An in Vitro triple screen model for human mineralocorticoid receptor activity.

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18ß-glycyrrhetinic acid (18ß-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

TRAF6-mediated ubiquitination of AKT1 in the nucleus occurs in a ß-arrestin2-dependent manner upon insulin stimulation.

AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.

Repetitive Transcranial Magnetic Stimulation and Non-convulsive Electric Shock in the Treatment of Depression: A Meta-analysis.

Background: Depression is an affective mental disorder that seriously endangers the physical and psychological health of human beings. This study attempted to systematically evaluate and compare the clinical efficacy and onset time of repetitive transcranial magnetic stimulation and non-convulsive electroshock in the treatment of Depression through the method of evidence-based medicine. Methods: As of December 2022, we have selectively searched domestic and foreign databases by computer, including English databases PubMed, ScienceDir ETC (Elsevier), Embase, wiley, and Chinese databases HowNet (CNKI), Wanfang (WanFang), VIP (VIP), Chinese Medical Association, CBM (sinomed) Chinese biomedical literature database, etc., collected randomized controlled studies on repetitive transcranial magnetic stimulation and non-convulsive electric shock in the treatment of Depression, and included 21 documents in total. Two researchers independently screened the literature, comprehensively evaluated the retrieved literature according to the established inclusion and exclusion criteria, extracted valid data, and used Review Manager 5.4 software for quantitative statistical analysis. The clinical effective rate and Hamilton depression Rating scale (Hamilton depression scale, HAMD) and onset time were used as outcome indicators for evaluation. Results: A total of 12 literatures were included in this study for Meta-analysis, involving a total of 678 subjects. The results of Meta-analysis showed that the HAMD score of the study group was higher than that of the control group after treatment, MD=2.01, 95%CI (0.59-3.68), P < .05; there was no statistically significant difference in clinical efficacy between the study group and the control group, OR = 0.88, 95%CI (0.31-1.92), Z = 1.16, P = .29; the onset time of the study group was shorter than that of the control group, MD = 2.01, 95%CI (0.59-3.68), Z = 3.31, P = .001. Conclusion: Repetitive transcranial magnetic stimulation is superior to non-convulsive electroconvulsive shock in the treatment of Depression. However, further research is needed to verify its long-term efficacy in the treatment of such diseases. Future studies could focus on investigating the sustainability of treatment effects, exploring potential predictors of treatment response, and comparing the cost-effectiveness of rTMS and NCES in clinical practice. Such research would provide valuable insights for optimizing treatment strategies for depression.

The impact of PET/CT and brain MRI for metastasis detection among patients with clinical T1-category lung cancer: Findings from a large-scale cohort study.

PURPOSE: [18F]-FDG PET/CT and brain MRI are common approaches to detect metastasis in patients of lung cancer. Current guidelines for the use of PET/CT and MRI in clinical T1-category lung cancer lack risk-based stratification and require optimization. This study stratified patients based on metastatic risk in terms of the lesions' size and morphological characteristics. METHODS: The detection rate of metastasis was measured in different sizes and morphological characteristics (solid and sub-solid) of tumors. To confirm the cut-off value for discriminating metastasis and overall survival (OS) prediction, the receiver operating characteristic (ROC) analysis was performed based on PET/CT metabolic parameters (SUVmax/SUVmean/SULpeak/MTV/TLG), followed by Kaplan-Meier analysis for survival in post-operation patients with and without PET/CT plus MRI. RESULTS: 2,298 patients were included. No metastasis was observed in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm. The cut-off of PET/CT metabolic parameters on discriminating metastasis were 1.09 (SUVmax), 0.26 (SUVmean), 0.31 (SULpeak), 0.55 (MTV), and 0.81 (TLG), respectively. Patients undergoing PET/CT plus MRI exhibited longer OS compared to those who did not receive it in solid nodules ≥ 8.0 mm & sub-solid nodules ≥ 10.0 mm (HR, 0.44; p < 0.001); in solid nodules ≥ 8.0 mm (HR, 0.12; p<0.001) and in sub-solid nodules ≥ 10.0 mm (HR; 0.61; p=0.075), respectively. Compared to patients with metabolic parameters lower than cut-off values, patients with higher metabolic parameters displayed shorter OS: SUVmax (HR, 12.94; p < 0.001), SUVmean (HR, 11.33; p <0.001), SULpeak (HR, 9.65; p < 0.001), MTV (HR, 9.16; p = 0.031), and TLG (HR, 12.06; p < 0.001). CONCLUSION: The necessity of PET/CT and MRI should be cautiously evaluated in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm, however, these examinations remained essential and beneficial for patients with solid nodules ≥ 8.0 mm and sub-solid nodules ≥ 10.0 mm.

Identification of 10 differentially expressed and cuproptosis-related genes in immune infiltration and prognosis of thyroid carcinoma.

The association between the cuproptosis-related genes and the immune infiltration and their prognostic value in thyroid carcinoma is still unexplored. Bioinformatics analyses were performed with data obtained from the TCGA dataset. The aberrantly expressed genes were selected. KEGG and GO analyses were conducted to explore the enriched pathways of the up-regulated or down-regulated genes in thyroid carcinoma. Totally 1495 genes were differentially expressed (691 up-regulated, 804 down-regulated) in thyroid carcinoma (p<0.05). The 10 cuproptosis-related RNAs (DLD, LIAS, LIPT1, FDX1, DLAT, MTF1, PDHA1, CDKN2A, GLS and PDHB) were also demonstrated to be aberrantly expressed in thyroid carcinoma patients tissues. FDX1 expression was correlated with the overall survival in thyroid carcinoma patients (HR=0.4995, 95% CI: 0.2688-0.9285, p=0.0282). Further multivariate cox regression analysis revealed that DLD (HR=24.8869, 95% CI: 4.48772-138.01181, p=0.00024), and LIAS (HR=7.74092, 95% CI: 1.12194-53.40898, p=0.03783) were associated with the survival of thyroid carcinoma patients. The immune infiltration analysis demonstrated that significant correlation between the 10 cuproptosis-related genes and immune infiltration in thyroid carcinoma (p<0.01). We presented the expression profiles of dysregulated genes in thyroid carcinoma. The findings of our study highlighted the potential of cuproptosis-related genes as prognostic biomarkers for thyroid carcinoma.

The Spectrum of Dysregulated Aldosterone Production: An International Human Physiology Study.

CONTEXT: Primary aldosteronism is a form of low-renin hypertension characterized by dysregulated aldosterone production. OBJECTIVE: To investigate the contributions of renin-independent aldosteronism, and ACTH-mediated aldosteronism, in individuals with a low-renin phenotype representing the entire continuum of blood pressure. . DESIGN/PARTICIPANTS: Human physiology study of 348 participants with a low-renin phenotype with severe and/or resistant hypertension, hypertension with hypokalemia, elevated blood pressure and stage I/II hypertension, and normal blood pressure. SETTING: 4 international centers. . INTERVENTIONS/MAIN OUTCOME MEASURES: Saline suppression test (SST) to quantify the magnitude of renin-independent aldosteronism; dexamethasone suppression and ACTH-stimulation tests to quantify the magnitude of ACTH-mediated aldosteronism; adrenal venous sampling to determine lateralization. RESULTS: There was a continuum of non-suppressible and renin-independent aldosterone production following SST that paralleled the magnitude of the blood pressure continuum and transcended conventional diagnostic thresholds. In parallel, there was a full continuum of ACTH-mediated aldosteronism wherein post-SST aldosterone levels were strongly correlated with ACTH-stimulated aldosterone production (r = 0.75, P < 0.0001) and non-suppressible aldosterone production post-dexamethasone (r = 0.40, P < 0.0001). Beyond participants who met criteria for primary aldosteronism (post-SST aldosterone of ≥10 ng/dL or ≥277 pmol/L), the continuum of non-suppressible and renin-independent aldosterone production persisted below this diagnostic threshold, wherein 15% still had lateralizing aldosteronism amenable to surgical adrenalectomy, and the remainder were treated with mineralocorticoid receptor antagonists. CONCLUSIONS: In the context of a low-renin phenotype, there is a continuum of dysregulated aldosterone production that is prominently influenced by ACTH. A large proportion of individuals with low-renin have dysregulated aldosterone production and may benefit from aldosterone-directed therapy.

The role of type VI secretion system genes in antibiotic resistance and virulence in Acinetobacter baumannii clinical isolates.

Introduction: The type VI secretion system (T6SS) is a crucial virulence factor in the nosocomial pathogen Acinetobacter baumannii. However, its association with drug resistance is less well known. Notably, the roles that different T6SS components play in the process of antimicrobial resistance, as well as in virulence, have not been systematically revealed. Methods: The importance of three representative T6SS core genes involved in the drug resistance and virulence of A. baumannii, namely, tssB, tssD (hcp), and tssM was elucidated. Results: A higher ratio of the three core genes was detected in drug-resistant strains than in susceptible strains among our 114 A. baumannii clinical isolates. Upon deletion of tssB in AB795639, increased antimicrobial resistance to cefuroxime and ceftriaxone was observed, alongside reduced resistance to gentamicin. The ΔtssD mutant showed decreased resistance to ciprofloxacin, norfloxacin, ofloxacin, tetracycline, and doxycycline, but increased resistance to tobramycin and streptomycin. The tssM-lacking mutant showed an increased sensitivity to ofloxacin, polymyxin B, and furazolidone. In addition, a significant reduction in biofilm formation was observed only with the ΔtssM mutant. Moreover, the ΔtssM strain, followed by the ΔtssD mutant, showed decreased survival in human serum, with attenuated competition with Escherichia coli and impaired lethality in Galleria mellonella. Discussion: The above results suggest that T6SS plays an important role, participating in the antibiotic resistance of A. baumannii, especially in terms of intrinsic resistance. Meanwhile, tssM and tssD contribute to bacterial virulence to a greater degree, with tssM being associated with greater importance.

Multifocal, Asymmetric Bilateral Primary Aldosteronism Cannot be Excluded by Strong Adrenal Vein Sampling Lateralization: An International Retrospective Cohort Study.

BACKGROUND: Primary aldosteronism (PA) has been broadly dichotomized into unilateral and bilateral forms. Adrenal vein sampling (AVS) lateralization indices (LI) ≥2 to 4 are the standard-of-care to recommend unilateral adrenalectomy for presumed unilateral PA. We aimed to assess the rates and characteristics of residual PA after AVS-guided adrenalectomy. METHODS: We conducted an international, retrospective, cohort study of patients with PA from 7 referral centers who underwent unilateral adrenalectomy based on LI≥4 on baseline and/or cosyntropin-stimulated AVS. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue. RESULTS: The cohort included 283 patients who underwent AVS-guided adrenalectomy, followed for a median of 326 days postoperatively. Lack of PA cure was observed in 16% of consecutive patients, and in 22 patients with lateralized PA on both baseline and cosyntropin-stimulated AVS. Among patients with residual PA postoperatively, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue (versus 23% in those cured), wherein CACNA1D mutations were most prevalent (63% versus 33% in those cured). In adjusted regression models, independent predictors of postoperative residual PA included Black versus White race (odds ratio, 5.10 [95% CI, 1.45-17.86]), AVS lateralization only at baseline (odds ratio, 8.93 [95% CI 3.00-26.32] versus both at baseline and after cosyntropin stimulation), and CT-AVS disagreement (odds ratio, 2.75 [95% CI, 1.20-6.31]). CONCLUSIONS: Multifocal, asymmetrical bilateral PA is relatively common, and it cannot be excluded by robust AVS lateralization. Long-term postoperative monitoring should be routinely pursued, to identify residual PA and afford timely initiation of targeted medical therapy.

Mogroside V alleviates inflammation response by modulating miR-21-5P/SPRY1 axis.

Mogroside V (MV) is a natural sweetener extracted from the edible plant Siraitia grosvenorii that possesses anti-inflammatory bioactivity. It has been reported that microRNAs (miRNAs) play an important role in the inflammation response suppression by natural agents. However, whether the anti-inflammation effect of mogroside V is related to miRNAs and the underlying mechanism remains unclear. Our study aimed to identify the key miRNAs important for the anti-inflammation effect of MV and reveal its underlying mechanisms. Our results showed that MV effectively alleviated lung inflammation in ovalbumin-induced (OVA-induced) asthmatic mice. miRNA-seq and mRNA-seq combined analysis identified miR-21-5p as an important miRNA for the inflammation inhibition effect of MV and it predicted SPRY1 to be a target gene of miR-21-5p. We found that MV significantly inhibited the production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), and nitric oxide (NO), as well as the protein expression of p-P65/P65, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) in OVA-induced asthmatic mice and LPS-treated RAW 264.7 cells. Moreover, the release of ROS increased in LPS-stimulated RAW 264.7 cells but was mitigated by MV pretreatment. In the meantime, the expression of miR-21-5p was decreased by MV, leading to an increase in the expression of SPRY1 in RAW 264.7 cells. Furthermore, miR-21-5p overexpression or SPRY1 knockdown reversed MV's protective effect on inflammatory responses. Conversely, miR-21-5p inhibition or SPRY1 overexpression enhanced MV's effect on inflammatory responses in LPS-exposed RAW 264.7 cells. Therefore, the significant protective effect of mogroside V on inflammation response is related to the downregulation of miR-21-5p and upregulation of SPRY1 in vitro and in vivo, MiR-21-5p/SPRY1 may be novel therapeutic targets of MV for anti-inflammation treatment.

MR imaging findings of stage I intravenous leiomyomatosis: a retrospective single-center study in 19 cases.

OBJECTIVES: The aim was to explore the magnetic resonance imaging (MRI) features of stage-I intravenous leiomyomatosis (IVL). MATERIALS AND METHODS: From January 2019 to January 2023, clinical, pathological, and MRI data were collected from 19 cases confirmed by surgical pathology. Two radiologists retrospectively measured the tumor sizes, T1WIs, T2WIs, and ADC values and evaluated contrast-enhanced T1WIs, DWIs, complications and parauterine infiltrations. The number of tumor cells and the total nuclear area were measured. The percentage of tumor cell area out of the total area was used as the tumor cell density. RESULTS: Nineteen patients with stage-I IVL aged 33 to 66 years (mean age: 46 ± 7.6 years) were included in this study. All 19 cases were located in the myometrium or parametrium, with a mean diameter of 11.2 ± 4.8 cm. Among these cases, 14 (73.6%) were associated with leiomyoma, and six (31.6%) involved the broad ligament. Isointensity was observed in the T1WIs of 12 cases (63.2%), while slight hypointensity was seen in five patients (26.3%). Isointensity was observed in the on T2WIs of four cases (21.1%), and iso- or slight hyperintensity was observed in 15 cases (78.9%). A significant difference was detected between the normalized T2WIs of IVL and myometrium (p < 0.001). A Pearson correlation test showed demonstrated a negative correlation between the ADC and tumor cell density values (r = - 0.946, p < 0.001). Tortuous vessels were present in 17 cases (89.5%) within or next to the lesions, and multiple winding cord-like filling defects were seen in 11 cases (57.9%) within the tortuous vessels on the T2WIs. CONCLUSION: Identifying the characteristic MRI features of stage-I IVL helped improve the diagnostic accuracy achieves for this rare tumor. Stage-I IVL often presents as a large mass accompanied by leiomyoma, and it easily invades the broad ligament. TIWI signals exhibited isointensity, and T2WI signals contained iso- or slight hyperintensity. Tortuous vessels were present within or next to the lesions, and multiple winding cord-like filling defects were observed within the tortuous vessels on the T2WIs.

ß1-blockers in the reduction of bleeding risk in patients prescribed with potent dual antiplatelet therapy after acute coronary syndrome or percutaneous coronary intervention.

BACKGROUND: ß1-blockers could improve clinical outcomes in patients with coronary artery disease by lowering the heart rate, blood pressure, and myocardial contractility. Moreover, recent studies have suggested that ß1-blockers may also have the potential to reduce bleeding risk. OBJECTIVES: This study aimed to evaluate the association between ß1-blockers and bleeding risk in the patients prescribed with potent dual antiplatelet therapy (DAPT) after acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI). METHODS: Patients with ACS or undergoing PCI treated by DAPT of ticagrelor and aspirin were consecutively recruited. Follow-up for all eligible patients was conducted for 1 year. Major bleeding outcomes were defined as events that were type ≥2 based on the Bleeding Academic Research Consortium (BARC) criteria. RESULTS: A total of 1,113 eligible ticagrelor-treated patients were recruited. During the 1-year follow-up interval, 142 (12.6%) patients experienced BARC ≥2 bleedings including 23 patients (2.1%) suffering BARC ≥3 bleedings, with the most common site of bleeding located in the gastrointestinal tract. ß1-blockers treatment was associated with a lower risk of BARC ≥2 bleedings (11.2% vs. 23.3%, adjusted HR: 0.42, 95% CI: 0.28-0.62, P < 0.01). Moreover, metoprolol (11.1% vs. 23.3%, adjusted HR: 0.56, 95% CI: 0.37-0.83, P < 0.01) and bisoprolol (11.3% vs. 23.3%, adjusted HR: 0.56, 95% CI: 0.33-0.96, P = 0.04) had similar effects on the reduction of bleeding risk. CONCLUSION: ß1-blockers might be beneficial for the reduction of bleeding risk in potent dual antiplatelet therapy patients with ACS or undergoing PCI.

Research on the crisis propagation in the global coal trade under the Russia-Ukraine conflict.

The outbreak of the 2022 Russia-Ukraine conflict exacerbated the natural gas supply shortage in European countries. European countries restarted coal-fired power plants to maintain economic and social operations. The uneven distribution of coal resources in the world makes coal international trade inevitable. The intricate trade relations between trading countries have formed a coal trade network. When a country's coal exports are limited due to geopolitical factors, it will cause coal supply risks. The risk will spread to more countries along the trade network, eventually leading to the collapse of the trade network. This paper builds a crisis propagation model of the coal supply under the Russia-Ukraine conflict using the cascading failure model. The results showed that the Czech Republic, Ireland, Portugal, and Bulgaria become abnormal as the proportion of coal exports ß increases. When the Russian Federation reduced its coal exports by 80% and countries maintained only 10% coal exports against crisis, 23 European countries were the worst. Iceland, Ireland, Turkey and other countries were spread by the indirect risk and became abnormal countries. The Czech Republic and Bulgaria were spread by multiple risk and became abnormal countries.

Identification of T-cell exhaustion-related gene signature for predicting prognosis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumour with a poor prognosis in adults. Identifying biomarkers that can aid in the molecular classification and risk stratification of GBM is critical. Here, we conducted a transcriptional profiling analysis of T-cell immunity in the tumour microenvironment of GBM patients and identified two novel T cell exhaustion (TEX)-related GBM subtypes (termed TEX-C1 and TEX-C2) using the consensus clustering. Our multi-omics analysis revealed distinct immunological, molecular and clinical characteristics for these two subtypes. Specifically, the TEX-C1 subtype had higher infiltration levels of immune cells and expressed higher levels of immune checkpoint molecules than the TEX-C2 subtype. Functional analysis revealed that upregulated genes in the TEX-C1 subtype were significantly enriched in immune response and signal transduction pathways, and upregulated genes in the TEX-C2 subtype were predominantly associated with cell fate and nervous system development pathways. Notably, patients with activated T-cell activity status in the TEX-C1 subgroup demonstrated a significantly worse prognosis than those with severe T cell exhaustion status in the TEX-C2 subgroup. Finally, we proposed a machine-learning-derived novel gene signature comprising 12 TEX-related genes (12TexSig) to indicate tumour subtyping. In the TCGA cohort, the 12TexSig demonstrated the ability to accurately predict the prognosis of GBM patients, and this prognostic value was further confirmed in two independent external cohorts. Taken together, our results suggest that the TEX-derived subtyping and gene signature has the potential to serve as a clinically helpful biomarker for guiding the management of GBM patients, pending further prospective validation.

Comparative study of the molecular mechanisms underlying the G protein and ß-arrestin-dependent pathways that lead to ERKs activation upon stimulation by dopamine D2 receptor.

Dopamine D2 receptor (D2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or ß-arrestin. However, there has not been a systematic study of the regulatory process of D2 R-mediated ERKs activation by G protein- versus ß-arrestin-dependent signaling since D2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, ß-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and ß-arrestin-dependent pathway-mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3-kinase inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and ß-Arr2 played a main role in ß-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, ß-Arr2, and importinß1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes.

Interplay of Cortisol, Testosterone, and Abdominal Fat Mass in Normal-weight Women With Polycystic Ovary Syndrome.

Context: Ovarian and adrenal steroidogenesis underlie endocrine-metabolic dysfunction in polycystic ovary syndrome (PCOS). Adipocytes express aldo-keto reductase 1C3 and type 1 11ß-hydroxysteroid dehydrogenase, which modulate peripheral androgen and cortisol production. Objectives: To compare serum adrenal steroids, including 11-oxygenated androgens (11-oxyandrogens), cortisol, and cortisone between normal-weight women with PCOS and body mass index- and age-matched ovulatory women with normal-androgenic profiles (controls), and assess whether adrenal steroids associate with abdominal adipose deposition. Design: Prospective, cross-sectional, cohort study. Setting: Academic medical center. Patients: Twenty normal-weight women with PCOS and 20 body mass index-/age-matched controls. Interventions: Blood sampling, IV glucose tolerance testing, and total-body dual-energy x-ray absorptiometry. Main Outcome Measures: Clinical characteristics, hormonal concentrations, and body fat distribution. Results: Women with PCOS had higher serum total/free testosterone (T) and androstenedione (A4) levels and a greater android/gynoid fat mass than controls (androgens P < .001; android/gynoid fat mass ratio, P = .026). Serum total/free T and A4 levels correlated positively with android/gynoid fat mass ratio in all women combined (P < .025, all values). Serum 11ß-hydroxyA4, 11-ketoA4, 11ß-hydroxyT, 11-ketoT, cortisol, and cortisone levels were comparable between female types and unrelated to body fat distribution. Serum 11-oxyandrogens correlated negatively with % total body fat, but lost significance adjusting for cortisol. Serum cortisol levels, however, correlated inversely with android fat mass (P = .021), with a trend toward reduced serum cortisol to cortisone ratio in women with PCOS vs controls (P = .075), suggesting diminished 11ß-hydroxysteroid dehydrogenase activity. Conclusion: Reduced cortisol may protect against preferential abdominal fat mass in normal-weight PCOS women with normal serum 11-oxyandrogens.

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Mixed legume-grass seeding and nitrogen fertilizer input enhance forage yield and nutritional quality by improving the soil enzyme activities in Sichuan, China.

Information regarding relationships between forage yield and soil enzymes of legume-grass mixtures under nitrogen (N) fertilization can guide the decision-making during sustainable forage production. The objective was to evaluate the responses of forage yield, nutritional quality, soil nutrients, and soil enzyme activities of different cropping systems under various N inputs. Alfalfa (Medicago sativa L.), white clover (Trifolium repens L.), orchardgrass (Dactylis glomerata L.), and tall fescue (Festuca arundinacea Schreb.) were grown in monocultures and mixtures (A1: alfalfa, orchardgrass, and tall fescue; A2: alfalfa, white clover, orchardgrass, and tall fescue) under three N inputs (N1: 150 kg ha-1; N2, 300 kg ha-1; and N3: 450 kg ha-1) in a split plot arrangement. The results highlight that A1 mixture under N2 input had a greater forage yield of 13.88 t ha-1 year-1 than the other N inputs, whereas A2 mixture under N3 input had a greater forage of 14.39 t ha-1 year-1 than N1 input, but it was not substantially greater than N2 input (13.80 t ha-1 year-1). The crude protein (CP) content of grass monocultures and mixtures significantly (P < 0.05) increased with an increase in the rate of N input, and A1 and A2 mixtures under N3 input had a greater CP content of 18.91% and 18.94% dry matter, respectively, than those of grass monocultures under various N inputs. The A1 mixture under N2 and N3 inputs had a substantially greater (P < 0.05) ammonium N content of 16.01 and 16.75 mg kg-1, respectively, whereas A2 mixture under N3 had a greater nitrate N content of 4.20 mg kg-1 than the other cropping systems under various N inputs. The A1 and A2 mixtures under N2 input had a substantial higher (P < 0.05) urease enzyme activity of 0.39 and 0.39 mg g-1 24 h-1 and hydroxylamine oxidoreductase enzyme activity of 0.45 and 0.46 mg g-1 5 h-1, respectively, than the other cropping systems under various N inputs. Taken together, growing legume-grass mixtures under N2 input is cost-effective, sustainable, and eco-friendly, which provide greater forage yield and improved nutritional quality by the better utilization of resources.

The Role of Introgression During the Radiation of Endemic Fishes Adapted to Living at Extreme Altitudes in the Tibetan Plateau.

Recent genomic analyses of evolutionary radiations suggest that ancient introgression may facilitate rapid diversification and adaptive radiation. The loach genus Triplophysa, a genus with most species endemic to Tibetan Plateau, shows ecological diversity and rapid evolution and represents a potential example of adaptive radiation linked to the uplift of the Tibetan Plateau. Here, we interrogate the complex evolutionary history of Triplophysa fishes through the analysis of whole-genome sequences. By reconstructing the phylogeny of Triplophysa, quantifying introgression across this clade, and simulating speciation and migration processes, we confirm that extensive gene flow events occurred across disparate Triplophysa species. Our results suggest that introgression plays a more substantial role than incomplete lineage sorting in underpinning phylogenetic discordance in Triplophysa. The results also indicate that genomic regions affected by ancient gene flow exhibit characteristics of lower recombination rates and nucleotide diversity and may associate with selection. Simulation analysis of Triplophysa tibetana suggests that the species may have been affected by the Gonghe Movement in the third uplift of the Tibetan Plateau, resulting in founder effects and a subsequent reduction in Ne.