Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

UNLABELLED: Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro-organisms in-situ, thereby improving resource management and public health decision-making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in-situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source-tracking marker (human-specific Bacteriodales) and a HAB species (Psuedo-nitzschia spp.) over a 45-day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in-situ collected water samples. The in-situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction(-1) . No differences were observed in the concentrations of enterococci, the human-specific marker in Bacteroidales spp., and P. australis between in-situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet-accessible within hours of sample collection, demonstrating the feasibility of same-day public notification of current water quality conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first report of in-situ qPCR enumeration of both faecal indicators and harmful algal species in coastal marine waters. We utilize a robotic device for in-situ quantification of enterococci, the human-specific marker in Bacteriodales and Pseudo-nitzschia spp. from the same water samples collected and processed in-situ. The results demonstrate that rapid, in-situ monitoring can be utilized to identify and quantify multiple health-relevant micro-organisms important in water quality monitoring and that this monitoring can be used to inform same-day notifications.

Effects of intravenous administration of umbilical cord blood CD34(+) cells in a mouse model of neonatal stroke.

Neonatal stroke occurs in approximately 1/4000 live births and results in life-long neurological impairments: e.g., cerebral palsy. Currently, there is no evidence-based specific treatment for neonates with stroke. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment in rodent models of neonatal brain injury. However, all of the studies examined the effects of administering either the UCB mononuclear cell fraction or UCB-derived mesenchymal stem cells in neonatal rat models. The objective of this study was to examine the effects of human UCB CD34(+) cells (hematopoietic stem cell/endothelial progenitor cells) in a mouse model of neonatal stroke, which we recently developed. On postnatal day 12, immunocompromized (SCID) mice underwent permanent occlusion of the left middle cerebral artery (MCAO). Forty-eight hours after MCAO, human UCB CD34(+) cells (1×10(5)cells) were injected intravenously into the mice. The area in which cerebral blood flow (CBF) was maintained was temporarily larger in the cell-treated group than in the phosphate-buffered saline (PBS)-treated group at 24h after treatment. With cell treatment, the percent loss of ipsilateral hemispheric volume was significantly ameliorated (21.5±1.9%) compared with the PBS group (25.6±5.1%) when assessed at 7weeks after MCAO. The cell-treated group did not exhibit significant differences from the PBS group in either rotarod (238±46s in the sham-surgery group, 175±49s in the PBS group, 203±54s in the cell-treated group) or open-field tests. The intravenous administration of human UCB CD34(+) cells modestly reduced histological ischemic brain damage after neonatal stroke in mice, with a transient augmentation of CBF in the peri-infarct area.

Pathological role of angiostatin in heart failure: an endogenous inhibitor of mesenchymal stem-cell activation.

OBJECTIVE: Recently, a clinical trial was initiated to evaluate the efficacy of transendocardial transplantation of autologous bone marrow-derived mesenchymal stem cells (MSC) for the treatment of heart failure (HF). Because some HF patient-derived sera did not induce proliferation of autologous MSC, the present study aimed to elucidate humoral factors in sera that attenuate MSC activation and to investigate the role of these humoral factors in the pathogenesis of HF. METHODS AND RESULTS: Inhibitory effects present in serum were analysed by culturing human MSC with sera from 10 HF patients (FS <25%, BNP >100 pg/ml) and four healthy control subjects. Among the patients, two sera from HF patients showed significant inhibitory activity on MSC proliferation. Protein array and ELISA analysis revealed that these sera contained high levels of angiostatin as well as the active form of matrix metalloproteinase (MMP)-9, which generates angiostatin. Angiostatin significantly inhibited the proliferation and migration of cultured human MSC and increased their apoptosis in a dose-dependent manner. In a rat HF model, serum levels of angiostatin and MMPs increased, but treatment with an MMP inhibitor suppressed these increases. CONCLUSIONS: The results suggest that angiostatin, which can attenuate the activity of MSC, might play a role in the progression of HF.

Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide.

BACKGROUND: Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft. METHODS AND RESULTS: Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery. Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days. More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81+/-0.05 versus 0.30+/-0.14, P<0.01) and at 28 days (0.96+/-0.01 versus 0.45+/-0.08, P<0.001). The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts. Neointimal thickening was significantly suppressed in the Ad.CNP group. The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration. CONCLUSIONS: Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.

C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization.

We recently reported that C-type natriuretic peptide (CNP) occurs in vascular endothelial cells and acts as a vascular-type natriuretic peptide. In the present study, we stimulated the cGMP cascade in proliferating smooth muscle cells (SMCs), in which particulate guanylate cyclase-B, the specific receptor for CNP, is predominantly expressed, by use of an adenovirus encoding rat CNP cDNA (Ad.CNP). In the Ad.CNP-treated cultured SMCs, CNP caused the growth inhibition of SMCs at G(1) phase with an early increase of p21(CIP1/WAF1) expression and subsequent upregulation of p16(INK4a). The expression of smooth muscle myosin heavy chain-2, which is the molecular marker of highly differentiated SMCs, was reinduced in the Ad.CNP-treated SMCs. The Ad.CNP-treated SMCs also reexpressed particulate guanylate cyclase-A, which shows high affinity to atrial and brain natriuretic peptide and is exclusively expressed in well-differentiated SMCs. CNP, which was overexpressed in rabbit femoral arteries in vivo at the time of balloon injury, significantly suppressed neointimal formation. Furthermore, an enhancement of the expression of smooth muscle myosin heavy chain-2 occurred in the residual neointima. In addition, early regeneration of endothelial cells was observed in the Ad.CNP-infected group. Thus, stimulation of cGMP cascade in proliferating dedifferentiated SMCs can induce growth inhibition and redifferentiation of SMCs with accelerated reendothelialization.

Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin.

OBJECTIVE: Excess oxidative stress is one of the major metabolic abnormalities on vascular walls in hypertension and atherosclerosis. In order to further elucidate the endothelial function under oxidative stress, the effect of hydrogen peroxide (H2O2) on expression of two novel endothelium-derived vasorelaxing peptides, C-type natriuretic peptide (CNP) and adrenomedullin (AM) from bovine carotid artery endothelial cells (BCAECs) was examined. METHODS: BCAECs were treated with H2O2 (0.1-1.0 mmol/ l) and/or an antioxidant, N-acetylcysteine (NAC) (5-10 mmol/l), and incubated for 48 h. The concentrations of CNP and AM were measured with the specific radioimmuno assays that we originally developed. CNP and AM mRNA expressions were also examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Treatment of BCAECs with 0.5 and 1 mmol/l H2O2 induced 9-and 10-fold increases of CNP concentration in the media. Addition of 10 mmol/l NAC significantly suppressed the effect of H2O2 by 52%. RT-PCR analysis showed that CNP mRNA expression in BCAECs was also rapidly augmented within 1 h with H2O2 (1 mmol/l) treatment, and reached a peak at 3 h to show a 10-fold increase. AM secretion from BCAECs also increased to two-fold with exposure to 0.5 mmol/l H2O2, accompanied with the augmented level of AM mRNA. NAC 10 mmol/l completely suppressed the effect of H2O2 on AM secretion. CONCLUSIONS: In this study, it has been demonstrated that H2O2 augments endothelial secretion of the two endothelium-derived relaxing peptides, CNP and AM. Our findings suggest the increased secretion of CNP and AM from endothelium under oxidative stress may function to compensate the impaired nitric oxide-dependent vasorelaxation in hypertension and atherosclerosis.