Binding of transmembrane mucins to galectin-3 limits herpesvirus 1 infection of human corneal keratinocytes.

Epithelial cells lining mucosal surfaces impose multiple barriers to viral infection. At the ocular surface, the carbohydrate-binding protein galectin-3 maintains barrier function by cross-linking transmembrane mucins on the apical glycocalyx. Despite these defense mechanisms, many viruses have evolved to exploit fundamental cellular processes on host cells. Here, we use affinity assays to show that herpes simplex virus type 1 (HSV-1), but not HSV-2, binds human galectin-3. Knockdown of galectin-3 in human corneal keratinocytes by small interfering RNA significantly impaired HSV-1 infection, but not expression of nectin-1, indicating that galectin-3 is a herpesvirus entry mediator. Interestingly, exposure of epithelial cell cultures to transmembrane mucin isolates decreased viral infectivity. Moreover, HSV-1 failed to elute the biological counterreceptor MUC16 from galectin-3 affinity columns, suggesting that association of transmembrane mucins to galectin-3 provides protection against viral infection. Together, these results indicate that HSV-1 exploits galectin-3 to enhance virus attachment to host cells and support a protective role for transmembrane mucins under physiological conditions by masking viral entry mediators on the epithelial glycocalyx.

Interfacial interaction between transmembrane ocular mucins and adhesive polymers and dendrimers analyzed by surface plasmon resonance.

PURPOSE: Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. METHODS: The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). RESULTS: While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH(2)(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)>> CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). CONCLUSIONS: The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and dendrimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability.

Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families.

INTRODUCTION: A strong family history of breast and/or ovarian cancer can often be explained by small insertions, deletions, or substitutions in BRCA1 or BRCA2 and large genomic rearrangements in BRCA1. However, there is little evidence that genomic rearrangements are a major factor in BRCA2 associated breast cancer and the frequencies of rearrangements in BRCA1 in large clinic based populations are unknown. OBJECTIVE: To investigate the frequency of large genomic rearrangements in BRCA1 and BRCA2 in a large clinic based population at high risk of developing breast and/or ovarian cancer. METHODS: Multiplex ligation dependent probe amplification was used to comprehensively screen BRCA1 and/or BRCA2 in 312 index cases. RESULTS: Three novel deletions detected in BRCA2 were found exclusively in families with at least one case of male breast cancer. Novel rearrangements in BRCA1 were detected mostly in families with both breast and ovarian cancer. Families with these mutations were significantly younger at average age of cancer diagnosis. CONCLUSION: Screening for large genomic rearrangements in both BRCA1 and BRCA2 is strongly supported by this study, in particular in multiple case breast/ovarian families with a young age of onset (BRCA1) and families containing at least one case of male breast cancer (BRCA2).

Cellular distributions of the ERM proteins in MDCK epithelial cells: regulation by growth and cytoskeletal integrity.

The highly homologous ERM (ezrin/radixin/moesin) proteins, molecular cross-linkers which connect the cell membrane with the underlying cytoskeleton, have molecular weights of 81, 80 and 78 kDa respectively. We present data which shows significant variation in the molecular weight and presence of multiple forms of ERM proteins in different cell lines, such that specific antibodies to each protein are essential for unambiguous detection. Biochemical fractionation of MDCK cells demonstrates that although the individual ERM fractionation patterns are unaltered by cell density, the multiple forms of moesin each associate with different subcellular fractions. Since ERM proteins can exist in dormant or active conformations corresponding to their phosphorylation state, we propose that the partitioning of ERM proteins between subcellular compartments may depend on their activation status. In addition, we show that when the co-localization between ezrin and F-actin is disrupted by cytochalasin D, MDCK cells undergo a dramatic morphology change during which long, branching, ezrin-rich protrusions are formed. Consistent with other workers, our data suggest that maintenance of ezrin:F-actin interactions are required for the maintenance of normal cellular morphology.

Histometrics: improvement of the dynamic range of fluorescently stained proteins resolved in electrophoretic gels using hyperspectral imaging.

Most image-based analyses, using absorbance or fluorescence of the spatial distribution of identifiable structures in complex biological systems, use only a very small number of dimensions of possible spectral data for the generation and interpretation of the image. We here extend the concepts of hyperspectral imaging, being developed in remote sensing, into analytical biotechnology. The massive volume of information contained in hyperspectral spectroscopic images requires multivariate analysis in order to extract the chemical and spatial information contained within the data. We here describe the use of multivariate statistical methods to map and quantify common protein staining fluorophores (SYPRO Red, Orange and Tangerine) in electrophoretic gels. Specifically, we find (a) that the 'background' underpinning limits of detection is due more to proteins that have not migrated properly than to impurities or to ineffective destaining, (b) the detailed mechanisms of staining of SYPRO red and orange are apparently not identical, and in particular (c) that these methods can provide two orders of magnitude improvement in the detection limit per pixel, to levels well below the limit observable optically.

Non-linear dielectric spectroscopy: antifouling and stabilisation of electrodes by a polymer coating.

Non-linear dielectric spectroscopy (NLDS) has previously been shown to produce quantitative information that is indicative of the metabolic state of various organisms, by modeling the non-linear effects of their membranous enzymes on an applied oscillating electromagnetic field using supervised multivariate analysis methods. However, the instability of the characteristics of the measuring apparatus rendered the process temperamental at best in the laboratory and impractical for field use. The main practical problem, of the non-stationarity of the electrode-solution interface and the ease with which the electrode surfaces are subject to protein fouling. It is addressed by applying a thin, electrically transparent antifouling coat to the electrodes. This reduces the interminable cleaning procedures previously required to prepare the electrodes for use, increases their usable lifetime before recleaning, and also improves the precision and linearity of multivariate models on NLDS data.

Rapid analysis of high-dimensional bioprocesses using multivariate spectroscopies and advanced chemometrics.

There are an increasing number of instrumental methods for obtaining data from biochemical processes, many of which now provide information on many (indeed many hundreds) of variables simultaneously. The wealth of data that these methods provide, however, is useless without the means to extract the required information. As instruments advance, and the quantity of data produced increases, the fields of bioinformatics and chemometrics have consequently grown greatly in importance. The chemometric methods nowadays available are both powerful and dangerous, and there are many issues to be considered when using statistical analyses on data for which there are numerous measurements (which often exceed the number of samples). It is not difficult to carry out statistical analysis on multivariate data in such a way that the results appear much more impressive than they really are. The authors present some of the methods that we have developed and exploited in Aberystwyth for gathering highly multivariate data from bioprocesses, and some techniques of sound multivariate statistical analyses (and of related methods based on neural and evolutionary computing) which can ensure that the results will stand up to the most rigorous scrutiny.

Genetic programming as an analytical tool for non-linear dielectric spectroscopy.

By modelling the non-linear effects of membranous enzymes on an applied oscillating electromagnetic field using supervised multivariate analysis methods, Non-Linear Dielectric Spectroscopy (NLDS) has previously been shown to produce quantitative information that is indicative of the metabolic state of various organisms. The use of Genetic Programming (GP) for the multivariate analysis of NLDS data recorded from yeast fermentations is discussed, and GPs are compared with previous results using Partial Least Squares (PLS) and Artificial Neural Nets (NN). GP considerably outperforms these methods, both in terms of the precision of the predictions and their interpretability.

Laparoscopic retroperitoneal repair of recurrent postoperative lumbar hernia.

A patient who previously underwent an attempt at open mesh repair of a lumbar hernia that occurred following iliac crest bone harvesting was managed successfully by laparoscopic retroperitoneal fixation of the prosthetic material to the iliac crest using bone screws. A review of the literature supports the use of such a novel approach to manage this often vexing problem.

The incidence of secondary hernias diagnosed during laparoscopic total extraperitoneal inguinal herniorrhaphy.

During a 24-month period beginning in July of 1995, laparoscopic total extraperitoneal inguinal herniorrhaphy was attempted in 53 patients. All procedures were performed at a single institution, by senior-level general surgery residents, with the same attending surgeon functioning as first assistant. Three patients required conversion to an "open" procedure (all had a prior history of herniorrhaphy or lower abdominal surgery), leaving 50 patients for analysis. Preoperatively, a unilateral hernia was evident on clinical grounds in 29 patients, the remaining 21 presenting with signs of a bilateral hernia; of the total, 11 had a history of prior hernia repair on the presently affected side. At surgery, a total of 115 hernia defects (indirect, direct, femoral) were identified, 38% of which were discovered only at the time of surgery. Sixty-four percent of patients were found to have at least one of these "secondary" hernias. After reduction of the hernia(s), all defects were covered with polypropylene mesh secured with spiral tacks. There were 10 perioperative complications, one of which required corrective surgical intervention. Over 70% of patients were discharged on the day of surgery; 92% returned home within 23 h of their operation. The most common reason for delay of hospital discharge was urinary retention. There have been no recurrences in short-term follow-up. Most patients were pleased with the recovery time from and the cosmetic results of their surgery. These results suggest that laparoscopic total extraperitoneal herniorrhaphy represents a safe, effective, cosmetically appealing alternative to open hernia repair. Moreover, this approach may provide an added advantage insofar as identifying additional hernia defects that, when repaired, may ultimately yield a lower recurrence rate than might otherwise have been expected.

An acute dystonic reaction with long-term use of ranitidine in an intensive care unit patient.

Dystonic reactions produce twisting and repetitive movements or abnormal posturing; this sign is considered an extrapyramidal sequela that is most typically thought to arise from decreased dopamine activity in the basal ganglia. Severe dystonic reactions have been shown to occur in concert with numerous medications. Although most commonly described with anti-psychotic agents such as haloperidol and phenothiazine, dystonic reactions have been observed in those who have used fluoxetine, erythromycin, crack cocaine, phenobarbital, cisapride, and buspirone. This report details the case of a patient who developed an acute dystonic reaction while taking ranitidine for peptic ulcer prophylaxis, a complication that, to our knowledge, has yet to be described with the use of this agent.

Genetic programming: a novel method for the quantitative analysis of pyrolysis mass spectral data.

A technique for the analysis of multivariate data by genetic programming (GP) is described, with particular reference to the quantitative analysis of orange juice adulteration data collected by pyrolysis mass spectrometry (PyMS). The dimensionality of the input space was reduced by ranking variables according to product moment correlation or mutual information with the outputs. The GP technique as described gives predictive errors equivalent to, if not better than, more widespread methods such as partial least squares and artificial neural networks but additionally can provide a means for easing the interpretation of the correlation between input and output variables. The described application demonstrates that by using the GP method for analyzing PyMS data the adulteration of orange juice with 10% sucrose solution can be quantified reliably over a 0-20% range with an RMS error in the estimate of ∼1%.

Oscillatory, stochastic and chaotic growth rate fluctuations in permittistatically controlled yeast cultures.

We describe a continuous culture system related to the turbidostat, but using a feedback system based on biomass estimation from the dielectric permittivity of the cell suspension rather than its optical density. It is shown that this system provides an excellent method of maintaining a constant biomass level within a fermentor. The computer-controlled system was able to effect the essentially continuous registration of growth rate by monitoring the rate of medium addition via the time-dependent activity of the pump. At some biomass setpoints for aerobically grown cultures of baker's yeast substantial time-dependent fluctuations in the growth rate of the culture were thereby observed. At some biomass setpoints, however, or under anaerobic conditions, or when using a non-Crabtree yeast, the growth rate was constant, indicating that the fluctuations were inherent to the biological system and not simply a property of the fermentor and control system. A variety of time series analyses (Fourier transformations, Hurst and Lyapunov exponents, the determination of embedding dimension, and non-linear time series predictions based on the methodology of Sugihara and May) were used to demonstrate, for the first time, that as well as stochastic and periodic components these fluctuations exhibited deterministic chaos. 'Trivial predictors' were unable to give accurate predictions of the growth rate in these cultures. The growth rate fluctuations were studied further by means of offline measurements of changes in percentage viability, bud count, and in the external ethanol and glucose concentrations; these data and other evidence suggested that the growth rate fluctuations were closely linked to the primary respiro-fermentative metabolism of this organism. The identification of chaotic growth rates in cell cultures suggests that there may be novel methods for controlling the growth of such cultures.

Barriers to mental health care for Hispanic Americans: a literature review and discussion.

The Hispanic American population, the second largest and fastest growing minority population in the United States, faces barriers to access to both medical health and mental health care. This paper examines both financial and cultural barriers to utilization of mental health care services; it is a broad review of the literature and is not intended to be comprehensively detailed. The research review suggests that the financial barrier is a major determinant of mental health service access for Hispanic American populations. Also, nonfinancial barriers such as acculturation are examined. A two-part plan is suggested to reduce both financial and nonfinancial barriers. Very little literature on utilization of substance abuse services was found; suggestions for further research are thus proposed.

Confirmation by using mutant strains that the membrane-bound H(+)-ATPase is the major source of non-linear dielectricity in Saccharomyces cerevisiae.

Non-linear dielectric spectroscopy is a novel technique for determining the activity of (predominantly) membranous enzymes as their ability to generate harmonics when excited with a sinusoidal electrical field. In washed suspensions of yeast cells, the ability to generate harmonics is inhibited by low concentrations of sodium vanadate, suggesting that the vanadate-sensitive H(+)-ATPase is the major source of the non-linear dielectricity. This conclusion is greatly strengthened by the demonstration herein that the generation of harmonics by a strain containing a vandate-resistant H(+)-ATPase is also highly resistant to sodium metavanadate.