Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.     

Acute vasoconstriction after subarachnoid hemorrhage

OBJECTIVE: Decreased cerebral blood flow (CBF) and cerebral ischemia occurring immediately after subarachnoid hemorrhage (SAH) may be caused by acute microvascular constriction. However, CBF can also be influenced by changes in intracranial pressure (ICP) and cerebral perfusion pressure (CPP). The goal of these experiments was to assess the significance of acute vasoconstriction after SAH and its relationship to changes in CBF, ICP, CPP, and extracellular glutamate concentrations. METHODS: Three experiments were performed using the endovascular filament technique to produce SAH. In the first experiment, CBF, ICP, and CPP were measured for 60 minutes after SAH (n = 21) and were correlated with the 24-hour mortality rate. In the second experiment, rats undergoing SAH (n = 23) or a sham procedure (n = 7) were perfused 60 minutes after SAH for measurement of the circumference and wall thickness of the internal carotid and anterior cerebral arteries and correlation with CBF, ICP, and CPP. In the third experiment (n = 11), extracellular glutamate concentrations determined by hippocampal and cortical microdialysis and high performance liquid chromatography were correlated with physiological changes. RESULTS: CBF reductions to less than 40% of baseline for 60 minutes after SAH predicted 24-hour mortality with 100% accuracy and were used to define "lethal" SAH. In contrast, ICP and CPP 60 minutes after SAH were not correlated with the mortality rate. The vascular circumference was significantly smaller in lethal than in sublethal SAH or sham-operated rats (P < 0.001). Vessel measurements were correlated with both CBF and hemorrhage size (P < 0.01). Extracellular glutamate concentration increased to 600% of baseline after lethal SAH in both hippocampus and cortex and was inversely correlated with CBF (r = 0.9, P < 0.001) but did not increase after sublethal SAH. CONCLUSION: Acute vasoconstriction after SAH occurs independently of changes in ICP and CPP and is associated with decreased CBF, larger hemorrhage size, persistent elevations of extracellular glutamate, and poor outcome. Acute vasoconstriction seems to contribute directly to ischemic brain injury after SAH. Further evaluations of pharmacological agents with the potential to reverse acute vasoconstriction may increase CBF and improve outcome.     

The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2

Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.     

Sensitivity of the retinal pigment epithelium to spindle poisons

PURPOSE: The function of RPE is well known in PVR. Pharmacological agents have been extensively studied both experimentally and clinically. Few reports have detailed the interactions of antimitotic drugs on the microtubule network. The aim of this study is to visualize by indirect immunofluorescence the effects of colchicine and paclitaxel on the microtubule network of cultured pig RPE cells in interphase. METHODS: Pigs were killed at the slaughter-house, their eyes were enucleated. RPE cells were isolated and cultured. RPE cells were plated onto glass cover-slips at a density of 2,000,000 cells/ml, cultured and treated with the drugs during 4 and 24 hours at 37 degrees C at different concentrations. Immunofluorescence reaction was developped using antitubulin and fluoresceinated anti-mouse antibodies. The cytoskeletons were visualized employing a Zeiss photomicroscope equipped with epiilumination, a 63 x lens and appropriate filters for fluoresceine. RESULTS: The cytoplasmic microtubules of RPE cells were disrupted in a concentration and time-dependant manner by colchicine. Between 10 and 100 nm Veveral degrees of depolymarization of the microtubule network were observed. Paclitaxel between 1 micron and 10 microns was found to induce several degrees of microtubule "bundling" after 4 and 24 hours of incubation. Actin network was modified neither by colchicine and paclitaxel used in the same conditions. CONCLUSIONS: The results show that low doses of antimitotic drugs inhibit the microtubule network formation by depolymerization (colchicine) or stabilize it (paclitaxel). These actions inhibit cell division, which is one of the mechanisms implicated in PVR.     

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.     

Minimum‐variance control of astronomical adaptive optic systems with actuator dynamics under synchronous and asynchronous sampling

Adaptive optic (AO) systems are now routinely used in ground‐based telescopes to counter the effects of atmospheric turbulence. A deformable mirror (DM) generates a correction wavefront, which is subtracted from the turbulent wavefront using measurements of the residual phase provided by a wavefront sensor (WFS). Minimizing the variance of the residual phase defines a sampled data control problem combining a continuous time minimum‐variance (MV) performance criterion with a discrete‐time controller. For a fairly general class of linear time‐invariant DM and turbulence WFS models, this control problem can be transformed into an equivalent discrete‐time LQ optimization problem involving a set of (discrete‐time) control‐sufficient statistics of the incoming continuous‐time turbulence. This paper shows how to constructively solve this MV problem in the presence of DM's dynamics, starting from continuous‐time models of DM and turbulence. This result is extended to the case of asynchronous DM/WFS sampling. An illustrative application to optimal control of tip‐tilt turbulent modes for the European extremely large telescope in the presence of first‐order DM's dynamics is presented. Copyright © 2010 John Wiley & Sons, Ltd.     

Transmission power and data rate aware routing on wireless networks

Wireless networks can vary both the transmission power and modulation of links. Existing routing protocols do not take transmission power control (TPC) and modulation adaptation (also known as rate adaptation – RA) into account at the same time, even though the performance of wireless networks can be significantly improved when routing algorithms use link characteristics to build their routes. This article proposes and evaluates extensions to routing protocols to cope with TPC and RA. The enhancements can be applied to any link state or distance vector routing protocols. An evaluation considering node density, node mobility and link error show that TPC- and RA-aware routing algorithms improve the average latency and the end-to-end throughput, while consuming less energy than traditional protocols.     

Digitally-controlled array of solid-state microcoolers for use in surgery

This paper presents an approach for generating a well-defined cooling pattern over an area of tissue. An array of solid-state microcoolers is used, which could be included in a probe that provides local cooling. This medical instrument can be used for removal of scar tissue in the eye or for the rapid stopping of bleeding due to micro-cuts, which makes it a useful tool to medical doctors and could make surgery more secure to the patient. The array of microcoolers is composed of 64 independent thermo-electric elements, each controlled using an integrated circuit designed in CMOS. The independent control allows the flexible programming of the surface temperature profile. This type of control is very suitable in case abrupt temperature steps should be avoided. Cooling by lateral heat flow was selected in order to minimize the influence of heat by dissipation from the electronic circuits. Moreover, a thermo-electric component with lateral heat allows fabrication of the cooling elements using planar thin-film technology, lithography and wet etching on top of the silicon wafer. This approach is potentially CMOS compatible, which would allow for the fabrication of the thermo-electric elements on top of a pre-fabricated CMOS wafer as a post-process step. Each pixel is composed of thin-films of n-type bismuth telluride, Bi₂Te₃ and p-type antimony telluride, Sb₂Te₃, which are electrically interconnected as thermocouple. These materials have excellent thermoelectric characteristics, such as thermoelectric figures-of-merit, ZT, at room temperatures of 0.84 and 0.5, respectively, which is equivalent to power-factors, PF, of 3.62 × 10⁻³ W K⁻¹ m⁻² and 2.81 × 10⁻³ W K⁻¹ m⁻², respectively. The theoretical study presented here demonstrates a cooling capability of 15°C at room temperature (300 K ≈ 27°C). This cooling performance is sufficient to maintain a local tissue temperature at 25°C, which makes it suitable for the intended application. A first prototype was successfully fabricated to demonstrate the concept.     

Video editing with pen-based technology

Multimedia Tools and Applications - The manipulation of video content is still a difficult task due to its complexity and richness. This paper applies pen-based technology to video editing, with...