of the key emergency response technologies for abrupt air pollution accidents around the globe with emphasis on the major achievements that China has obtained in recent years. With decades of effort, China has made significant progress in emergency monitoring technologies and equipment, source estimation technologies, pollutant dispersion simulation technologies and others. Many effective domestic emergency monitoring instruments (e.g., portable DOAS/FT-IR systems, portable FID/PID systems, portable GC-MS systems, scanning imaging remote sensing systems, and emergency monitoring vehicles) had been developed which can meet the demands for routine emergency response activities. A monitoring layout technique combining air dispersion simulation, fuzzy comprehensive evaluation, and a post-optimality analysis was proposed to identify the optimal monitoring layout scheme under the constraints of limited monitoring resources. Multiple source estimation technologies, including the forward method and the inversion method, have been established and evaluated under various scenarios. Multi-scale dynamic pollution dispersion simulation systems with high temporal and spatial resolution were further developed. A comprehensive emergency response platform integrating database support, source estimation, monitoring schemes, fast monitoring of pollutants, pollution predictions and risk assessment was developed based on the technical idea of "source identification - model simulation - environmental monitoring" dynamic interactive feedback. It is expected that the emergency response capability for abrupt air pollution accidents will gradually improve in China.

Generating odour imagery enhances brain activity in individuals with low subjective olfactory imagery ability.

Olfactory imagery (OI) is defined as the generation of odour images in the mind. There are large inter-individual differences regarding OI abilities. However, the neural representations of OI among individuals with high or low OI abilities are less understood. Participants with high or low OI abilities evaluated using the Vividness of Olfactory Imagery Questionnaire were recruited in this study. Brain activations were measured during a word cueing OI and visual imagery (VI) tasks using functional magnetic resonance imaging (fMRI). In addition, the OI task was divided into two parts. In one part, OI was performed for 8 s (long imagery generation time) and in the other part for 2 s (short imagery generation time). Ratings of the overall imagery vividness were collected at the end of each task. The vividness of OI during short OI was lower among participants with low OI abilities compared to participants with high OI abilities. Brain imaging results showed that participants with low OI ability had stronger brain activation of the supplementary motor area and the superior frontal cortex, compared to participants with higher OI abilities during the short versus long imagery generation time conditions. These results suggest that when generating odour images in a relatively short period of time (e.g., 2 s), people with either high or low OI abilities may have adopted different approaches, resulting in diverse brain activation.

Prognostic significance of soluble CD25 in patients with sepsis: a prospective observational study.

OBJECTIVES: The diagnosis of sepsis is challenging, the need for sensitive and specific diagnostic and prognostic biomarkers has not been met. Soluble CD25 (sCD25) is a readily available biomarker reported to represent the severity of the disease. This study aimed to assess the association between sCD25 and mortality in patients with sepsis. METHODS: In total, 329 adult patients with sepsis were screened through a prospective, observational study. We investigated the severity scores and sCD25 levels at admission to the intensive care unit (ICU), defined by sepsis (sepsis-3). The prognostic value of sCD25 was assessed using receiver operating characteristic (ROC) curves and binary logistic regression models in predicting unfavourable outcome. The correlations between variables and severity of disease were analysed by Spearman correlation tests. RESULTS: After entering the ICU, the sCD25 level and sequential organ failure assessment (SOFA) score were significantly higher in nonsurvivors than in survivors. The prognostic values estimated by the ROC curves were 0.678 for sCD25 and 0.945 for SOFA score at ICU admission. sCD25 had a modest ability to predict poor outcome. Logistic regression showed that increased levels of sCD25 were independently associated with unfavourable outcome. Spearman correlation tests showed that sCD25 levels were positively correlated with disease severity. CONCLUSIONS: In sepsis patients, increased sCD25 levels were independently associated with poor clinical outcomes. Further research is needed to improve the understanding of the pathophysiology of this relationship.

Olfactory metacognition and memory in individuals with different subjective odor imagery abilities.

Imagery vividness is one of the key indicators to evaluate the ability to generate mental images. There is large inter-individual variability in olfactory imagery (OI) abilities, however, little is known about the underlying factors for individual OI abilities. Using a word cueing imagery paradigm and the trial-by-trial imagery vividness rating method, participants with high or low OI abilities (differentiated by the Vividness of Olfactory Imagery Questionnaire) completed two OI tasks with either shorter (2 s) or longer (8 s) image generation time. Participants' olfactory function, olfactory-related working memory and episodic recognition memory were measured using validated methods. Moreover, olfactory metacognition was assessed using the Odor Awareness Scale (OAS) and the Importance of Olfaction Questionnaire (IOQ). Compared to participants with high OI abilities, those with low OI abilities reported less vivid odor images during OI tasks. For participants with low OI abilities, the imagery vividness significantly improved as the image generation time increased. There was no difference regarding olfactory perception or olfactory-related memory performances between the high and the low OI ability groups. However, participants with higher OI abilities had significant higher scores on the OAS and the IOQ, indicating a superior olfactory-related metacognition. These results provide evidences supporting the underlying factors that related to variances of subjective ability of generating vivid odor mental images.

Global regulatory factor VeA upregulates the production of antitumor substances in endophytic Fusarium solani.

A number of studies have demonstrated that endophytic fungi have the potential to produce antitumor active substances with novel structures and significant activities. In our previous studies, we isolated a Fusarium strain from the stem of the medicinal plant Nothapodytes pittosporoides (Oliv.). In this study, we identified this strain as Fusarium solani and found that its crude extract has significant antitumor activity against human alveolar adenocarcinoma cells (A549). We overexpressed the global regulatory factor VeA in F. solani (VeAOE), resulting in a significant increase in antitumor activity. The MTT assay results showed that the inhibition rate of the VeAOE mutant extract on A549 cancer cells was significantly higher than that of the WT extract, as the IC50 decreased from 369.22 to 285.89 µg/mL, and the apoptosis ratio was significantly increased by approximately 4.86-fold. In VeAOE, accumulation of alkaloids, terpenoids, carboxylic acid derivatives, phenols and flavonoid metabolites with potential antitumor activity was significantly increased compared with WT based on metabolomic analysis. Additionally, transcriptome analysis found that the expression patterns of 48 genes related to antitumor activity were significantly changed in VeAOE, mainly involving glycosyl hydrolases, the Zn(2)-Cys(6) class, cytochrome P450 monooxygenase, 3-isopropylmalate dehydratase, and polyketide synthases. These results suggested that VeA mediated the antitumor activity of the metabolites in F. solani HB1-J1 by regulating multiple metabolic pathways.

A phase 1 double-blind, placebo-controlled study of zuranolone (SAGE-217) in a phase advance model of insomnia in healthy adults.

OBJECTIVE: To evaluate single zuranolone (SAGE-217) 30 or 45 mg doses in a 5-h phase advance insomnia model. METHODS: In this double-blind, three-way crossover study, healthy adults received placebo (n = 41), zuranolone 30 mg (n = 44), and zuranolone 45 mg (n = 42) across three treatment periods. Sleep was assessed by polysomnography and a postsleep questionnaire. Next-day residual effects and safety/tolerability were evaluated. RESULTS: Compared with placebo, zuranolone resulted in significant improvements in median sleep efficiency (30 mg, 84.6%; 45 mg, 87.6%; placebo, 72.9%; p < 0.001 for both doses), wake after sleep onset (WASO; 30 mg, 55.0 min; 45 mg, 42.5 min; placebo, 113.0 min; p < 0.001 for both doses), duration of awakenings (30 mg, 4.2 min, p < 0.001; 45 mg, 3.7 min, p = 0.001; placebo, 7.4 min), and total sleep time (TST; 30 mg, 406.3 min; 45 mg, 420.3 min; placebo, 350.0 min; p < 0.001 for both doses). Subjective endpoints (WASO, TST, sleep latency, sleep quality) also improved relative to placebo. Zuranolone was generally well tolerated, and the most common adverse events (≥2 participants, any period) were headache and fatigue. CONCLUSION: Zuranolone improved sleep measures versus placebo in a phase advance model of insomnia in healthy adults, supporting future studies in patients with insomnia disorder.

Overexpression of the global regulator FnVeA upregulates antitumor substances in endophytic Fusarium nematophilum.

The special niche of endophytic fungi promotes their potential to produce antitumor compounds with novel structure and significant bioactivity for screening of new antitumor drugs. In our previous studies, we isolated a Fusarium strain from the roots of the medicinal plant Nothapodytes pittosporoides and identified it as Fusarium nematophilum. We found that the crude extract of F. nematophilum had significant antitumor activity on A549 cancer cells, and overexpressing the global regulatory factor FnVeA (the VeA gene of the fungus F. nematophilum) resulted in a significant increase in the antitumor activity, which was approximately fivefold higher than wild strain for relative inhibition rate. In FnVeAOE, the accumulation of indole, alkene, alkaloid, steroid, and flavonoid metabolites with potential antitumor activity was significantly upregulated compared with wild type via metabolomic analysis. Moreover, the transcriptome analysis showed that 134 differential genes were considered to be closely related to the biosynthesis of antitumor substances, of which 59 differential genes were considered as candidate key genes, and related to tryptophan dimethylallyltransferase, cytochrome P450 monooxygenase, polyketide synthases, and transcription factors. Taken together, we suggest that FnVeA may regulate the biosynthesis of antitumor substances by mediating the expression of genes related to secondary metabolic pathways in F. nematophilum.

Global regulatory factor AaLaeA upregulates the production of antitumor substances in the endophytic fungus Alternaria alstroemeria.

The global regulatory factor LaeA has been shown to be involved in the biosynthesis of secondary metabolites in various fungi. In a previous work, we isolated an endophytic fungus from Artemisia annua, and its extract had a significant inhibitory effect on the A549 cancer cell line. Phylogenetic analysis further identified the strain as Alternaria alstroemeria. Overexpression of AalaeA gene resulted in significantly increased antitumor activity of this strain's extract. The 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay results showed that the inhibition rate of the AalaeAOE29 mutant extract on A549 cancer cells was significantly higher than that of the WT extract, as the IC50 decreased from 195.0 to 107.4 µg/ml, and the total apoptosis rate was enhanced. Overexpression of the AalaeA gene significantly increased the contents of myricetin, geraniol, ergosterol, and 18 other antitumor compounds as determined by metabolomic analysis. Transcriptomic analysis revealed significant changes in 95 genes in the mutant strain, including polyketide synthases, nonribosomal peptide synthases, cytochrome P450s, glycosyltransferases, acetyl-CoA acetyltransferases, and others. These results suggested that AaLaeA mediated the antitumor activity of the metabolites in A. alstroemeria by regulating multiple metabolic pathways.

Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

Measurement of HONO flux using the aerodynamic gradient method over an agricultural field in the Huaihe River Basin, China.

To investigate nitrous acid (HONO) levels and potential HONO sources above crop rotation fields. The HONO fluxes were measured by the aerodynamic gradient (AG) method from 14 December 2019 to 2 January 2020 over an agricultural field in the Huaihe River Basin. The ambient HONO levels were measured at two different heights (0.15 and 1.5 m), showing a typical diurnal cycle with low daytime levels and high nighttime levels. The upward HONO fluxes were mostly observed during the day, whereas deposition dominated at night. The diurnal variation of HONO flux followed solar radiation, with a noontime maximum of 0.2 nmol/(m2∙sec). The average upward HONO flux of 0.06 ± 0.17 nmol/(m2∙sec) indicated that the agricultural field was a net source for atmospheric HONO. The higher HONO/NO2 ratio and NO2-to-HONO conversion rate close to the surface suggested that nocturnal HONO was formed and released near the ground. The unknown HONO source was derived from the daytime HONO budget analysis, with an average strength of 0.31 ppbV/hr at noontime. The surface HONO flux, which was highly correlated with the photolysis frequency J(NO2) (R2 = 0.925) and the product of J(NO2) × NO2 (R2 = 0.840), accounted for ∼23% of unknown daytime HONO source. The significant correlation between HONO fluxes and J(NO2) suggests a light-driven HONO formation mechanism responsible for the surface HONO flux during daytime.

BACE1 SUMOylation increases its stability and escalates the protease activity in Alzheimer's disease.

Amyloid beta (Aß) is a major pathological marker in Alzheimer's disease (AD), which is principally regulated by the rate-limiting ß-secretase (i.e., BACE1) cleavage of amyloid precursor protein (APP). However, how BACE1 activity is posttranslationally regulated remains incompletely understood. Here, we show that BACE1 is predominantly SUMOylated at K501 residue, which escalates its protease activity and stability and subsequently increases Aß production, leading to cognitive defect seen in the AD mouse model. Compared with a non-SUMOylated K501R mutant, injection of wild-type BACE1 significantly increases Aß production and triggers cognitive dysfunction. Furthermore, overexpression of wild-type BACE1, but not non-SUMOylated K501R mutant, facilitates senile plaque formation and aggravates the cognitive deficit seen in the APP/PS1 AD mouse model. Together, our data strongly suggest that K501 SUMOylation on BACE1 plays a critical role in mediating its stability and enzymatic activity.