Activation of the ACE2/Angiotensin-(1-7)/Mas Receptor Axis Enhances the Reparative Function of Dysfunctional Diabetic Endothelial Progenitors

We tested the hypothesis that activation of the protective arm of the renin angiotensin system, the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(1-7) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(1-7) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(1-7) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(1-7)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Successful medical treatment of Acanthamoeba keratitis

Purpose: To describe the outcome of a series of Acanthamoeba keratitis treated with a similar regimen. Methods: All cases diagnosed with Acanthamoeba keratitis in a referral centre from June 1994 through June 1997 were included. Diagnosis of Acanthamoeba keratitis was based in clinical presentation and laboratory results. Positive laboratory identification of Acanthamoeba from corneal scraping or contact lens was required, unless the patient had very characteristic symptoms (severe pain) and signs of the infection, including perineural infiltrates. Initial intensive treatment included topical polyhexamethylene biguanide (PHMB) 0.02%, propamidine isothionate 0.1% and broad-spectrum antibiotics. The treatment was gradually tapered. After documented response to anti-acanthamoeba therapy, topical steroids were introduced; they were discontinued before cessation of the antiAcanthamoeba regimen. Results: Six males and four females, with a mean age of 30.0 ± 7.4 years were included in this study. All cases weared contact lenses. On presentation all cases had severe pain, and epitheliopathy was associated with stromal infiltrate in most (seven of ten) cases. Four patients had anterior uveitis. Perineural infiltrates were present in three cases and ring infiltrate in one patient. Anti-amoebic treatment was started 12.7 ± 7.2 days after beginning of symptoms. The clinical response to therapy was very satisfactory in all patients. Within two to three weeks all patients had remarkable lessening of pain and photophobia, and improvement of clinical signs. At two to three months, visual acuity had improved in all patients. Two patients required penetrating keratoplasty for visual rehabilitation. Conclusion: The use of PHMB and propamidine cured all cases of Acanthamoeba keratitis. Cautious introduction of steroids was associated with expedited resolution of inflammation and provided symptomatic relief.

Amniotic membrane transplantation for ocular surface reconstruction

Aims - To evaluate the efficacy of amniotic membrane transplantation (AMT) for ocular surface reconstruction. Methods - 10 consecutive patients who underwent AMT were included. The indications were: group A, cases with persistent epithelial defect after corneal abscess (n = 1), radiation (n = 1), or chemical burn (n = 3); group B, cases with epithelial defect and severe stromal thinning and impending or recent perforation, due to chemical burn (two patients, three eyes) or corneal abscess (n = 2); group C, to promote corneal epithelium healing and prevent scarring after symblepharon surgery with extensive corneo-conjunctival adhesion (n = 1). Under sterile conditions amniotic membrane was prepared from a fresh placenta of a seronegative pregnant woman and stored at -70°C. This technique involved the use of amniotic membrane to cover the entire cornea and perilimbal area in groups A and B, and the epithelial defect only in group C. Results - The cornea healed satisfactorily in four of five patients in group A, but the epithelial defect recurred in one of these patients. After AMT three patients underwent limbal transplantation and one penetrating keratoplasty and cataract extraction. In group B amniotic membrane transplantation was not helpful, and all cases underwent an urgent tectonic corneal graft. Surgery successfully released the symblepharon, promoted epithelialisation and prevented adhesions in the case of group C. Conclusion - AMT was effective to promote corneal healing in patients with persistent epithelial defect, and appeared to be helpful after surgery to release corneo-conjunctival adhesion. Most surgery for further surface rehabilitation. Amniotic membrane used as a patch was not effective to prevent tectonic corneal graft in cases with severe stromal thinning and impending or recent perforation.

The Origin of Phragmoplast Asymmetry

The phragmoplast coordinates cytokinesis in plants [1]. It directs vesicles to the midzone, the site where they coalesce to form the new cell plate. Failure in phragmoplast function results in aborted or incomplete cytokinesis leading to embryo lethality, morphological defects, or multinucleate cells [2, 3]. The asymmetry of vesicular traffic is regulated by microtubules [1, 4, 5, 6], and the current model suggests that this asymmetry is established and maintained through treadmilling of parallel microtubules. However, we have analyzed the behavior of microtubules in the phragmoplast using live-cell imaging coupled with mathematical modeling and dynamic simulations and report that microtubules initiate randomly in the phragmoplast and that the majority exhibit dynamic instability with higher turnover rates nearer to the midzone. The directional transport of vesicles is possible because the majority of the microtubules polymerize toward the midzone. Here, we propose the first inclusive model where microtubule dynamics and phragmoplast asymmetry are consistent with the localization and activity of proteins known to regulate microtubule assembly and disassembly.

Ser6 in the maize actin-depolymerizing factor, ZmADF3, is phosphorylated by a calcium-stimulated protein kinase and is essential for the control of functional activity

Maize actin-depolymerizing factor, ZmADF, binds both G- and F-actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal-responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium-stimulated protein kinase present in a 40-70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3-4) does not bind G- or F-actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.

Early Holocene M similar to 6 explosive eruption from Plosky volcanic massif (Kamchatka) and its tephra as a link between terrestrial and marine paleoenvironmental records

We report tephrochronological and geochemical data on early Holocene activity from Plosky volcanic massif in the Kliuchevskoi volcanic group, Kamchatka Peninsula. Explosive activity of this volcano lasted for similar to 1.5 kyr, produced a series of widely dispersed tephra layers, and was followed by profuse low-viscosity lava flows. This eruptive episode started a major reorganization of the volcanic structures in the western part of the Kliuchevskoi volcanic group. An explosive eruption from Plosky (M similar to 6), previously unstudied, produced tephra (coded PL2) of a volume of 10-12 km(3) (11-13 Gt), being one of the largest Holocene explosive eruptions in Kamchatka. Characteristic diagnostic features of the PL2 tephra are predominantly vitric sponge-shaped fragments with rare phenocrysts and microlites of plagioclase, olivine and pyroxenes, medium- to high-K basaltic andesitic bulk composition, high-K, high-Al and high-P trachyandesitic glass composition with SiO2 = 57.5-59.5 wt%, K2O = 2.3-2.7 wt%, Al2O3 = 15.8-16.5 wt%, and P2O5 = 0.5-0.7 wt%. Other diagnostic features include a typical subduction-related pattern of incompatible elements, high concentrations of all REE (> 10x mantle values), moderate enrichment in LREE (La/Yb similar to 5.3), and non-fractionated mantle-like pattern of LILE. Geochemical fingerprinting of the PL2 tephra with the help of EMP and LA-ICP-MS analyses allowed us to map its occurrence in terrestrial sections across Kamchatka and to identify this layer in Bering Sea sediment cores at a distance of > 600 km from the source. New high-precision C-14 dates suggest that the PL2 eruption occurred similar to 10,200 cal BP, which makes it a valuable isochrone for early Holocene climate fluctuations and permits direct links between terrestrial and marine paleoenvironmental records. The terrestrial and marine C-14 dates related to the PL2 tephra have allowed us to estimate an early Holocene reservoir age for the western Bering Sea at 1,410 +/- A 64 C-14 years. Another important tephra from the early Holocene eruptive episode of Plosky volcano, coded PL1, was dated at 11,650 cal BP. This marker is the oldest geochemically characterized and dated tephra marker layer in Kamchatka to date and is an important local marker for the Younger Dryas-early Holocene transition. One more tephra from Plosky, coded PL3, can be used as a marker northeast of the source at a distance of similar to 110 km.

Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as

Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia

Background Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS.

Methods Adult sheep (30–40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×10¹¹ CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringer's solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×10⁶ hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×10⁶ hMSCs/kg, n=4.

Results By 24 h, the PaO₂/FiO₂ ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO₂/FiO₂ of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9–5.8] vs control: 6.7 g wet/g dry [IQR 6.4–7.5] (p=0.01)). The hMSCs had no adverse effects.

Conclusions Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS.

Biocompatibility of Calcium Phosphate Bone Cement with Optimised Mechanical Properties

The broad aim of this work was to investigate and optimise the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen and alkaline phosphatase activity assays respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7 and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.

Epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in ovarian adult granulosa cell tumours

AIMS: Adult granulosa cell tumours (AGCTs) are uncommon ovarian sex cord-stromal tumours which recur following surgical removal in up to 50% of patients. Treatment options for recurrent and advanced stage AGCTs are limited, with poor response to chemotherapy and radiotherapy. We aimed to assess epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in AGCTs with a view to investigating whether or not these receptors might be potential therapeutic targets in these neoplasms.

METHODS AND RESULTS: Immunohistochemical staining for EGFR, HER2 and IGF-1R was undertaken in 31 AGCTs. Tumour DNA was also analysed for mutations in the tyrosine kinase domain of EGFR (exons 18-21) by Cobas mutation RT-PCR. Twenty-three of 31 (74%) AGCTs showed some degree of EGFR expression, generally with cytoplasmic or mixed membranous and cytoplasmic staining of variable intensity. Eleven of 27 (41%) cases exhibited strong membranous and cytoplasmic expression of IGF-1R. HER2 expression was not seen. No mutations were found in exons 18-21 of the EGFR gene in hot-spots of therapeutic relevance.

CONCLUSIONS: This study raises the possibility that anti-EGFR and/or anti-IGF-1R therapies may be of potential benefit in ovarian AGCTs, and this requires further study. Lack of known mutations within the tyrosine kinase domain of EGFR suggests that EGFR-related tyrosine kinase inhibitors may not be useful therapeutically.

What sources of feeding advice do parents use in the first year of life?

There has been restructuring of health visiting services from universal services for all children to targeting families in need. Also, United Kingdom (UK) recommendations on infant feeding have recently changed. With the many sources of information available on feeding babies, it is important to know where parents get feeding advice and which sources they find valuable. In this study, 215 mothers of one-year-old infants were interviewed about where they had obtained feeding advice in the first year of their infant’s life and how useful they found this information. The health visitor was the most commonly cited source of information (70 %), followed by grandparents (53 %). Ten % of mothers relied solely on health visitor advice. This study highlights the importance placed by mothers on the health visitors, which may have implications for the service in the midst of the reorganization of the health visitor’s role.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

One-Electron Reduction of 2-Nitrotoluene, Nitrocyclopentane, and 1-Nitrobutane in Room Temperature Ionic Liquids: A Comparative Study of Butler–Volmer and Symmetric Marcus–Hush Theories Using Microdisk Electrodes

The voltammetry for the reduction of 2-nitrotoluene at a gold microdisk electrode is reported in two ionic liquids: trihexyltetradecylphosphonium tris(pentafluoroethyl)trifluorophosphate ([P-14,P-6,P-6,P-6][FAP]) and 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][NTf2]). The reduction of nitrocyclopentane (NCP) and 1-nitrobutane (BuN) was investigated using voltammetry at a gold microdisk electrode in the ionic liquid [P-14,P-6,P-6,P-6][FAP]. Simulated voltammograms, generated through the use of ButlerVolmer theory and symmetric MarcusHush theory, were compared to experimental data, with both theories parametrizing the data similarly well. An experimental value for the Marcusian parameter, 1 was also determined in all cases. For the reduction of 2-nitrotoluene, this was 0.5 +/- 0.1 eV in both solvents, while for NCP and BuN in [P-14,P-6,P-6,P-6][FAP], it was 2 +/- 0.1 and 5 +/- 0.1 eV, respectively. This is attributed to the localization of charge on the nitro group and the primary nitro alkyls increased interaction with the environment, resulting in a larger reorganization energy.

Application of Asymmetric Marcus-Hush Theory to Voltammetry in Room-Temperature Ionic Liquids

Asymmetric MarcusHush (AMH) theory is applied for the first time in ionic solvents to model the voltammetric reduction of oxygen in 1-butyl-1-methylpyrrolidinium bis-(trifluoromethylsulfonyl)-imide and of 2-nitrotoluene (2-NT), nitrocyclopentane (NCP), and 1-nitro-butane (BuN) in trihexyltetradecylphosphonium tris(pentafluoroethyl)trifluorophosphate on a gold microdisc electrode. An asymmetry parameter, gamma, was estimated for all systems as -0.4 for the reduction of oxygen and -0.05, 0.25, and 0 +/- 0.05 for the reductions of 2-NT, NCP, and BuN, respectively, which suggests equal force constants of reactants and products in the case of 2-NT and BuN and unequal force constants for oxygen and NCP where the force constants of the oxidized species are greater than the reduced species in the case of oxygen and less than the reduced species in the case of NCP. Previously measured values for a, the Butler-Volmer transfer coefficient, reflect this in each case. Where appreciable asymmetry occurs, AMH theory was seen to parametrize the experimental data better than either Butler-Volmer or symmetric Marcus-Hush theory, allowing additionally the extraction of reorganization energy. This is the first study to provide key physical insights into electrochemical systems in room-temperature ionic liquids using AMH theory, allowing elucidation of the reorganization energies and the relative force constants of the reactants and products in each reaction.