A dielectrophoretic method of discrimination between normal oral epithelium, and oral and oropharyngeal cancer in a clinical setting.

Despite the accessibility of the oral cavity to clinical examination, delays in diagnosis of oral and oropharyngeal carcinoma (OOPC) are observed in a large majority of patients, with negative impact on prognosis. Diagnostic aids might help detection and improve early diagnosis, but there remains little robust evidence supporting the use of any particular diagnostic technology at the moment. The aim of the present feasibility first-in-human study was to evaluate the preliminary diagnostic validity of a novel technology platform based on dielectrophoresis (DEP). DEP does not require labeling with antibodies or stains and it is an ideal tool for rapid analysis of cell properties. Cells from OOPC/dysplasia tissue and healthy oral mucosa were collected from 57 study participants via minimally-invasive brush biopsies and tested with a prototype DEP platform using median membrane midpoint frequency as main analysis parameter. Results indicate that the current DEP platform can discriminate between brush biopsy samples from cancerous and healthy oral tissue with a diagnostic sensitivity of 81.6% and a specificity of 81.0%. The present ex vivo results support the potential application of DEP testing for identification of OOPC. This result indicates that DEP has the potential to be developed into a low-cost, rapid platform as an assistive tool for the early identification of oral cancer in primary care; given the rapid, minimally-invasive and non-expensive nature of the test, dielectric characterization represents a promising platform for cost-effective early cancer detection.

Diversity of Foliar Phytophthora Species on Rhododendron in Oregon Nurseries.

The genus Phytophthora contains some of the most notorious plant pathogens affecting nursery crops. Given the recent emergence of the sudden oak death pathogen Phytophthora ramorum, particularly in association with Rhododendron spp., characterization of Phytophthora communities associated with this host in nursery environments is prudent. Many taxa may present symptoms similar to P. ramorum but we do not necessarily know their identity, frequency, and importance. Here, we present a survey of Phytophthora taxa observed from seven nurseries in the U.S. state of Oregon. Incidence and diversity of Phytophthora communities differed significantly among nurseries and among seasons within nursery. The taxa P. syringae and P. plurivora were widespread and detected at most of the nurseries sampled. Nine other taxa were also detected but were found either in a single nursery or were shared among only a few nurseries. Characterization of the Phytophthora communities present in nurseries is an important step toward understanding the ecology of these organisms as well as an aid to nursery managers in determining what risks may be present when symptomatic plants are observed. This study builds on an increasing literature, which characterizes Phytophthora community structure in nurseries.

Varying degrees of amplification of the N-ras oncogene in the human breast cancer cell line MCF-7.

The oncogene N-ras has been found to be amplified (congruent to 20 copies) in the human breast carcinoma cell line MCF-7. The amplified sequences have been localized to a marker chromosome by in situ hybridization. Sublines of MCF-7, serially passaged in different laboratories, have marked variation in the degree of N-ras amplification. The differing degrees of amplification of N-ras are further evidence of heterogeneity within MCF-7 subclones. The phenomenon may not have general relevance for breast cancer, since other breast cancer cell lines and DNA from patient biopsies failed to show evidence of N-ras amplification.

Relationship of low vitamin D status with positive, negative and cognitive symptom domains in people with first-episode schizophrenia.

AIM: Deficient vitamin D levels are very common among Americans of all ages and ethnicities, but little is known about its prevalence or associated problems among those with schizophrenia. METHODS: Stored plasma from 20 recent onset schizophrenia subjects and 20 matched healthy comparison subjects were analysed for 25 OH vitamin D, and related to measures of symptom severity and neurocognition. RESULTS: There was no significant difference in mean 25 OH vitamin D between the schizophrenia and the healthy comparison subjects (28.2 standard deviation (SD) 12.6 ng mL(-1) vs. 29.9 SD 14.3 ng mL(-1) ), and about half the subjects in each group had insufficient levels (<30 ng mL(-1) ). Among psychosis subjects, greater severity of negative symptoms was correlated with lower vitamin D status (r = -0.55, P = 0.012); the correlations of overall symptom severity and positive symptom severity with 25 OH vitamin D levels approached significance (r = -0.42, P = 0.07 and r = -0.36, P = 0.12, respectively). There was no relationship of vitamin D with depressive symptoms. Among the schizophrenia subjects, lower 25 OH vitamin D levels were associated with more severe overall cognitive deficits (r = 0.56, P = 0.019). CONCLUSION: This study found that lower vitamin D levels in schizophrenia subjects were associated with more severe negative symptoms and overall cognitive deficits. However, the cross-sectional design precludes any conclusions about whether low vitamin D status in fact causes more severe negative symptoms and cognitive impairments. No relationship was found between lower vitamin D levels and depressive symptoms.

Acid-base regulation in peritoneal dialysis.

To prevent increased protein degradation and renal bone disease, optimal correction of acidosis should therefore be a priority in the management of CAPD patients. Initial results suggest that bicarbonate solutions may be more biocompatible than lactate, and one would expect increasing use over the next decade. Manufacturers could help to improve acid-base regulation in CAPD by providing a range of solutions with varying lactate and bicarbonate concentration, so that correction of acidosis could be optimized individually.

The relation of perception and motor action: ideomotor compatibility and interference in divided attention.

Ideomotor theory is one variation of the general position that perception and motor action are related. According to this theory, a perceptual representation of the goal of a response must be generated prior to response initiation. Ideomotor theory can be extended by assuming that generation of this representation, which usually is required prior to motor action, is not needed if the stimulus itself corresponds to the response goal, that is, if the stimulus and response are ideomotor compatible. Because processing to generate the response representation is not needed with ideomotor compatibility, it should be possible to control two responses simultaneously without mutual interference if at least one stimulus-response relation is ideomotor compatible. Although supported in previous work involving discrete responses, this prediction of perfect time-sharing was found not to hold in the experiments reported here. These experiments, unlike those showing perfect time-sharing, involved continuous responses. We propose an alternative version of ideomotor compatibility, in which perfect time-sharing can occur if an integrated stimulus is provided to match the continuous and integrated response.

Pre and interdialytic acid-base balance in hemodialysis patients.

In an observational study, nine hemodialysis patients using 35 mmol/l bicarbonate dialysate were studied over a 44-hour interdialytic interval. Serum bicarbonate was measured at regular intervals at home and the mean time averaged concentration was 27.0+/-1.2 mmol/l. Seven of the nine patients showed a slow linear decline in bicarbonate whilst in two patients levels were unchanged. In 8 of 9 patients the average of the post and predialysis bicarbonate accurately predicted the time-averaged (area under the curve) bicarbonate concentration. In addition, predialysis serum bicarbonate was measured in 46 patients after both a 2 and 3-day interdialytic interval. The serum bicarbonate was significantly lower after the 3-day interdialytic interval (3-day, 22.1+/-0.6 vs 2-day, 23.0+/-0.5 mmol/l, p<0.05). The results from this study emphasize the importance of standardization of bicarbonate measurement in order to avoid spurious acidosis.

Inorganic aerosols and their role in catalyzing sulfuric acid production in furnaces.

Submicrometer ash and sulfur oxides are important pollutants formed during pulverized coal combustion. The submicrometer ash contains known catalysts for sulfur dioxide oxidation as well as alkaline species that will react with sulfur oxides. This work was an investigation to determine if submicrometer ash-sulfur oxide interactions can have a significant impact on the fate of sulfur. The submicrometer-ash-sulfur oxide interaction studies were carried out using submicrometer ash of controlled composition generated from the combustion of synthetic chars. The submicrometer ash was either exposed to SO2 in the combustion chamber or immobilized on a quartz-fiber filter for later exposure to SO2. Submicrometer-ash catalysis of SO2 oxidation was found to contribute significantly to the SO3 formed in the convective passes of a utility boiler, depending on the coal, combustion conditions, and temperature profile in the convective section. The calcium within the freshly formed submicrometer ash appears to sulfate more rapidly than would be predicted from rates published in the literature and is expected to sulfate completely in the convective section of a utility boiler.

Differential transport of cytosine-containing nucleosides by recombinant human concentrative nucleoside transporter protein hCNT1.

The transportability of cytosine-containing nucleosides by recombinant hCNT1 was investigated in transfected mammalian cells. Apparent K(m) values for hCNT1-mediated transport of uridine, cytidine and deoxycytidine were, respectively, 59, 140 and 150 microM. Uridine transport was inhibited 89, 32 and 11%, respectively, by 500 microM gemcitabine, cytarabine and lamivudine, demonstrating that, unlike gemcitabine (a high-affinity hCNT1 permeant), cytarabine and lamivudine are poor hCNT1 permeants.

Human oral cancer cells with increasing tumorigenic abilities exhibit higher effective membrane capacitance.

OBJECTIVE: Although cells with tumorigenic/stem cell-like properties have been identified in many cancers, including oral squamous cell carcinoma (OSCC), their isolation and characterisation is still at early stages. The aim of this study is to characterise the electrophysiological properties of OSCC cells with different tumorigenic properties in order to establish if a correlation exists between tumorigenicity and cellular electrical characteristics. MATERIALS AND METHODS: Rapid adherence to collagen IV was used as a non-invasive, functional method to isolate subsets of cells with different tumorigenic abilities from one oral dysplastic and three OSCC-derived cell lines. The cell subsets identified and isolated using this method were further investigated using dielectrophoresis, a label-free method to determine their electrophysiological parameters. Cell membrane morphology was investigated using scanning electron microscopy (SEM) and modulated by use of 4-methylumbelliferone (4-MU). RESULTS: Rapid adherent cells (RAC) to collagen IV, enriched for increased tumorigenic ability, had significantly higher effective membrane capacitance than middle (MAC) and late (LAC) adherent cells. SEM showed that, in contrast to MAC and LAC, RAC displayed a rough surface, extremely rich in cellular protrusions. Treatment with 4-MU dramatically altered RAC membrane morphology by causing loss of filopodia, and significantly decreased their membrane capacitance, indicating that the highest membrane capacitance found in RAC was due to their cell membrane morphology. CONCLUSION: This is the first study showing that OSCC cells with higher tumour formation ability exhibit higher effective membrane capacitance than cells that are less tumorigenic. OSSC cells with different tumorigenic ability possessed different electrophysiological properties mostly due to their differences in the cell membrane morphology. These results suggest that dielectrophoresis could potentially used in the future for reliable, label-free isolation of putative tumorigenic cells.

Sustained expression of interleukin-1beta in mouse hippocampus impairs spatial memory.

Glial activation and neuroinflammation occur in neurodegenerative disease and brain injury, however their presence in normal brain aging suggests that chronic neuroinflammation may be a factor in age-related dementia. Few studies have investigated the impact of sustained elevation of hippocampal interleukin-1beta, a pro-inflammatory cytokine upregulated during aging and Alzheimer's disease, on cognition in mice. We utilized the IL-1beta(XAT) transgenic mouse to initiate bilateral hippocampal overexpression of interleukin-1beta to determine the influence of sustained neuroinflammation independent of disease pathology. Fourteen days following transgene induction, adult male and female IL-1beta(XAT) mice were tested on non-spatial and spatial versions of the Morris water maze. For the spatial component, one retention trial was conducted 48 h after completion of a 3 day acquisition protocol (eight trials per day). Induction of IL-1beta did not impact non-spatial learning, but was associated with delayed acquisition and decreased retention of the spatial task. These behavioral impairments were accompanied by robust reactive gliosis and elevated mRNA expression of inflammatory genes in the hippocampus. Our results suggest that prolonged neuroinflammation response per se may impact mnemonic processes and support the future application of IL-1beta(XAT) transgenic mice to investigate chronic neuroinflammation in age- and pathology-related cognitive dysfunction.

Sodium butyrate induces differentiation in breast cancer cell lines expressing the estrogen receptor.

Addition of sodium butyrate (NaB) to 6 cultured human breast carcinoma cell lines results in a dose and time-dependent growth inhibition. Kinetic evidence, related to the growth of a minority cell population which decreases in size with time of exposure, is presented to indicate that the NaB effect is reversible. In those cell lines that express the estrogen receptor (ER), growth inhibition is accompanied by a more differentiated phenotype, which is characterized by increased accumulation of lipid and milk-fat globule membrane glycoproteins. The potential for differentiation is not blocked by tamoxifen, indicating that the relationship to ER expression is likely secondary to the association of ER expression with a particular stage of secretory cell differentiation that is susceptible to NaB induction. Of the 3 lines shown to respond in this way (MCF-7, ZR-75-1, and MDA-134), ZR-75-1 is an extreme example that may serve as a model for studies of gene expression during human mammary epithelial cell differentiation.

Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical in manifestation of viral virulence.

Myxoma virus (MYX) induces extensive immunosuppression in infected rabbits and is associated with high levels of mortality. The virus encodes multiple gene products designed to circumvent the cellular immune response to the viral infection. Deletion analysis has shown that the M11L open reading frame (ORF) is an important virulence factor which downregulates leukocyte infiltration of MYX-induced tumors. To investigate the role of the M11L protein in viral pathogenesis, we sequenced the MYX M11L ORF and showed that the sequence has motifs consistent with a 166-aa class III membrane-spanning molecule possessing a single transmembrane helix near the C-terminus and a 142-aa N-terminal extracellular domain that has six cysteine residues plus two consensus N-glycosylation sites. Transcription analysis indicates that M11L is expressed as an early gene, and surface immunofluorescence studies with anti-M11L antibodies reveal that M11L protein is transported to the infected cell surface. Immunoprecipitation analysis of an attenuated viral recombinant, vMYX-GF-delta M11L, indicates that an M11L variant protein with an altered C-terminus is synthesized at about 45% of wild type levels; however, it is not detectable on the cell surface, suggesting that proper M11L function requires localization at the infected cell membrane. We propose that M11L is a virulence factor whose function is to recognize an extracellular ligand essential for the cellular inflammatory response.

A freeze-fracture analysis of intramembrane particle densities on dystrophic hamster heart sarcolemma.

The intramembrane particle (IMP) profile of control and dystrophic (Bio 14.6) hamster cardiac muscle plasma membrane was assessed in freeze-fracture replicas to determine whether this animal model of muscular dystrophy exhibits the same membrane characteristics found in skeletal muscle from other more thoroughly studied dystrophic animals, and to test the hypothesis that the plasma membrane of the cardiac muscle cell is the site of a defect associated with the disease. Samples of cardiac muscle tissue from hamsters ranging in age from 1 to 13 months were freeze-fractured. Intramembrane particle numbers were determined for all tissue samples by counting randomly selected areas of P- and E-face surfaces. Up to the age of 1 month, the particle density was the same in both strains of hamster, after which time, the population of IMPs was about 30% lower in dystrophic than in normal heart sarcolemma. This 30% difference in particle frequency in dystrophic hamster heart membrane is consistent with values published for cell membrane from other muscular dystrophies and supports the theory that there is a defect in the plasma membrane of dystrophic cells. In addition, this study has shown for the first time that a presumed membrane defect related to muscular dystrophy (reduced number of IMPs) may be present throughout the life of the animal (1-13 months), and expressed in every cell sampled.

Recovery of 13C in breath from infused NaH13CO3 increases during euglycaemic hyperinsulinaemia.

1. The effect of euglycaemic hyperinsulinaemia on the recovery of 13C in expired CO2 has been assessed in six normal subjects. Each was studied on three occasions: once with a 6 h primed constant infusion of NaH13CO3 combined with a euglycaemic hyperinsulinaemic clamp for the last 3 h (study 1), once with a 6 h primed constant infusion of NaH13CO3 alone (study 2) and once with a 6 h infusion of normal saline combined with a hyperinsulinaemic clamp for the last 3 h (study 3). Measurements of 13C enrichment of expired CO2 were made in the third and sixth hour of each infusion. 2. There was no significant increase in enrichment during study 3 (3 h 0.00047 +/- 0.00016 versus 6 h 0.00069 +/- 0.00028 atom per cent excess) with potato-starch-derived D-glucose used to maintain euglycaemia. 13C recovery increased in the sixth hour of both study 1 and 2 (study 1: 3 h 74.4 +/- 2.0 versus 6 h 85.5 +/- 2.6%, P < 0.01; study 2: 3 h 72.1 +/- 2.4 versus 6 h 81.7 +/- 1.4%, P < 0.01). There was no significant difference in recovery between studies 1 and 2. 3. These results suggest that increased recovery during a sequential euglycaemic clamp is predominantly time-dependent. Studies which use this technique to examine the effect of insulin on substrate oxidation should take this into account.

Correction of acidosis in hemodialysis decreases whole-body protein degradation.

Correction of acidosis in hemodialysis (HD) decreases protein degradation. The effect of the correction of chronic metabolic acidosis in chronic renal failure patients treated with HD was determined from the kinetics of infused L-[1-(13)C]leucine. Six HD patients were studied before (acid) and after (bicarbonate) correction of acidosis (pH: acid 7.36 +/- 0.01, bicarbonate 7.40 +/- 0.01, P < 0.005). Leucine appearance from body protein (PD) and leucine disappearance into body protein (PS) decreased significantly with correction of acidosis (PD: acid 180.6 +/- 7.3, bicarbonate 130.9 +/- 7.2 mumol.kg-1.h-1, P < 0.005; PS: acid 172.3 +/- 6.8, bicarbonate 122.0 +/- 6.8 mumol.kg-1.h-1, P < 0.005). There was no significant change in leucine oxidation or plasma amino acid concentrations. These results demonstrate that optimal correction of acidosis in HD is beneficial in terms of protein turnover and may improve long-term nutritional status in HD.

Correction of acidosis in hemodialysis patients increases the sensitivity of the parathyroid glands to calcium.

Correction of acidosis in hemodialysis patients increases the sensitivity of the parathyroid glands to calcium. In this study, the parathyroid response to the correction of acidosis in eight hemodialysis patients was determined by performing dynamic assessment of parathyroid function before and after the correction of acidosis. The parathyroid response to intravenous calcitriol before and after the correction of acidosis was also assessed. After optimal correction of acidosis, there were no significant changes in blood pH, ionized calcium, phosphate, or alkaline phosphatase values, but the level of venous total CO2 increased significantly. Parathyroid hormone/ionized calcium curves were displaced downward after correction of acidosis, but not after the administration of intravenous calcitriol. The correction of metabolic acidosis in hemodialysis patients with secondary hyperparathyroidism can suppress parathyroid hormone secretion by increasing the sensitivity of the parathyroid glands to ionized calcium.

Sodium bicarbonate treatment and ubiquitin gene expression in acidotic human subjects with chronic renal failure.

BACKGROUND: In chronic renal failure, metabolic acidosis is associated with increased whole body protein degradation. In rats this effect of acidosis occurs in skeletal muscle and is associated with increased ubiquitin mRNA expression. This has not been demonstrated in humans. MATERIALS AND METHODS: Six patients with chronic renal failure and acidosis underwent muscle biopsy before and after 1 month's treatment with sodium bicarbonate. RNA was extracted from the biopsy, and the expression of the genes for ubiquitin and the proteasome component, C2, were measured by Northern blotting. RESULTS AND CONCLUSIONS: There was no significant difference in the expression of ubiquitin or C2 after bicarbonate treatment. This is contrast with results from animal models of acidosis and some other catabolic conditions in humans. This may reflect the complexity of the ubiquitin-dependent pathway, and it may be that changes in ubiquitin expression are only seen with more severe and/or acute changes in pH.

The use of restriction fragment polymorphisms to identify the cell line MCF-7.

We have recently documented variations in the level of N-ras gene amplification in independently passaged MCF-7 cells. To establish if these differences are due to a lack of genetic identity between current MCF-7 passages and the original cell line, we have performed karyological and restriction fragment length polymorphism (RFLP) analyses. Documentation of a combination of restriction fragment patterns which are characteristic of MCF-7 has been facilitated by an analysis of DNA from an early-passage of MCF-7 from the Michigan Cancer Foundation (MCF). In addition, such early passage cells and other MCF-7 lines also share a unique amplification of the N-ras oncogene. Using these criteria and karyotypic analysis it can be shown that a sample of MCF-7 obtained from the American Type Culture Collection (ATCC) was derived from a different individual than was the original MCF-7 cell line. It is important that researchers verify the relationship of current cell lines to the original MCF-7. Furthermore, the techniques described here provide a powerful tool which may be used to assess the identity of cell stocks.