Establishment and Reliability Evaluation of Prognostic Models in Diabetic Foot.

Purpose: To study the risk factors affecting amputation and survival in patients with diabetic foot (DF) and to construct a predictive model using the machine learning technique for DF foot amputation and survival and evaluate its effectiveness. Materials and Methods: A total of 200 patients with DF hospitalized in the First Affiliated Hospital of Shantou University Medical College in China were selected via cluster analysis screening, Kaplan-Meier survival calculation, amputation rate and Cox proportional hazards model investigation of risk factors associated with amputation and death. In addition, we constructed various models, including Cox proportional hazards regression analysis, the deep learning method convolution neural network (CNN) model, backpropagation (BP) neural network model, and backpropagation neural network prediction model after optimizing the genetic algorithm. The accuracy of the 4 prediction models for survival and amputation was assessed, and we evaluated the reliability of these computational models based on the size of the area under the ROC curve (AUC), sensitivity and specificity. Results: We found that the 1-year survival rate in patients with DF was 88.5%, and the 1-year amputation rate was 12.5%. Wagner's Classification of Diabetic Foot Ulcers grade, ankle-brachial index (ABI), low-density lipoprotein (LDL), and percutaneous oxygen partial pressure (TcPO2) were independent risk factors for amputation in patients with DF, while cerebrovascular disease, Sudoscan sweat gland function score, glycated hemoglobin (HbA1c) and peripheral artery disease (PAD) were independent risk factors for death in patients with DF. In addition, our results showed that in the case of amputation, the COX regression predictive model revealed an AUC of 0.788, sensitivity of 74.1% and specificity of 83.6%. The BP neural network predictive model identified an AUC of 0.874, sensitivity of 87.0% and specificity of 87.7%. An AUC of 0.909, sensitivity of 90.7% and specificity of 91.1% were found after optimizing the BP neural network prediction model via genetic algorithm. In the deep learning CNN model, the AUC, sensitivity and specificity were 0.939, 92.6%, and 95.2%, respectively. In the analysis of risk factors for death, the COX regression predictive model identified the AUC, sensitivity and specificity as 0.800, 74.1% and 85.9%, respectively. The BP neural network predictive model revealed an AUC, sensitivity and specificity of 0.937, 93.1% and 94.4%, respectively. Genetic algorithm-based optimization of the BP neural network predictive model identified an AUC, sensitivity and specificity of 0.932, 91.4% and 95.1%, respectively. The deep learning CNN model found the AUC, sensitivity and specificity to be 0.861, 82.8% and 89.4%, respectively. Conclusion: To identify risk factors for death, the BP neural network predictive model and genetic algorithm-based optimizing BP neural network predictive model have higher sensitivity and specificity than the deep learning method CNN predictive model and COX regression analysis.

Relationships among the Dosage of Erythropoiesis-Stimulating Agents, Erythropoietin Resistance Index, and Mortality in Maintenance Hemodialysis Patients.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) constitute an important treatment option for anemia in hemodialysis (HD) patients. We investigated the relationships among the dosage of ESA, erythropoietin resistance index (ERI) scores, and mortality in Chinese MHD patients. METHODS: This multicenter observational retrospective study included MHD patients from 16 blood purification centers (n = 824) who underwent HD in 2011-2015 and were followed up until December 31, 2016. We collected demographic variables, HD parameters, laboratory values, and ESA dosages. Patients were grouped into quartiles according to ESA dosage to study the effect of ESA dosage on all-cause mortality. The ERI was calculated as follows: ESA (IU/week)/weight (kg)/hemoglobin levels (g/dL). We also compared outcomes among the patients stratified into quartiles according to ERI scores. We used the Cox proportional hazards model to measure the relationships between the ESA dosage, ERI scores, and all-cause mortality. Using propensity score matching, we compared mortality between groups according to ERI scores, classified as either > or ≤12.80. RESULTS: In total, 824 patients were enrolled in the study; 200 (24.3%) all-cause deaths occurred within the observation period. Kaplan-Meier analyses showed that patients administered high dosages of ESAs had significantly worse survival than those administered low dosages of ESAs. A multivariate Cox regression identified that high dosages of ESAs could significantly predict mortality (ESA dosage >10,000.0 IU/week, HR = 1.59, 95% confidence intervals (CIs) (1.04, 2.42), and p = 0.031). Our analysis also indicated a significant increase in the risk of mortality in patients with high ERI scores. Propensity score matching-analyses confirmed that ERI > 12.80 could significantly predict mortality (HR = 1.56, 95% CI [1.11, 2.18], and p = 0.010). CONCLUSIONS: Our data suggested that ESA dosages >10,000.0 IU/week in the first 3 months constitute an independent predictor of all-cause mortality among Chinese MHD patients. A higher degree of resistance to ESA was related to a higher risk of all-cause mortality.

Complete nucleotide sequence of a novel partitivirus infecting the plant-pathogenic fungus Phomopsis vexans.

Here, we report the molecular characterization of a novel partitivirus from Phomopsis vexans strain PvHZ002, a plant-pathogenic fungus infecting eggplant. The virus was designated "Phomopsis vexans partitivirus 1" (PvPV1). PvPV1 contains two dsRNA segments, dsRNA1 and dsRNA2, which are 1,662 bp and 1,628 bp long, respectively. Each segment contains a single open reading frame, putatively encoding RNA-dependent RNA polymerase (dsRNA 1) and capsid protein (dsRNA 2). A homology search and phylogenetic analysis showed that PvPV1 clustered with viruses of the genus Deltapartitivirus of the family Partitiviridae.

N-acetyl-seryl-aspartyl-lysyl-proline treatment protects heart against excessive myocardial injury and heart failure in mice.

Myocardial infarction (MI) in mice results in cardiac rupture at 4-7 days after MI, whereas cardiac fibrosis and dysfunction occur later. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory, anti-fibrotic, and pro-angiogenic properties. We hypothesized that Ac-SDKP reduces cardiac rupture and adverse cardiac remodeling, and improves function by promoting angiogenesis and inhibiting detrimental reactive fibrosis and inflammation after MI. C57BL/6J mice were subjected to MI and treated with Ac-SDKP (1.6 mg/kg per day) for 1 or 5 weeks. We analyzed (1) intercellular adhesion molecule-1 (ICAM-1) expression; (2) inflammatory cell infiltration and angiogenesis; (3) gelatinolytic activity; (4) incidence of cardiac rupture; (5) p53, the endoplasmic reticulum stress marker CCAAT/enhancer binding protein homology protein (CHOP), and cardiomyocyte apoptosis; (6) sarcoplasmic reticulum Ca2+ ATPase (SERCA2) expression; (7) interstitial collagen fraction and capillary density; and (8) cardiac remodeling and function. Acutely, Ac-SDKP reduced cardiac rupture, decreased ICAM-1 expression and the number of infiltrating macrophages, decreased gelatinolytic activity, p53 expression, and myocyte apoptosis, but increased capillary density in the infarction border. Chronically, Ac-SDKP improved cardiac structures and function, reduced CHOP expression and interstitial collagen fraction, and preserved myocardium SERCA2 expression. Thus, Ac-SDKP decreased cardiac rupture, ameliorated adverse cardiac remodeling, and improved cardiac function after MI, likely through preserved SERCA2 expression and inhibition of endoplasmic reticulum stress.

Overexpression of prostaglandin E2 EP4 receptor improves cardiac function after myocardial infarction.

BACKGROUND: Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We recently reported that PGE2 via its EP3 receptor could reduce cardiac contractility of isolated myocytes and the working heart preparation. We thus hypothesized that there is an imbalance in the EP3/EP4 ratio towards EP3 in the failing heart and that overexpression of EP4 in a mouse model of heart failure would improve cardiac function. METHODS AND RESULTS: Our hypothesis was tested in a mouse model of myocardial infarction (MI) with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the cardiac myocytes. Echocardiography was performed to assess cardiac function. We found that overexpression of EP4 improved shortening fraction (p = 0.0025), ejection fraction (p = 0.0003), and reduced left ventricular dimension at systole (p = 0.0013). Overexpression of EP4 also significantly reduced indices of cardiac hypertrophy and interstitial collagen fraction. Animals treated with AAV9-EP4 also had a significant decrease in TNFα mRNA expression and in the number of macrophages and T cells migrated post MI coupled with a reduction in the expression of iNOS. CONCLUSION: Overexpression of EP4 improves cardiac function post MI. This may be mediated through reductions in adverse cardiac remodeling or via inhibition of cytokine/chemokine production.

Ac-SDKP suppresses TNF-α-induced ICAM-1 expression in endothelial cells via inhibition of IκB kinase and NF-κB activation.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that, in angiotensin II-induced hypertension, Ac-SDKP decreased the activation of nuclear transcription factor NF-κB, whereas, in experimental autoimmune myocarditis and hypertension animal models, it also reduced the expression of endothelial leukocyte adhesion molecule ICAM-1. However, the mechanisms by which Ac-SDKP downregulated ICAM-1 expression are still unclear. TNF-α is a proinflammatory cytokine that induces ICAM-1 expression in various cell types via TNF receptor 1 and activation of the classical NF-κB pathway. We hypothesized that in endothelial cells Ac-SDKP suppresses TNF-α-induced ICAM-1 expression by decreasing IKK phosphorylation that as a consequence leads to a decrease of IκB phosphorylation and NF-κB activation. To test this hypothesis, human coronary artery endothelial cells were treated with Ac-SDKP and then stimulated with TNF-α. We found that TNF-α-induced ICAM-1 expression was significantly decreased by Ac-SDKP in a dose-dependent manner. Ac-SDKP also decreased TNF-α-induced NF-κB translocation from cytosol to nucleus, as assessed by electrophoretic mobility shift assay, which correlated with a decrease in IκB phosphorylation. In addition, we found that Ac-SDKP decreased TNF-α-induced IKK phosphorylation and IKK-ß expression. However, Ac-SDKP had no effect on TNF-α-induced phosphorylation of p38 MAP kinase or ERK. Thus we conclude that Ac-SDKP inhibition of TNF-α activation of canonical, i.e., IKK-ß-dependent, NF-κB pathway and subsequent decrease in ICAM-1 expression is achieved via inhibition of IKK-ß.

Cardiac-deleterious role of galectin-3 in chronic angiotensin II-induced hypertension.

Galectin-3 (Gal-3), a member of the ß-galactoside lectin family, has an important role in immune regulation. In hypertensive rats and heart failure patients, Gal-3 is considered a marker for an unfavorable prognosis. Nevertheless, the role and mechanism of Gal-3 action in hypertension-induced target organ damage are unknown. We hypothesized that, in angiotensin II (ANG II)-induced hypertension, genetic deletion of Gal-3 prevents left ventricular (LV) adverse remodeling and LV dysfunction by reducing the innate immune responses and myocardial fibrosis. To induce hypertension, male C57BL/6J and Gal-3 knockout (KO) mice were infused with ANG II (3 µg·min-1·kg-1 sc) for 8 wk. We assessed: 1) systolic blood pressure by plethysmography, 2) LV function and remodeling by echocardiography, 3) myocardial fibrosis by histology, 4) cardiac CD68+ macrophage infiltration by histology, 5) ICAM-1 and VCAM-1 expression by Western blotting, 6) plasma cytokines, including interleukin-6 (IL-6), by enzyme-linked immunosorbent assay, and 7) regulatory T (Treg) cells by flow cytometry as detected by their combined expression of CD4, CD25, and FOXP3. Systolic blood pressure and cardiac hypertrophy increased similarly in both mouse strains when infused with ANG II. However, hypertensive C57BL/6J mice suffered impaired ejection and shortening fractions. In these mice, the extent of myocardial fibrosis and macrophage infiltration was greater in histological sections, and cardiac ICAM-1, as well as plasma IL-6, expression was higher as assessed by Western blotting. However, all these parameters were blunted in Gal-3 KO mice. Hypertensive Gal-3 KO mice also had a higher number of splenic Treg lymphocytes. In conclusion, in ANG II-induced hypertension, genetic deletion of Gal-3 prevented LV dysfunction without affecting blood pressure or LV hypertrophy. This study indicates that the ANG II effects are, in part, mediated or triggered by Gal-3 together with the related intercellular signaling (ICAM-1 and IL-6), leading to cardiac inflammation and fibrosis.

Increased 4-hydroxy-2-nonenal-induced proteasome dysfunction is correlated with cardiac damage in streptozotocin-injected rats with isoproterenol infusion.

Increase in 4-hydroxy-2-nonenal (4HNE) due to oxidative stress has been observed in a variety of cardiac diseases such as diabetic cardiomyopathy. 4HNE exerts a damaging effect in the myocardium by interfering with subcellular organelles like mitochondria by forming adducts. Therefore, we hypothesized that increased 4HNE adduct formation in the heart results in proteasome inactivation in isoproterenol (ISO)-infused type 1 diabetes mellitus (DM) rats. Eight-week-old male Sprague Dawley rats were injected with streptozotocin (STZ, 65 mg kg(-1) ). The rats were infused with ISO (5 mg kg(-1) ) for 2 weeks by mini pumps, after 8 weeks of STZ injection. We studied normal control (n = 8) and DM + ISO (n = 10) groups. Cardiac performance was assessed by echocardiography and Millar catheter at the end of the protocol at 20 weeks. Initially, we found an increase in 4HNE adducts in the hearts of the DM + ISO group. There was also a decrease in myocardial proteasomal peptidase (chymotrypsin and trypsin-like) activity. Increases in cardiomyocyte area (446 ± 32·7 vs 221 ± 10·83) (µm(2) ), per cent area of cardiac fibrosis (7·4 ± 0·7 vs 2·7 ± 0·5) and cardiac dysfunction were also found in DM + ISO (P < 0·05) relative to controls. We also found increased 4HNE adduct formation on proteasomal subunits. Furthermore, reduced aldehyde dehydrogenase 2 activity was observed in the myocardium of the DM + ISO group. Treatment with 4HNE (100 µM) for 4 h on cultured H9c2 cardiomyocytes attenuated proteasome activity. Therefore, we conclude that the 4HNE-induced decrease in proteasome activity may be involved in the cardiac pathology in STZ-injected rats infused with ISO. Copyright © 2016 John Wiley & Sons, Ltd.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline: mechanisms of renal protection in mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease characterized by the development of auto antibodies against a variety of self-antigens and deposition of immune complexes that lead to inflammation, fibrosis, and end-organ damage. Up to 60% of lupus patients develop nephritis and renal dysfunction leading to kidney failure. N-acetyl-seryl-aspartyl-lysyl-proline, i.e., Ac-SDKP, is a natural tetrapeptide that in hypertension prevents inflammation and fibrosis in heart, kidney, and vasculature. In experimental autoimmune myocarditis, Ac-SDKP prevents cardiac dysfunction by decreasing innate and adaptive immunity. It has also been reported that Ac-SDKP ameliorates lupus nephritis in mice. We hypothesize that Ac-SDKP prevents lupus nephritis in mice by decreasing complement C5-9, proinflammatory cytokines, and immune cell infiltration. Lupus mice treated with Ac-SDKP for 20 wk had significantly lower renal levels of macrophage and T cell infiltration and proinflammatory chemokine/cytokines. In addition, our data demonstrate for the first time that in lupus mouse Ac-SDKP prevented the increase in complement C5-9, RANTES, MCP-5, and ICAM-1 kidney expression and it prevented the decline of glomerular filtration rate. Ac-SDKP-treated lupus mice had a significant improvement in renal function and lower levels of glomerular damage. Ac-SDKP had no effect on the production of autoantibodies. The protective Ac-SDKP effect is most likely achieved by targeting the expression of proinflammatory chemokines/cytokines, ICAM-1, and immune cell infiltration in the kidney, either directly or via C5-9 proinflammatory arm of complement system.

Activation of angiotensin II type 2 receptor suppresses TNF-α-induced ICAM-1 via NF-кB: possible role of ACE2.

ANG II type 2 receptor (AT2) and ANG I-converting enzyme 2 (ACE2) are important components of the renin-ANG system. Activation of AT2 and ACE2 reportedly counteracts proinflammatory effects of ANG II. However, the possible interaction between AT2 and ACE2 has never been established. We hypothesized that activation of AT2 increases ACE2 activity, thereby preventing TNF-α-stimulated ICAM-1 expression via inhibition of NF-κB signaling. Human coronary artery endothelial cells were pretreated with AT2 antagonist PD123319 (PD) or ACE2 inhibitor DX600 and then stimulated with TNF-α in the presence or absence of AT2 agonist CGP42112 (CGP). We found that AT2 agonist CGP increased both ACE2 protein expression and activity. This effect was blunted by AT2 antagonist PD. ICAM-1 expression was very low in untreated cells but greatly increased by TNF-α. Activation of AT2 with agonist CGP or with ANG II under concomitant AT1 antagonist reduced TNF-α-induced ICAM-1 expression, which was reversed by AT2 antagonist PD or ACE2 inhibitor DX600 or knockdown of ACE2 with small interfering RNA. AT2 activation also suppressed TNF-α-stimulated phosphorylation of inhibitory κB (p-IκB) and NF-κB activity. Inhibition of ACE2 reversed the inhibitory effect of AT2 on TNF-α-stimulated p-IκB and NF-κB activity. Our findings suggest that stimulation of AT2 reduces TNF-α-stimulated ICAM-1 expression, which is partly through ACE2-mediated inhibition of NF-κB signaling.

Fractalkine neutralization improves cardiac function after myocardial infarction.

NEW FINDINGS: What is the central question of this study? What is the cardioprotective role of fractalkine neutralization in heart failure and what are the mechanisms responsible? What is the main finding and its importance? The concentration of fractalkine is increased in the left ventricle of mice with myocardial infarction, similar to the increases in plasma from heart failure patients. The present study shows a clear beneficial effect of neutralizing fractalkine in a model of myocardial infarction, which results in increased survival. Such an approach may be worthwhile in human patients. Concentrations of the chemokine fractalkine (FKN) are increased in patients with chronic heart failure, and our previous studies show that aged mice lacking the prostaglandin E2 EP4 receptor subtype (EP4-KO) have increased cardiac FKN, with a phenotype of dilated cardiomyopathy. However, how FKN participates in the pathogenesis of heart failure has rarely been studied. We hypothesized that FKN contributes to the pathogenesis of heart failure and that anti-FKN treatment prevents heart failure induced by myocardial infarction (MI) more effectively in EP4-KO mice. Male EP4-KO mice and wild-type littermates underwent sham or MI surgery and were treated with an anti-FKN antibody or control IgG. At 2 weeks post-MI, echocardiography was performed and hearts were excised for determination of infarct size, immunohistochemistry and Western blot of signalling molecules. Given that FKN protein levels in the left ventricle were increased to a similar extent in both strains after MI and that anti-FKN treatment improved survival and cardiac function in both strains, we subsequently used only wild-type mice to examine the mechanisms whereby anti-FKN is cardioprotective. Myocyte cross-sectional area and interstitial collagen fraction were reduced after anti-FKN treatment, as were macrophage migration and gelatinase activity. Activation of ERK1/2 and p38 MAPK were reduced after neutralization of FKN. In vitro, FKN increased fibroblast proliferation. In conclusion, increased FKN contributes to heart failure after MI. This effect is not exacerbated in EP4-KO mice, suggesting that there is no link between FKN and lack of EP4. Overall, inhibition of FKN may be important to preserve cardiac function post-MI.

Flexibility Analysis of Bacillus thuringiensis Cry1Aa.

OBJECTIVE: To investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa. METHODS: The graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail. RESULTS: The decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II. CONCLUSION: Although several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

Combination treatment with N-acetyl-seryl-aspartyl-lysyl-proline and tissue plasminogen activator provides potent neuroprotection in rats after stroke.

BACKGROUND AND PURPOSE: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), an endogenously produced circulating peptide in humans and rodents, exerts anti-inflammatory and cardioprotective activities in various cardiovascular diseases. METHODS: The present study evaluated the neuroprotective effect of AcSDKP alone and in combination with thrombolytic therapy in a rat model of embolic focal cerebral ischemia. RESULTS: We found that treatment with AcSDKP alone at 1 hour or the combination treatment with AcSDKP and tissue plasminogen activator (tPA) at 4 hours after stroke onset substantially increased AcSDKP levels in plasma and cerebrospinal fluid and robustly reduced infarct volume and neurological deficits, without increasing the incidence of brain hemorrhage compared with ischemic rats treated with saline, AcSDKP alone at 4 hours, and tPA alone at 4 hours. Moreover, the combination treatment considerably reduced the density of nuclear transcription factor-κB (NF-κB), transforming growth factor ß (TGF-ß), and plasminogen activator inhibitor-1 (PAI-1) positive cerebral blood vessels in the ischemic brain, all of which were associated with reduced microvascular fibrin extravasation and platelet accumulation compared with tPA monotherapy. In vitro, AcSDKP blocked fibrin-elevated TGF-ß1, PAI-1, and NF-κB proteins in primary human brain microvascular endothelial cells. CONCLUSIONS: Our data indicate that AcSDKP passes the blood-brain barrier, and that treatment of acute stroke with AcSDKP either alone at 1 hour or in combination with tPA at 4 hours of the onset of stroke is effective to reduce ischemic cell damage in a rat model of embolic stroke. Inactivation of TGF-ß and NF-κB signaling by AcSDKP in the neurovascular unit may underlie the neuroprotective effect of AcSDKP.

Thymosin-ß4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction.

Thymosin-ß4 (Tß4) promotes cell survival, angiogenesis, and tissue regeneration and reduces inflammation. Cardiac rupture after myocardial infarction (MI) is mainly the consequence of excessive regional inflammation, whereas cardiac dysfunction after MI results from a massive cardiomyocyte loss and cardiac fibrosis. It is possible that Tß4 reduces the incidence of cardiac rupture post-MI via anti-inflammatory actions and that it decreases adverse cardiac remodeling and improves cardiac function by promoting cardiac cell survival and cardiac repair. C57BL/6 mice were subjected to MI and treated with either vehicle or Tß4 (1.6 mg·kg(-1)·day(-1) ip via osmotic minipump) for 7 days or 5 wk. Mice were assessed for 1) cardiac remodeling and function by echocardiography; 2) inflammatory cell infiltration, capillary density, myocyte apoptosis, and interstitial collagen fraction histopathologically; 3) gelatinolytic activity by in situ zymography; and 4) expression of ICAM-1 and p53 by immunoblot analysis. Tß4 reduced cardiac rupture that was associated with a decrease in the numbers of infiltrating inflammatory cells and apoptotic myocytes, a decrease in gelatinolytic activity and ICAM-1 and p53 expression, and an increase in the numbers of CD31-positive cells. Five-week treatment with Tß4 ameliorated left ventricular dilation, improved cardiac function, markedly reduced interstitial collagen fraction, and increased capillary density. In a murine model of acute MI, Tß4 not only decreased mortality rate as a result of cardiac rupture but also significantly improved cardiac function after MI. Thus, the use of Tß4 could be explored as an alternative therapy in preventing cardiac rupture and restoring cardiac function in patients with MI.

N-acetyl-seryl-aspartyl-lysyl-proline reduces cardiac collagen cross-linking and inflammation in angiotensin II-induced hypertensive rats.

We have reported previously that Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) reduces fibrosis and inflammation (in macrophages and mast cells). However, it is not known whether Ac-SDKP decreases collagen cross-linking and lymphocyte infiltration; lymphocytes modulate both collagen cross-linking and ECM (extracellular matrix) formation in hypertension. Thus we hypothesized that (i) in AngII (angiotensin II)-induced hypertension, Ac-SDKP prevents increases in cross-linked and total collagen by down-regulating LOX (lysyl oxidase), the enzyme responsible for cross-linking, and (ii) these effects are associated with decreased pro-fibrotic cytokine TGFß (transforming growth factor ß) and the pro-inflammatory transcription factor NF-κB (nuclear factor κB) and CD4+/CD8+ lymphocyte infiltration. We induced hypertension in rats by infusing AngII either alone or combined with Ac-SDKP for 3 weeks. Whereas Ac-SDKP failed to lower BP (blood pressure) or LV (left ventricular) hypertrophy, it did prevent AngII-induced increases in (i) cross-linked and total collagen, (ii) LOX mRNA expression and LOXL1 (LOX-like 1) protein, (iii) TGFß expression, (iv) nuclear translocation of NF-κB, (v) CD4+/CD8+ lymphocyte infiltration, and (vi) CD68+ macrophages infiltration. In addition, we found a positive correlation between CD4+ infiltration and LOXL1 expression. In conclusion, the effect of Ac-SDKP on collagen cross-linking and total collagen may be due to reduced TGFß1, LOXL1, and lymphocyte and macrophage infiltration, and its effect on inflammation could be due to lower NF-κB.

The mutual interactions based on amphipathic tetraoxacalix[2]arene[2]triazine: recognition cases of anion and cation investigated by a computational study.

The nature of anion···π (anion X1-4(-) = SCN(-), PF6(-), BF4(-) and NO3(-), respectively) interactions with electron-deficient and cavity self-tunable macrocyclic host tetraoxacalix[2]arene[2]triazine 1 as electron-acceptor (J. Am. Chem. Soc., 2013, 135, 892) have been theoretically investigated with the density functional theory (B3LYP, M06-2X, M06-L, M06, M05-2X, M05, DFT-D3) and the second-order Møller-Plesset perturbation theory (MP2) using a series of basis sets. The binding energies calculated are in good quantitative agreement with the experiments. The LMO-EDA (local molecular orbital energy decomposition analysis) results show that the major contributors of anion···π are electrostatic. The alkali metal cations M(+) (Na(+), K(+)) and alkaline earth metal cations M(2+) (Mg(2+), Ca(2+)) can also interact with 1 and, the cation···π binding of M(2+)···1 is stronger than that of M(+)···1, as well as their strength is gradually decreased along with an increase in the radius of M(+,2+). The investigation of interplay between the anion···π and the cation···π shows that the interactions among three-body, X(-), 1 and M(+) is varied with different phases. The polar solvent can strongly reduce the strength of the interaction, and the more increased the solvent polarity, the more reduced is the binding energy.

[Posture evoked response detecting in normal lower limb muscle and its influencing factors].

OBJECTIVE: To analyze the latency of posture evoked response of normal lower limb muscle in different stimulations and explore its influencing factors. METHODS: The normal lower limb was induced to produce postural evoked response by the dynamic posturography through two kinds of perturbations, the supporting surface rotation stimulation (Toes-up and Toes-down) and the horizontal perturbation stimulation (Forward and Backward). The latencies of tibialis anterior muscle and gastrocnemius muscle were recorded by surface electromyography acquisition system. The differences of the left and right limb, gender and height on the latency of postural evoked response were analyzed. RESULTS: (1) Under the Toes-up and Backward perturbation, the latency of tibialis anterior muscle was longer than gastrocnemius muscle; under the Toes-down and Forward perturbation, the latency of gastrocnemius muscle was longer than tibialis anterior muscle. (2) The latencies of left limb and right limb had no significant difference. (3) The latency in male was longer than that in female. (4) The latency gradually increased with the increase of height. CONCLUSION: In the postural evoked response, different perturbations, gender and height have significant impacts on the latency of posture evoked response of lower limb muscle. However, the effect of height and gender should be not considered referring to the same individual.

A label-free activatable biosensor for in situ detection of exosomal microRNAs based on DNA-AgNCs and hairpin type nucleic acid probes.

Exosomal microRNA (miRNA) is a potential biomarker for cancer diagnosis, metastasis, and treatment. In situ detection of exosomal miRNA is an attractive option due to its simplicity and high accuracy. However, in situ exosomal miRNA detection has encountered challenges because of the low target abundance of targets and limited probe permeability. Herein, a label-free and activatable biosensor was developed for in situ exosomal miRNA assays by utilizing hairpin-shaped nucleic acid probes and DNA-hosted silver nanoclusters (DNA-AgNCs). The probe is directly internalized into the exosomes, and then hybridized with the target miRNA-21. Subsequently, the DNA-AgNCs are pulled closer to the G-rich sequence, ultimately leading to in situ red fluorescence activation. The biosensor not only can detect exosomal miRNA-21 but also distinguish cancer cells from normal cells. Under optimal reaction conditions, the detection limit (LOD) of exosomal miRNA-21 is 1.53 × 107 particles per mL. Furthermore, DNA-AgNCs are used as label-free signal elements for in situ detection of exosomal miRNAs for the first time, expanding the application of nanomaterials in this field. This strategy does not require tedious RNA extraction steps and expensive instruments, and may develop into a non-invasive diagnostic tool for ovarian cancer.

Scavenger receptor-BI is a receptor for lipoprotein(a).

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

Activation of Stat3 in endothelial cells following hypoxia-reoxygenation is mediated by Rac1 and protein Kinase C.

Stat3 is an important transcription factor that regulates both proinflammatory and anit-apoptotic pathways in the heart. This study examined the mechanisms of activation of Stat3 in human endothelial cells following hypoxia/reoxygenation (H/R). By expression of constitutively active Rac1 mutant protein, and by RNA silencing of Rac1, we found that Stat3 forms a multiprotein complex with Rac1 and PKC in an H/R-dependent manner, which at least in part, appears to regulate Stat3 S727 phosphorylation. Selective inhibition of PKC with calphostin C produces a marked suppression of Stat3 S727 phosphorylation. The association of Stat3 with Rax1 occurs predominantly at the cell membrane, but also inside the nucleus, and occurs through the binding of the coiled-coil domain of Stat3 to the 54 NH(2)-terminal residues of Rac1. Transfection with a peptide comprising the NH(2)-terminal 17 amino acid residues of Rac1-dependent signaling pathways resulting in physical association between Rac1 and Stat3 and the formation of a novel multiprotein complex with PKC.