Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer`s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Cross-mating experiments with geographically different populations of Amblyomma cajennense (Acari: Ixodidae)

The present study evaluated the reproductive compatibility of the crosses between adult ticks of the following three geographically different populations of Amblyomma cajennense: State of So Paulo (SP), southeastern Brazil; State of Rondnia (RO), northern Brazil; and Colombia (CO). In addition, crosses between A. cajennense ticks from Argentina (AR) and SP ticks were also performed. The Argentinean population (AR) was compatible with SP because their crosses resulted in high % egg hatching (mean values ranging from 71.5 to 93.5%), similarly to all homologous (intrapopulational) crosses. In contrast, the tick populations SP, RO, and CO were shown to be incompatible with each other, since their heterologous (interpopulational) crosses always resulted in very low % egg hatching (range: 0-5%). The F(1) larval offspring derived from some of these females that yielded 5% egg hatching were reared until the F(1) adult stage. In all cases, only adult females molted from engorged nymphs. These F(1) females were likely to be a product of thelytokous parthenogenesis of the SP, RO, and CO females that were used in the heterologous crosses. Reproductive incompatibility is not expected to occur between different populations of a single species. Thus, our results suggest that the taxon A. cajennense might be represented by a complex of different species, whereas SP and AR ticks might represent a single species. Further populational genetic studies, coupled with extensive morphological analyses, are needed to clarify and determine a possible complex of valid species that might have been classified under the taxon A. cajennense.

Nucleophosmin, p53, and Ki-67 expression patterns on an oral squamous cell carcinoma tissue microarray

Oral cancer is the eighth most prevalent cancer worldwide. It causes significant mortality and morbidity rates, which have motivated the search for prognostic factors to better tailor the individual management of oral squamous cell carcinoma patients. Nucleophosmin is a multifunctional protein that is involved in many cellular activities, such as, regulation of the tumor suppressor genes TP53 and p14(ARF). and is associated with proliferative and growth suppressive roles in the cell. Nucleophosmin is overexpressed in many solid tumors in human, including tumors of the colon, liver, stomach, ovary, and prostate. In this study, we analyzed the expression of nucleophosmin, Ki-67, and p53 by immunohistochemistry in oral squamous cell carcinomas. Less than 10% of nuclear staining was observed in 90.3%, 50.6%, and 65.3% of the cases for nucleophosmin, p53, and Ki-67, respectively. Expression of p53 was not significantly associated with any of the clinicopathologic parameters analyzed. Increased expression of Ki-67 was associated with the presence of lymph node metastasis (P < .0001), advanced stages of disease (P = .0030), tumors occurring in the floor of mouth (P = .0018), and moderately/well-differentiated tumors (P = .0287). Local recurrence was associated with higher expression of nucleophosmin (P = .0233), and disease-free survival rate was significantly better in patients with low expression of nucleophosmin. Multivariate analysis suggested that expression of nucleophosmin could be an independent prognostic factor for oral squamous cell carcinoma patients. (C) 2010 Elsevier Inc. All rights reserved.

Oral squamous cell carcinoma: status of tight junction claudins in the different histopathological patterns and relationship with clinical parameters. A tissue-microarray-based study of 136 cases

Aims Claudins are integral transmembrane proteins of the tight junctions, critical for maintaining cell adhesion and polarity. Alterations in the expression of individual claudins have been detected in carcinomas and appear to correlate with tumour progression. Methods In this study, a panel of anti-claudin antibodies (anti-claudins 1, 2, 3, 4, 5 and 7) was employed to map claudin expression in 136 cases of oral squamous cell carcinoma (OSCC) organised in a tissue microarray. Results Claudins were expressed in a reticular pattern up to the prickle layer in normal mucosal epithelium. In OSCC, claudins were strongly present in well-differentiated tumours, they presented mild and low expression in moderately differentiated OSCC, and were negative in poorly differentiated OSCC; the absences of claudin 1 (p = 0.002) and claudin 4 (p<0.001) were associated with moderately/poorly differentiated tumours. Strong expression of claudin 4 was associated with decreased perineural infiltration (p = 0.024). Claudins 5 and 7 were mostly negative or weakly expressed in all cases studied. Expression of claudin 7 was associated with the early clinical stages of the disease, whereas loss of claudin 7 tended to be more frequent in advanced stages of OSCC (p = 0.054). Absence of claudin 7 was also associated with absent vascular infiltration (p = 0.045) and with presence of recurrence (p = 0.052). Conclusions Claudin expression patterns showed a strong correlation with histological type of OSCC; claudin expression was decreased in areas of invasion, and negative in poorly differentiated tumours. This pattern may be related to evolution and prognosis of these tumours, especially in the case of claudin 7, which seems to be associated with a poor prognosis in OSCC.

Using clusters of computers for large QU-GENE simulation experiments

The QU-GENE Computing Cluster (QCC) is a hardware and software solution to the automation and speedup of large QU-GENE (QUantitative GENEtics) simulation experiments that are designed to examine the properties of genetic models, particularly those that involve factorial combinations of treatment levels. QCC automates the management of the distribution of components of the simulation experiments among the networked single-processor computers to achieve the speedup.

A mixture model-based approach to the clustering of microarray expression data

Motivation: This paper introduces the software EMMIX-GENE that has been developed for the specific purpose of a model-based approach to the clustering of microarray expression data, in particular, of tissue samples on a very large number of genes. The latter is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. A feasible approach is provided by first selecting a subset of the genes relevant for the clustering of the tissue samples by fitting mixtures of t distributions to rank the genes in order of increasing size of the likelihood ratio statistic for the test of one versus two components in the mixture model. The imposition of a threshold on the likelihood ratio statistic used in conjunction with a threshold on the size of a cluster allows the selection of a relevant set of genes. However, even this reduced set of genes will usually be too large for a normal mixture model to be fitted directly to the tissues, and so the use of mixtures of factor analyzers is exploited to reduce effectively the dimension of the feature space of genes. Results: The usefulness of the EMMIX-GENE approach for the clustering of tissue samples is demonstrated on two well-known data sets on colon and leukaemia tissues. For both data sets, relevant subsets of the genes are able to be selected that reveal interesting clusterings of the tissues that are either consistent with the external classification of the tissues or with background and biological knowledge of these sets.

Evolutionary experiments on mate recognition in the Drosophila serrata species complex

It is becoming increasingly apparent that at least some aspects of the evolution of mate recognition may be amenable to manipulation in evolutionary experiments. Quantitative genetic analyses that focus on the genetic consequences of evolutionary processes that result in mate recognition evolution may eventually provide an understanding of the genetic basis of the process of speciation. We review a series of experiments that have attempted to determine the genetic basis of the response to natural and sexual selection on mate recognition in the Drosophila serrata species complex. The genetic basis of mate recognition has been investigated at three levels: (1) between the species of D. serrata and D. birchii using interspecific hybrids, (2) between populations of D. serrata that are sympatric and allopatric with respect to D. birchii, and (3) within populations of D. serrata. These experiments suggest that it may be possible to use evolutionary experiments to observe important events such as the reinforcement of mate recognition, or the generation of the genetic associations that are central to many sexual selection models.

Patterns of gene expression are altered in the frontal and motor cortices of human alcoholics

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin-related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol-responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.

Catheter effects in organ perfusion experiments

In a typical isolated organ perfusion experiment, a substance is injected upstream of an organ and then collected at some distance downstream. To reach the organ from the injection site, and then from the organ to the collector, a solute passes through catheters, usually tubes with circular cross-sections. Catheters cause distortion to the concentration-time profile of the perfusion. In this paper, we analyse catheter distribution kinetics from a mathematical point of view, develop the function most suitable for modeling this distribution and successfully apply this function to experimental data. (C) 2002 Academic Press.

Water table waves in an unconfined aquifer: Experiments and modeling

[1] Comprehensive measurements are presented of the piezometric head in an unconfined aquifer during steady, simple harmonic oscillations driven by a hydrostatic clear water reservoir through a vertical interface. The results are analyzed and used to test existing hydrostatic and nonhydrostatic, small-amplitude theories along with capillary fringe effects. As expected, the amplitude of the water table wave decays exponentially. However, the decay rates and phase lags indicate the influence of both vertical flow and capillary effects. The capillary effects are reconciled with observations of water table oscillations in a sand column with the same sand. The effects of vertical flows and the corresponding nonhydrostatic pressure are reasonably well described by small-amplitude theory for water table waves in finite depth aquifers. That includes the oscillation amplitudes being greater at the bottom than at the top and the phase lead of the bottom compared with the top. The main problems with respect to interpreting the measurements through existing theory relate to the complicated boundary condition at the interface between the driving head reservoir and the aquifer. That is, the small-amplitude, finite depth expansion solution, which matches a hydrostatic boundary condition between the bottom and the mean driving head level, is unrealistic with respect to the pressure variation above this level. Hence it cannot describe the finer details of the multiple mode behavior close to the driving head boundary. The mean water table height initially increases with distance from the forcing boundary but then decreases again, and its asymptotic value is considerably smaller than that previously predicted for finite depth aquifers without capillary effects. Just as the mean water table over-height is smaller than predicted by capillarity-free shallow aquifer models, so is the amplitude of the second harmonic. In fact, there is no indication of extra second harmonics ( in addition to that contained in the driving head) being generated at the interface or in the interior.

Surface stickiness of drops of carbohydrate and organic acid solutions during convective drying: Experiments and modeling

Drying kinetics of low molecular weight sugars such as fructose, glucose, sucrose and organic acid such as citric acid and high molecular weight carbohydrate such as maltodextrin (DE 6) were determined experimentally using single drop drying experiments as well as predicted numerically by solving the mass and heat transfer equations. The predicted moisture and temperature histories agreed with the experimental ones within 6% average relative (absolute) error and average difference of +/- 1degreesC, respectively. The stickiness histories of these drops were determined experimentally and predicted numerically based on the glass transition temperature (T-g) of surface layer. The model predicted the experimental observations with good accuracy. A nonsticky regime for these materials during spray drying is proposed by simulating a drop, initially 120 mum in diameter, in a spray drying environment.

Optical experiments beyond the quantum limit: Squeezing entanglement and teleportation

Results of experiments recently performed are reported, in which two optical parametric amplifiers were set up to generate two independently quadrature squeezed continuous wave laser beams. The transformation of quadrature squeezed states into polarization squeezed states and into states with spatial quantum correlations is demonstrated. By utilizing two squeezed laser beams, a polarization squeezed state exhibiting three simultaneously squeezed Stokes operator variances was generated. Continuous variable polarization entanglement was generated and the Einstein-Podolsky-Rosen paradox was observed. A pair of Stokes operators satisfied both the inseparability criterion and the conditional variance criterion. Values of 0.49 and 0.77, respectively, were observed, with entanglement requiring values below unity. The inseparability measure of the observed quadrature entanglement was 0.44. This value is sufficient for a demonstration of quantum teleportation, which is the next experimental goal of the authors.

Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.