Transcriptomic and physiological comparison of Shatangju (Citrus reticulata) and its late-maturing mutant provides insights into auxin regulation of citrus fruit maturation.

Previous studies have shown that abscisic acid (ABA) and ethylene are involved in pulp maturation and peel coloration in the nonclimacteric citrus fruits. There are also signs indicating that other plant hormones may play some roles in citrus fruit ripening. In this study, we compared profiles of genome-wide gene expression and changes in hormones and peel pigments between fruits of Shatangju mandarin (Citrus reticulata Blanco, designated WT) and its natural mutant, Yuenongwanju (designated MT). The MT fruit matures ~2 months later than the WT fruit. Significant differences in fruit diameter, total soluble solids, titratable acid content, chlorophylls and carotenoids were detected between the fruits of the two genotypes at the sampled time points. Genome-wide transcriptome profiling showed that many genes involved in auxin and ABA metabolism and/or signaling pathways were differentially expressed between the MT and the WT fruits. Importantly, the expression of CrYUCCA8 was significantly lower and the expression of CrNCED5 was significantly higher in WT than in MT fruits at 230 and 250 DPA, respectively. In addition, the indole-3-acetic acid (IAA) level in the MT fruit was significantly higher than that in the WT counterpart, whereas a significantly lower level of ABA was detected in the mutant. Treatment of the WT fruit with exogenous IAA significantly delayed fruit maturation. Our results provide experimental evidence supporting the notion that auxin is a negative regulator of fruit maturation in citrus.

Gut microbiota in hypertensive patients with versus without obstructive sleep apnea.

We investigated the alteration of gut microbiota and the associated metabolic risks in hypertensive patients with obstructive sleep apnea (OSA) comorbidity. Fecal and blood samples were collected from 52 hypertensive patients, who were divided into three groups: A (controls, apnea-hypopnea index[AHI] < 5, n = 15), B (mild OSA, 5 < AHI < 20, n = 17), and C (moderate-to-severe OSA, AHI > 20, n = 20). The composition of the gut microbiota was studied through 16s RNA sequencing of variable regions 3-4. Analysis of the results revealed that group C had a significant higher concentration of total cholesterol, low-density lipoprotein, and IL-1ß compared with group A. The Shannon index showed that bacterial biodiversity was lower in OSA patients. At the phylum level, the ratio of Firmicutes to Bacteroidetes (F/B) was significantly higher in group C than in groups A and B. At the genus level, the relative abundance of short-chain fatty acids (SCFA)-producing bacteria (e.g., Bacteroides and Prevotella) was lower while the number of inflammation-related bacteria (e.g., Lactobacillus) was increased in patients with OSA. We found that the IL-1ß level was negatively correlated with Bacteroidetes. The area under the receiver operating characteristic curve was .672 for F/B ratio in determining hypertensive patients with OSA. In patients with hypertension, OSA was associated with worse gut dysbiosis, as evidenced by decreased levels of short-chain fatty acids-producing bacteria and increased number of inflammation-related bacteria. The differences in gut microbiota discriminate hypertensive patients with OSA from those without and may result in an enhanced inflammatory response and increase the risk of metabolic diseases.

The Level of HbA1c Evaluates the Extent of Coronary Atherosclerosis Lesions and the Prognosis in Diabetes with Acute Coronary Syndrome.

Background: The level of HbA1c can reflect the average level of blood glucose over 3 months, which is the gold standard indicator for monitoring blood glucose. The relationship between the level of HbA1c and the extent of coronary atherosclerosis lesions or the prognosis in diabetes with acute coronary syndrome (ACS) remains poorly understood. Aims: To explore whether the level of HbA1c can evaluate the extent of coronary atherosclerosis lesions or the prognosis in diabetes with acute coronary syndrome (ACS) using the SYNTAX score, the Global Registry of Acute Coronary Events (GRACE) score, left ventricular function (LVEF), left ventricular end-diastolic volume (LVEDV), and major adverse cardiac events (MACEs) in the hospital and 12 months after discharge. Methods: This study was a prospective, randomized, open-label, and parallel group study. Patients with diabetes with ACS were recruited into this study indiscriminately, and all the participants were divided into two groups according to the level of HbA1c: HbA1c level ≤ 7%group and HbA1c level > 7%group. The followings were used as the evaluation indicators: SYNTAX score, GRACE score, LVEF, LVEDV, and MACEs in hospital and 12 months after discharge. Results: A total of 233 patients with diabetes and ACS were enrolled and assigned to two groups according to their level of HbA1c: the HbA1c ≤ 7%group (n = 92) and the HbA1c > 7%group (n = 141). The results showed that the proportion of STEMI was higher in the HbA1c ≤7% group (p < 0.05), while the proportion of NSTEMI has not significantly higher in the HbA1c >7% group (p > 0.05). Regression analysis indicated that HbA1c level was significantly positively correlated with GRACE score (r = 0.156, F = 5.784, p = 0.017, n = 233) and SYNTAX score (r = 0.237, F = 13.788, p < 0.001, n = 233), and there were no statistically significant differences in LVEDV and LVEF between the two groups (p > 0.05). The total MACEs rate showed no significant difference between the two groups during hospitalization (p > 0.05) but showed significant differences at 12 months after discharge (p < 0.05). Conclusions: This study shows that HbA1c level was positively correlated with the extent of coronary atherosclerosis lesions and the prognosis in diabetes with ACS. The higher the HbA1c level is, the more severe the coronary atherosclerotic lesion and the worse the prognosis in diabetes with ACS are.

Cholecystokinin-mediated pharmacological preconditioning effects on ischemic rat hearts: Possible signaling pathways.

BACKGROUND: Cholecystokinin (CCK-8) has been shown to exhibit pharmacological preconditioning and cardioprotective effects. However, the molecular mechanisms involved in CCK-8-induced pharmacological preconditioning have not yet been clarified. OBJECTIVES: The current study explored the molecular mechanisms involved in CCK-8-mediated pharmacological preconditioning effects on ischemic rat hearts. MATERIAL AND METHODS: Pharmacological preconditioning was induced in male Wistar rats by administration of CCK-8 (20 µg/kg) 24 h before heart isolation. The PI3K inhibitor LY294002 (10 mg/kg and 20 mg/kg) and the HIF-1α inhibitor YC-1 (1 mg/kg and 2 mg/kg) were administered 30 min before the administration of CCK-8. The hearts were subjected to ischemia-reperfusion (IR) injury using a Langendorff apparatus. Myocardial injury was quantified by measuring the release of LDH-1, CK-MB and cTnT. The levels of HIF-1α and p-Akt expression and the ratio of p-GSK-3ß/GSK-3ß, were assessed in the heart homogenates. RESULTS: Pharmacological preconditioning with CCK-8 reduced IR-induced increases in the release of LDH, CK-MB and cTnT. Moreover, it restored the expression of HIF-1α and p-Akt, and the p-GSK-3ß/GSK-3ß ratio. However, administration of LY294002 or YC-1 with CCK-8 significantly abolished the cardioprotective effects of pharmacological preconditioning. The PI3K and HIF-1α inhibitors also abolished the effects of CCK-8 preconditioning on HIF-1α, p-Akt and p-GSK-3ß/GSK-3ß. CONCLUSIONS: Based on these findings, it may be concluded that the molecular mechanisms participating in CCK-8-induced pharmacological preconditioning involve HIF-1α, PI3K, Akt, and GSK-3ß signaling pathways.

Renal denervation improves chronic intermittent hypoxia induced hypertension and cardiac fibrosis and balances gut microbiota.

AIMS: Investigate the effect of renal denervation (RDN) on chronic intermittent hypoxia (CIH) induced high blood pressure (BP) and cardiac injury, and explore whether the effect is associated with gut microbiota alteration and its product, trimethylamine N-oxide (TMAO). MATERIALS AND METHODS: Thirty six-week-old Sprague Dawley male rats were randomly divided into three groups: Control, CIH (20 cycles h-1, 7-8% at nadir, 8 h.day-1 for 6 weeks) and RDN group. Fecal samples, serum and heart tissue were collected at week 6. 16S rRNA gene sequencing was performed in fecal samples. KEY FINDINGS: Systolic BP in CIH group was significantly elevated compared with Control (164 ± 3 vs. 143 ± 4 mmHg, p = 0.004), while RDN treatment evidently reduced elevated systolic BP (133 ± 5 vs. 164 ± 3 mmHg, p < 0.001). CIH group featured significant cardiac perivascular fibrosis, compared with Control, whereas RDN treatment effectively attenuated perivascular fibrosis. Principal component analysis showed that CIH rats, but not RDN group were noticeably separated from Control. At phyla level, the structure of the biological community of RDN rats converged with that of control rats, which was apparently different in comparison to CIH rats. TMAO levels in the three groups were not significantly different. SIGNIFICANCE: RDN exerts beneficial effect on BP control and perivascular fibrosis in rats exposed to CIH. This effect is associated with its ability to revert the already skewed gut microbiota caused by CIH, but is not via regulation of TMAO.

A novel method to detect autoantibodies against platelets in patients with immune thrombocytopenia.

BACKGROUND AND OBJECTIVES: To examine anti-platelet autoantibodies in patients with immune thrombocytopenia (ITP) not only provides solid evidence for diagnosis, and also helps to select an individualized strategy for the treatment. The aim of this study is to develop a novel cell-based assay to detect autoantibodies in ITP patients. METHODS/PATIENTS: The DNA sequences of human platelet membrane protein GPIbα, GPIbß, GP IX, GPIIb and GPIIIa subunits were obtained from NCBI database and synthesized. The synthetic fragments were ligated into pcDNA 3.3 and constructed the recombinant plasmids and transfected into Chinese hamster ovary (CHO) cells to establish cell lines stable expressing GPIb-IX and/or GPIIb/IIIa complexes. One hundred and two ITP patients with different anti-platelet autoantibodies, 57 patients with other kinds of autoimmune diseases and 104 healthy control were selected to examine sensitivity, specificity and accuracy of this method. RESULTS: CHO cells stable expressing GPIb-IX and/or GPIIb/IIIa proteins were established. The cells were fixed with 4% paraformaldehyde and stored at -80 ℃, more than 80% of the cells were still expressed target proteins after 180 days of storage. The concentrations of target antibody from 0.1 to 100 µg/ml were detectable by this method, and 10-50 µg/ml antibody binding to the CHO cells yielded higher distinguishable fluorescent intensities. Inter-assay and intra-assay coefficients of variation and receiver operating characteristic curve analysis showed that this method had relatively higher reproducibility and specificity. Compared with Flow Cytometric Immunobead Array, this method has relatively higher specificity (95.2%) and accuracy (90.8%) in detection of 102 ITP patients. CONCLUSION: A novel cell-based assay to detect autoantibodies in ITP patients is established, which appears to be a promising method to diagnose ITP.

May Measurement Month 2018: an analysis of blood pressure screening results from China.

To further improve awareness, treatment, and control of hypertension, the May Measurement Month (MMM) campaign continued in 2018 in China. Study subjects were adults aged 18 years or more, ideally those who had not their blood pressure (BP) measured for at least a year. Blood pressure was measured three times consecutively with a 1-min interval in the sitting position, using automated BP monitors in 288 342 participants and transmitted to a central database by a smartphone app. Questionnaire data were collected with the same app. After imputation, the overall proportion of hypertension was 29.8%. Of those with hypertension, the rates of awareness, treatment, and control were 62.3%, 57.3%, and 35.9%, respectively. In analysis based on linear regression models, both systolic and diastolic BP were higher with cigarette smoking, alcohol intake, and overweight and obesity. Our study results suggest that hypertension management is improving in comparison with the data in MMM 2017 and the nationwide survey in 2012-15, and several known lifestyle factors are key to hypertension management.

Glabridin attenuates endothelial dysfunction and permeability, possibly via the MLCK/p-MLC signaling pathway.

Atherosclerosis is caused by various factors, and Glabridin may have protective effects on the cardiovascular system. The purpose of the present study was to evaluate the effects of Glabridin on atherosclerosis and evaluate whether Glabridin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light chain (MLC) kinase (MLCK)/phosphorylated (p)-MLC system via the mitogen activated protein kinase (MAPK) signaling pathway. Male New Zealand rabbits were randomly divided into 3 groups: The control group was administered an ordinary diet, whereas the high fat group and the Glabridin (2 mg/kg/d) intervention group were administered a high fat diet. Following 12 weeks, the blood lipid levels of rabbits, the morphological structure of the arterial wall, the arterial intimal permeability, the endothelial function and the mRNA levels of MLCK were measured. Western blot analysis was used to detect the levels of MLCK, p-c-Jun N-terminal kinase (JNK), p-extracellular signal regulated kinase (ERK), and p-p38. The high-fat diet group exhibited significantly increased total cholesterol and triglycerides, and endothelial dysfunction, which were attenuated by Glabridin treatment. Notably, the aortic endothelial permeability was increased in the high-fat diet group but was ameliorated in the Glabridin treatment group. Hyperlipidemia enhanced the expression of p-MLC and MLCK, which were associated with the increased phosphorylation of ERK, p38 and JNK. These changes were also ameliorated by Glabridin. In conclusion, the results of the present study suggested that atherosclerosis may be associated with upregulated MLCK expression and activity, which was downregulated by Glabridin. The mechanism of action of Glabridin was thought to proceed through modulating MAPK pathway signal transduction. However, further studies are required to adequately illuminate the exact regulatory mechanisms involved.

All­trans retinoic acid reduces endothelin­1 expression and increases endothelial nitric oxide synthase phosphorylation in rabbits with atherosclerosis.

All-trans retinoic acid (ATRA) is a natural derivative of vitamin A that ameliorates atherosclerosis (AS) by regulating inflammatory factors. However, studies concerning the role of retinoic acid in artery endothelial function are rare. Therefore, the present study investigated its role in regulating the production of endothelin­1 (ET­1) and nitric oxide (NO) in rabbits with AS. The rabbits were randomly divided into 3 groups: The control group was administered an ordinary diet, while the high fat group and the ATRA drug intervention group were administered a high fat diet. After 12 weeks, the blood lipid levels of rabbits, the morphological structure of the arterial wall, the arterial intimal permeability, the activity of blood endothelial nitric oxide synthase (eNOS) and the level of plasma NO were investigated. Western blot analysis was used to detect the levels of ET­1, eNOS and eNOS phosphorylation at Ser­1177 (p­eNOS), and a radioimmunoassay was performed to detect the level of ET­1 in the plasma. It was identified that plaque formation was alleviated in the ATRA group compared with the high fat group, as revealed by hematoxylin and eosin and oil red O staining, and a similar trend was reflected in the immunofluorescence results for endothelial permeability. Western blotting demonstrated significantly decreased ET­1 expression levels in the arterial tissue of rabbits in the ATRA group compared with the high fat group, together with increased p­eNOS level (P<0.05), however, no difference was observed in the expression of eNOS (P>0.05). The trends observed for ET­1 and the activity of eNOS in plasma were similar to those for arterial tissue. Therefore, the present study demonstrated that ATRA may regulate the grade of AS by the reduction of ET­1 secretion and increased NO formation via increased phosphorylation of eNOS. ATRA provides a potential novel method for the treatment of atherosclerosis.

Platelet Integrin αIIbß3 Inhibitor Rescues Progression of Apoptosis in Human Platelets.

BACKGROUND Apoptosis plays an important role in the physiology of platelet function. We aimed to detect the effect of the platelet integrin αIIbß3 inhibitor, tirofiban, on apoptotic events, including mitochondrial inner-membrane potential (ΔΨm), phosphatidylserine (PS) exposure on platelet surface, and the generation of reactive oxygen species (ROS), when washed platelets were stimulated with thrombin. MATERIAL AND METHODS The study included washed platelets from healthy humans, divided into 4 groups: vehicle, and tirofiban (0.05 µg/ml, 0.25 µg/ml, and 0.5 µg/ml). Platelets were pretreated with vehicle or tirofiban and incubated at 37°C with agitation for 6 h and 24 h. Before thrombin addition, the vehicle group divided into 2 equal groups. Except one vehicle group, the other 4 groups were all stimulated with thrombin (1 U/ml) for 30 min at 37°C. Using flow cytometry, we studied the DYm and PS exposure on platelet surfaces, and the generation of ROS in platelets. RESULTS We observed that at the time of 6 h and 24 h, thrombin-stimulated vehicle platelets induced significant depo-larization of ΔΨm, higher PS exposure, and increased ROS production compared with the vehicle group (P<0.01). However, the tirofiban group had significantly more recovery of DYm, PS exposure, and ROS production compared with the thrombin group (P<0.01). CONCLUSIONS The platelet integrin αIIbß3 inhibitor, tirofiban, inhibits the depolarization of DYm, PS exposure on platelet surface, and ROS production when stimulated with thrombin. These results suggest that αIIbß3 inhibitor inhibits the initiation of apoptosis in platelets, showing a potential clinical application of tirofiban as an apoptosis inhibitor.

IL-35 improves Treg-mediated immune suppression in atherosclerotic mice.

Interleukin (IL)-35 is an anti-inflammatory cytokine that may have a protective role in atherosclerosis (AS). However, the exact role of IL-35 in the disease, and the etiology of AS, remain incompletely understood. The present study aimed to investigate whether exogenous IL-35 was able to attenuate the formation of atherosclerotic lesions in apoE-/- mice, and analyze alterations in the expression levels of forkhead box protein 3 (Foxp3) in peripheral blood and the lesions during the progression of AS. ApoE-/- mice were randomly divided into two groups that received either a basal diet (negative control group) or a high-fat diet (HFD) for 4 weeks. The HFD group was further subdivided into groups that received IL-35, atorvastatin or no treatment for 12 weeks. Diagnostic enzyme assay kits were applied for the detection of plasma lipids, and hematoxylin and eosin staining was used to analyze the severity of atherosclerotic lesions in apoE-/- mice. Immunohistochemistry and flow cytometry were performed to analyze the expression of Foxp3 in the plasma and atherosclerotic plaques. As compared with the negative control group, the plasma lipids were significantly increased, and the lesions were obviously formed, in the HFD groups. Furthermore, the area of the lesion was reduced in IL-35- and atorvastatin-treated groups, as compared with the AS control group. In addition, Foxp3 expression was upregulated in the plasma and lesions of the IL-35- and atorvastatin-treated groups, as compared with the AS control group. The present study demonstrated that IL-35 improved Treg-mediated immune suppression in atherosclerotic mice, thus suggesting that IL-35 may be a novel therapeutic target for AS.

Fibrinogen facilitates atherosclerotic formation in Sprague-Dawley rats: A rodent model of atherosclerosis.

Fibrinogen (Fg) contributes to thrombosis and hemostasis and plays a role in inflammation. Fg is also known to play a significant role in atherosclerosis (AS). P-selectin has been associated with AS. The present study aimed to identify the role of Fg in AS and to examine the possible mechanisms behind the effects of fibrinogen on AS using Sprague-Dawley (SD) rats as a model system. Diet-induced atherosclerotic SD rats were adopted as the experimental models. Fg was transfused into these rats and the degree of atherosclerotic lesion development was compared with that of control rats. Blood was obtained from the common abdominal aorta and then the biochemical characteristics were measured and ELISA assays performed. The aortas were then carefully separated, removed and placed in 10% (w/v) neutral formalin for use at a later stage. The root of the aorta was cut and samples were washed, dehydrated, cleared, dipped in wax, embedded, sliced, coated, grilled and stained with HE. Pathological HE-stained sections were examined by light microscopic analysis and immunohistochemistry was performed for Fg and P-selectin on representative tissue sections. The Fg-transfused, high-fat diet-fed group developed atherosclerotic lesions more readily compared with the control group. Immunohistochemical analysis revealed that Fg expression was higher in the endarterium of the Fg-transfused, high-fat diet-fed rats. P-selectin expression was also found to be correlated with Fg expression. Fg actively promotes atherosclerotic lesion development; one possible mechanism behind this is the ability of Fg to enhance P-selectin expression, which is also able to facilitate the development of atherosclerotic lesions.

All-trans-retinoic acid ameliorated high fat diet-induced atherosclerosis in rabbits by inhibiting platelet activation and inflammation.

BACKGROUND: All-trans-retinoic acid (atRA) is effective for many proliferative diseases. We investigated the protective effects of atRA against atherosclerosis. METHODS: Rabbits were randomly allocated to receive basal diet or an HFD for 4 weeks. HFD group then received rosuvastatin (3 mg/day), atRA (5 mg/kg/day), or the same volume of vehicle, respectively, for next 8 weeks. RESULTS: HFD group showed increases in plasma lipids and aortic plaque formation. P-selectin expression and fibrinogen binding on platelets or deposition on the intima of the aorta also increased significantly as did the levels of TNF-α, IL-6, and fibrinogen in plasma. After 8 weeks of treatment with atRA, there was a significant decrease in plasma lipids and improvement in aortic lesions. AtRA also inhibited the expression of P-selectin and fibrinogen binding on platelets and deposition on the intima of the aorta. CONCLUSION: AtRA can ameliorate HFD-induced AS in rabbits by inhibiting platelet activation and inflammation.

Fibrinogen and P-selectin expression in atherosclerosis model of Sprague Dawley rat.

BACKGROUND: Platelet P-selectin plays an important role in inflammation and contributes to thrombosis and hemostasis. Fibrinogen may take part in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. This study aimed to discover the correlation between them in atherosclerosis model of Sprague Dawley (SD) rat. METHODS: Diet-induced atherosclerosis SD rats were adopted as experimental models. The blood from the common abdominal aorta of the rats was obtained to measure the biochemical characteristics and for the check of flow cytometry. Then the aortas were separated carefully, taken out, put into 10% (w/v) neutral formalin for later use. Then fibrinogen and P-selectin expression were detected by flow cytometry and immunohistochemistry. RESULTS: SD rats were induced to atherosclerosis model by high fat diet and vitamin D2 injected. It was discovered that the binding of fibrinogen and the expression of P-selectin on the platelet increase in atherosclerosis model (Group H) than in that in the control group (Group Z), there were closely interrelated. High levels of fibrinogen and P-selectin express on the artery of atherosclerosis rat model. CONCLUSIONS: Fibrinogen and P-selectin are concerned with atherosclerosis. Fibrinogen can interact with P-selectin in order to contribute to the development of atherosclerosis, high levels of fibrinogen and P-selectin can be regarded as risk factors for markers of atherosclerosis.