Circulating microRNAs in human obesity: a systematic review.

Context: Differential expression profiles of microRNAs have been reported in human obesity suggesting a miRNAs role in the development of obesity and associated disorders. Objective: To review circulating microRNAs (c-miRNAs) dysregulated in human obesity and to predict their possible target genes. Methods: We performed a systematic review on PubMed database (PROSPERO, CRD42017077742) for original works on c-miRNAs and human obesity and recorded c-miRNAs with differential expression profiles. Potential target genes and metabolic pathways for dysregulated miRNAs with at least two independent reports were searched using bioinformatic tools. Results: Twenty-two c-miRNAs are overexpressed, nine underexpressed and two c-miRNAs dysregulated in both directions in people with obesity compared to lean controls. Bioinformatic analyses suggest these c-miRNAs target on genes associated with fatty acid metabolism and PI3k/Akt pathway. Conclusion: Literature records 33 c-miRNAs confirmedly dysregulated in human obesity. Their predicted target genes are involved in pathways that could explain the development of obesity and its comorbidities. Further research will clarify the role of these miRNAs on metabolic diseases and their usefulness for the prognosis, prevention and treatment of obesity.

Inducible T-cell co-stimulators regulate the proliferation and invasion of human hepatocellular carcinoma HepG2 cells.

BACKGROUND: This study determined the regulatory effects of inducible T-cell co-stimulators (ICOS) in human hepatocellular carcinoma HepG2 cells using a RNA interference (RNAi) technique. METHODS: A RNAi technique was used to knockdown the expression of ICOS. ICOS expression after knockdown was detected as mRNA and protein levels by RT-PCR and Western blot, respectively. A MTT colorimetric assay was used to detect cell proliferation, and the Transwell assay was used to detect cell invasion. Western blot was carried out to detect the level of Bcl-2, AKT, and PI3K protein expression in different groups. RESULTS: The proliferation of HepG2 cells were significantly decreased after ICOS siRNA transfection (EG group). Similarly, the results of the Transwell experiment showed that invasion of HepG2 cells in the EG group was clearly reduced compared to the negative control (NC) and blank control groups (CON). Western blot analysis showed that knockdown of ICOS expression reduced the levels of Bcl-2 and AKT, and also significantly up-regulated the level of PI3K phosphorylation (P < 0.01). CONCLUSION: Down-regulating ICOS expression in HepG2 cells suppressed cell proliferation and invasion. The underlying mechanism may be related to the expression of the downstream factor, PI3K/AKT.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

PI3K, AKT, and P-AKT levels in thin endometrium.

The aim of this study was to explore the expression of PI3K, AKT, and P-AKT, and to investigate the role of PI3K/AKT signaling pathway in thin endometrium. We included 40 women treated in affiliated Shenzhen Nanshan People's Hospital of Guangdong Medical University for endometrial conditions between August 2013 and January 2015, 20 with a normal endometrium, and 20 with thin endometrium. The expression of PI3K, AKT, and P-AKT was evaluated by the immunohistochemical S-P method. The expression of PI3K, AKT, and P-AKT proteins was significantly lower in the thin endometrium group than in the normal endometrium group (P < 0.05). The expression of PI3K and AKT was positively correlated with the expression of P-AKT. The expression of PI3K, AKT, and P-AKT proteins in the thin endometrium decreases during the proliferative phase, and this process could be associated with PI3K/AKT signaling.

The role of YKL-40 in a cancerous process.

YKL-40 is a secretory protein secreted among others by tumor cells and tumor-associated macrophages. Due to the structural homology this protein was classified to chitinases family CLP (chitinase - like protein) and to 18 of glycosyl hydrolase family, but it has no catalytic function. Elevated levels of YKL-40 in serum is observed in the inflammatory diseases of various aetiology and in cancers, such as breast, ovarian, colon or lung. The results of many studies suggest a significant relationship of YKL-40 with progression of cancer: incidence of metastases, shorter relapse-free survival and shorter overall survival. It is believed that YKL-40 may be a prognostic factor of cancer development and the patient's response to the applied therapy. Elevated levels of protein in serum of cancer patients may play a role in angiogenesis, proliferation and migration of tumor cells. Probably the mechanism of this phenomenon is the result of YKL-40 action by FAK and PI3K/AKT signaling pathways. Results obtained so far are largely based on an analysis of the YKL-40 level in the patients' serum and on the assessment of changes in the expression level of this protein in studies in vitro and in vivo.

[Expression profiles of PI3K, NF-κB, and STAT1 in peripheral blood mononuclear cells in children with bronchial asthma].

OBJECTIVE: To study the expression profiles of PI3K, NF-κB, and STAT1 in peripheral blood mononuclear cells (PBMCs) in children with bronchial asthma, as well as their roles in the pathogenesis of asthma. METHODS: Thirty children with acute exacerbation of bronchial asthma were enrolled as the asthma group, and 20 healthy children were enrolled as the control group. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of PI3K, NF-κB, and STAT1 in PBMCs. A spirometer was used to compare the pulmonary function between the two groups. The correlations between the mRNA expression of PI3K, NF-κB, and STAT1 and pulmonary function in children with bronchial asthma were analyzed. RESULTS: The asthma group had significantly higher mRNA and protein expression levels of PI3K, NF-κB, and STAT1 than the control group (P<0.05). Compared with the control group, the asthma group showed significant reductions in pulmonary function indices such as FEV1%, FEV1/FVC, and PEF% (P<0.05). In children with bronchial asthma, the mRNA expression levels of PI3K, NF-κB, and STAT1 were negatively correlated with FEV1%, FEV1/FVC, and PEF% (P<0.05). CONCLUSIONS: The expression levels of PI3K, NF-κB, and STAT1 increase in children with asthma, and are negatively correlated with pulmonary function indices, suggesting that PI3K, NF-κB and STAT1 are involved in the development and progression of bronchial asthma in children.

Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial.

BACKGROUND: Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels. METHODS: We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323. FINDINGS: Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses. INTERPRETATION: BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations. FUNDING: Bayer HealthCare Pharmaceuticals.

Effects of oral N-acetylcysteine on walking capacity, leg reactive hyperemia, and inflammatory and angiogenic mediators in patients with intermittent claudication.

Increased oxidative stress and inflammation contribute to impaired walking capacity and endothelial dysfunction in patients with intermittent claudication (IC). The goal of the study was to determine the effects of oral treatment with the antioxidant N-acetylcysteine (NAC) on walking capacity, leg postocclusive reactive hyperemia, circulating levels of inflammatory mediators, and whole blood expression of angiogenic mediators in patients with IC. Following a double-blinded randomized crossover design, 10 patients with IC received NAC (1,800 mg/day for 4 days plus 2,700 mg before the experimental session) and placebo (PLA) before undergoing a graded treadmill exercise test. Leg postocclusive reactive hyperemia was assessed before and after the test. Blood samples were taken before and after NAC or PLA ingestions and 5 and 30 min after the exercise test for the analysis of circulating inflammatory and angiogenic markers. Although NAC increased the plasma ratio of reduced to oxidized glutathione, there were no differences between experimental sessions for walking tolerance and postocclusive reactive hyperemia. Plasma concentrations of soluble vascular cell adhesion protein-1, monocyte chemotactic protein-1, and endothelin-1 increased similarly following maximal exercise after PLA and NAC (P < 0.001). Whole blood expression of pro-angiogenic microRNA-126 increased after maximal exercise in the PLA session, but treatment with NAC prevented this response. Similarly, exercise-induced changes in whole blood expression of VEGF, endothelial nitric oxide synthase and phosphatidylinositol 3-kinase R2 were blunted after NAC. In conclusion, oral NAC does not increase walking tolerance or leg blood flow in patients with IC. In addition, oral NAC prevents maximal exercise-induced increase in the expression of circulating microRNA-126 and other angiogenic mediators in patients with IC.

PIK3CA mutations in serum DNA are predictive of recurrence in primary breast cancer patients.

We attempted to develop a highly sensitive and specific method for the detection of circulating tumor DNA (ctDNA) using a digital PCR (dPCR) assay for PIK3CA mutations (i.e., H1047R, E545K, and E542K) in primary breast cancer patients. The sensitivity of the dPCR assay for the mutant alleles was examined using cell lines with PIK3CA mutations and proved to be 0.01 %. Serum samples were collected pre-operatively from 313 stage I-III breast cancer patients, of whom 110 were found to have PIK3CA mutant tumors. The serum samples from these patients with PIK3CA mutant tumors were subjected to the dPCR assay, and 25 (22.7 %) were found to be positive. No PIK3CA mutant ctDNA was detected in the serum samples of 50 healthy women and 30 breast cancer patients with PIK3CA non-mutant tumors. The patients with PIK3CA mutant ctDNA were dichotomized into mutant ctDNA-high (ctDNA(high)) and ctDNA-low (ctDNA(low)) groups based on the median. The ctDNA(high) patients exhibited significantly shorter recurrence-free survival (RFS; P = 0.0002) and overall survival rates (OS; P = 0.0048) compared to those exhibited by the combined ctDNA(low) patient and ctDNA-free patient group. Multivariate analysis revealed that ctDNA(high) status significantly predicted poor RFS and OS and did so independently of conventional histological parameters. These results suggest that dPCR is a highly sensitive and specific method for the detection of PIK3CA mutant ctDNA and that ctDNA(high) but not ctDNA(low) status is a significant and independent prognostic factor for primary breast cancer patients.

Dysfunction of the PI3 kinase/Rap1/integrin α(IIb)ß(3) pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia.

Patients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca(2+) mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1,ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin α(IIb)ß(3) activation.

Neoadjuvant treatment of breast cancer: maximizing pathologic complete response rates to improve prognosis.

PURPOSE OF REVIEW: Neoadjuvant therapy is very often an adequate alternative to adjuvant therapy. This review summarizes the recent advances made in the area of neoadjuvant therapy in breast cancer. The focus will lie on recently published clinical trials, but will not further highlight surgical, imaging and radio-oncological issues related to neoadjuvant therapy. RECENT FINDINGS: Within the past year, it has been discussed if neoadjuvant treatment can be used as a faster way to get access to new therapies, based on new data in HER2+ breast cancer, suggesting a higher pathological complete response rate when a dual anti-HER2 therapy was used. Nevertheless, this higher pathological complete response rate does not necessarily always translate into a better survival. In triple negative breast cancer, carboplatin could be identified as an asset for patients, especially in patients with gBRCA mutations. However, mature long-term data are still missing. The neoadjuvant approach is ideal to identify new biomarkers which predict response or resistance to the given treatment. Tumour-infiltrating lymphocytes and PIK3CA mutations are amongst the most promising markers. SUMMARY: Neoadjuvant treatment should be considered for all patients with HER2-positive or triple negative breast cancer. Clinical trials in this setting are currently investigating new approaches.

Heterogeneity of epidermal growth factor receptor status and mutations of KRAS/PIK3CA in circulating tumor cells of patients with colorectal cancer.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC). METHODS: We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations. RESULTS: At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients. CONCLUSIONS: Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.

Thioacetamide promotes osteoclast transformation of bone marrow macrophages by influencing PI3K/AKT pathways.

BACKGROUND: Osteoclast cell increase is a major risk factor for osteoporosis and degenerative bone and joint diseases. At present, RANKL and M-CSF are commonly used to induce osteoclastogenesis. Thioacetamide (TAA) can lead to many types of liver and kidney damage, but less attention has been paid to the association of TAA with bone damage. In this work, we investigated the effects of TAA on the osteoclastogenesis and differentiation of bone marrow macrophages (BMMs). METHODS: BMMs of SD rat suckling mice were taken for primary culture. CCK-8 was used to detect the toxic effects of TAA on BMMs, and flow cytometry was used to detect the effects of TAA on the cell cycle, cell viability, apoptosis and intracytoplasmic Ca2+ concentration of BMMs. TRAP staining was used to detect the effect of RANKL and M-CSF and TAA on osteoclast differentiation of BMMs. Western Blot was used to detect the expression level of PI3K/AKT pathway and osteoclast-specific proteins (TRAP and cathepsin K). RESULTS: The results suggested that TAA inhibited the proliferation of BMMs, while enhancing osteoclastogenesis at 0.5 mg/mL and 1 mg/mL as assayed by TRAP staining. Exposed to TAA, BMMs could differentiate into osteoclast-like cells with overexpression of cathepsin K and TRAP proteins. Western blot results showed that TAA can activate the expression levels of P-PI3K, P-AKT, P-P38, and P-JNK, accompanied by apoptosis of BMMs and increase in intracellular Ca2+. CONCLUSION: TAA may induce osteoclast formation in BMMs by activating the expression of PI3K/AKT pathway proteins, which is comparable to the classic osteoclast differentiation inducer RANKL and M-CSF. This suggests that we may find a cheap osteoclast inducer.

Intake of red wine increases the number and functional capacity of circulating endothelial progenitor cells by enhancing nitric oxide bioavailability.

OBJECTIVE: Red wine (RW) consumption has been associated with a reduction of cardiovascular events, but limited data are available on potential mediating mechanisms. This study tested the hypothesis that intake of RW may promote the circulating endothelial progenitor cell (EPC) level and function through enhancement of nitric oxide bioavailability. METHODS AND RESULTS: Eighty healthy, young subjects were randomized and assigned to consume water (100 mL), RW (100 mL), beer (250 mL), or vodka (30 mL) daily for 3 weeks. Flow cytometry was used to quantify circulating EPC numbers, and in vitro assays were used to evaluate EPC functions. After RW ingestion, endothelial function determined by flow-mediated vasodilation was significantly enhanced; however, it remained unchanged after water, beer, or vodka intake. There were significantly increased numbers of circulating EPC (defined as KDR(+)CD133(+), CD34(+)CD133(+), CD34(+)KDR(+)) and EPC colony-forming units only in the RW group (all P<0.05). Only RW ingestion significantly enhanced plasma levels of nitric oxide and decreased asymmetrical dimethylarginine (both P<0.01). Incubation of EPC with RW (but not beer or ethanol) and resveratrol in vitro attenuated tumor necrosis factor-alpha-induced EPC senescence and improved tumor necrosis factor-alpha-suppressed EPC functions and tube formation. Incubation with nitric oxide donor sodium nitroprusside significantly ameliorated the inhibition of tumor necrosis factor-alpha on EPC proliferation, but incubation with endothelial nitric oxide synthase inhibitor l-NAME and PI3K inhibitor markedly attenuated the effect of RW on EPC proliferation. CONCLUSIONS: The intake of RW significantly enhanced circulating EPC levels and improved EPC functions by modifying nitric oxide bioavailability. These findings may help explain the beneficial effects of RW on the cardiovascular system. This study demonstrated that a moderate intake of RW can enhance circulating levels of EPC in healthy subjects by increasing nitric oxide availability. Direct incubation of EPC with RW and resveratrol can modify the functions of EPC, including attenuation of senescence and promotion of EPC adhesion, migration, and tube formation. These data suggest that RW ingestion may alter the biology of EPC, and these alterations may contribute to its unique cardiovascular-protective effect.

Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways.

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Plasma Metabolome Profiling by High-Performance Chemical Isotope-Labelling LC-MS after Acute and Medium-Term Intervention with Golden Berry Fruit (Physalis peruviana L.), Confirming Its Impact on Insulin-Associated Signaling Pathways.

PURPOSE: Golden berry (Physalis peruviana L.) is an exotic fruit exported from Colombia to different countries around the world. A review of the literature tends to demonstrate a hypoglycaemic effect with an improvement in insulin sensitivity after oral ingestion of fruit extracts in animal models. However, little is known about their potential effects in humans, and very little is known about the mechanisms involved. This study aimed at identifying discriminant metabolites after acute and chronic intake of golden berry. METHOD: An untargeted metabolomics strategy using high-performance chemical isotope-labelling LC-MS was applied. The blood samples of eighteen healthy adults were analysed at baseline, at 6 h after the intake of 250 g of golden berry (acute intervention), and after 19 days of daily consumption of 150 g (medium-term intervention). RESULTS: Forty-nine and 36 discriminant metabolites were identified with high confidence, respectively, after the acute and medium-term interventions. Taking into account up- and downregulated metabolites, three biological networks mainly involving insulin, epidermal growth factor receptor (EGFR), and the phosphatidylinositol 3-kinase pathway (PI3K/Akt/mTOR) were identified. CONCLUSIONS: The biological intracellular networks identified are highly interconnected with the insulin signalling pathway, showing that berry intake may be associated with insulin signalling, which could reduce some risk factors related to metabolic syndrome. Primary registry of WHO.

Circulating lncRNAs HIF1A-AS2 and LINLK-A: Role and Relation to Hypoxia-Inducible Factor-1α in Cerebral Stroke Patients.

Long noncoding RNAs (lncRNAs) have been recently recognized as key players of gene expression in cerebral pathogenesis. Thus, their potential use in stroke diagnosis, prognosis, and therapy is actively pursued. Due to the complexity of the disease, identifying stroke-specific lncRNAs remains a challenge. This study investigated the expression of lncRNAs HIF1A-AS2 and LINK-A, and their target gene hypoxia-inducible factor-1 (HIF-1) in Egyptian stroke patients. It also aimed to determine the molecular mechanism implicated in the disease. A total of 75 stroke patients were divided into three clinical subgroups, besides 25 healthy controls of age-matched and sex-matched. Remarkable upregulation of lncRNA HIF1A-AS2 and HIF1-α along with a downregulation of lncRNA LINK-A was noticed in all stroke groups relative to controls. Serum levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated-Akt (p-Akt), vascular endothelial growth factor (VEGF), and angiopoietin-1 (ANG1) as well as their receptors, malondialdehyde (MDA), and total antioxidant capacity (TAC) were significantly increased, whereas brain-derived neurotrophic factor (BDNF) levels were significantly decreased particularly in hemorrhagic stroke versus ischemic groups. Eventually, these findings support the role of lncRNAs HIF1A-AS2 and LINK-A as well as HIF1-α in activation of angiogenesis, neovascularization, and better prognosis of stroke, especially the hemorrhagic type.

LncRNA-SNHG16 promotes proliferation and migration of acute myeloid leukemia cells via PTEN/PI3K/AKT axis through suppressing CELF2 protein.

The silence of lncRNA small nucleolar RNA host gene 16 (SNHG16) suppressed acute lymphoblastic leukemia (ALL) cell proliferation and migration, whereas its role in acute myeloid leukemia (AML) still lacks clarity. This study showed that SNHG16 was upregulated in AML patients and cells. And SNHG16 overexpression remarkably enhanced the proliferation and migration capacities of HL60 and AML-193 cells, while SNHG16 knockdown acted the opposite way. Subsequently, we revealed that SNHG16 directly bound to CELF2 (CUGBP Elav-like family member 2) protein, and caused CELF2 mRNA unstably and proteins reducing. CELF2 was decreased both in AML patients and cells. CELF2 overexpression or interference weakened the effect of overexpressing or silencing SNHG16 on proliferation and migration. Moreover, the transfection of pcDNA-CELF2 elevated PTEN (phosphatase and tensin homolog) activity and hindered the phosphoinositide 3-kinase (PI3K)/AKT signaling. And SNHG16 reduced PTEN activity and promoted the PI3K/AKT pathway activation by restraining CELF2. Furthermore, GDC-0941 (a specific inhibitor of the PI3K/AKT pathway) impeded the effect of SNHG16 increase, and bpV(pic) (a specific PTEN inhibitor) declined the effect of SNHG16 decrease on cell proliferation and migration. Taken together, the present study indicated that SNHG16 promoted proliferation and migration of AML cells via PTEN/PI3K/AKT axis through suppressing CELF2 protein.

Circulating exosomal microRNAs in bipolar disorder.

INTRODUCTION: Emerging evidence suggests central roles of miRNAs in the pathogenesis of bipolar disorder (BD). Exosomes are membrane-bound vesicles acting as "biological cargo carriers" of various types of molecules including microRNAs. In this study, we aimed to investigate circulating exosomal microRNAs as potential diagnostic biomarkers for BD. METHODS: The exosomes were precipitated from plasma samples of patients with BD (n = 69; 15 depressed, 27 manic, 27 euthymic) and healthy controls (n = 41). Total RNA was extracted from the exosomes and the levels of miRNAs were assayed by qPCR. Dysregulated miRNAs were subjected to Kyoto Encyclopedia of Genes and Genomes" (KEGG) pathway analysis by DIANA-miRPath v3.0 to identify the predicted targets and the related pathways. RESULTS: Thirteen miRNAs showed significant differences between patients with BD and healthy individuals; among these, MiR-484, -652-3p, -142-3p remained significantly downregulated and miR-185-5p remained significantly upregulated after accounting for multiple comparisons and adjustments for potential confounders. There were no significant alterations among different states of BD. The KEEG analysis of four dysregulated miRNAs highlighted several target pathways including PI3K/Akt signaling, fatty acid biosynthesis/metabolism, extracellular matrix and adhesion pathways. CONCLUSION: Our findings suggest that dysregulation of miRNAs might be involved in the underlying pathophysiology of BD through several biological pathways; and highlight the importance of the exosomal miRNAs for biomarker research in BD. Further longitudinal studies may clarify the roles of exosomal miRNAs and their targets in the neurobiology of BD.