Techniques of radioautography for medical and biological research

Standard techniques for radioautography used in biological and medical research can be classified into three categories, i.e., macroscopic radioautography, light microscopic radioautography and electron microscopic radioautography. The routine techniques used in these three procedures are described. With regard to macroscopic radioautography, whole body radioautography is a standard technique which employs freezing and cryosectioning and can demonstrate organ distributions of both soluble and insoluble compounds. In contrast, in light and electron microscopic radioautography, soluble and insoluble techniques are separated. In order to demonstrate insoluble labeled compounds, conventional chemical fixations such as formalin for light microscopy or buffered glutaraldehyde and osmium tetroxide for both light and electron microscopy followed by dehydration, embedding and wet-mounting applications of radioautographic emulsions can be used. For the demonstration of soluble labeled compounds, however, cryotechniques such as cryofixation, cryosectioning, freeze-drying, freeze-substitution followed by dry-sectioning and dry-mounting radioautography should be employed both for light and electron microscopy. The outlines of these techniques, which should be utilized in various fields of biological and medical research, are described in detail

Complementarity of radioautographic and immunohistochemical techniques for localizing neuroreceptors at the light and electron microscopy level

To assess relationships between neuropeptide-binding sites and receptor proteins in rat brain, the distribution of radioautographically labeled somatostatin and neurotensin-binding sites was compared to that of immunolabeled sst2A and NTRH receptor subtypes, respectively. By light microscopy, immunoreactive sst2A receptors were either confined to neuronal perikarya and dendrites or diffusely distributed in tissue. By electron microscopy, areas expressing somatodendritic sst2A receptors displayed only low proportions of membrane-associated, as compared to intracellular, receptors. Conversely, regions displaying diffuse sst2A labeling exhibited higher proportions of membrane-associated than intracellular receptors. Furthermore, the former showed only low levels of radioautographically labeled somatostatin-binding sites whereas the latter contained high densities of somatostatin-binding suggesting that membrane-associated receptors are preferentially recognized by the radioligand. In the case of NTRH receptors, there was a close correspondence between the light microscopic distribution of NTRH immunoreactivity and that of labeled neurotensin-binding sites. Within the substantia nigra, the bulk of immuno- and autoradiographically labeled receptors were associated with the cell bodies and dendrites of presumptive DA neurons. By electron microscopy, both markers were detected inside as well as on the surface of labeled neurons. At the level of the plasma membrane, their distribution was highly correlated and characterized by a lack of enrichment at the level of synaptic junctions and by a homogeneous distribution along the remaining neuronal surface, in conformity with the hypothesis of an extra-synaptic action of this neuropeptide. Inside labeled dendrites, there was a proportionally higher content of immunoreactive than radiolabeled receptors. Some of the immunolabeled receptors not recognized by the radioligand were found in endosome-like organelles suggesting that, as in the case of sst2A receptors, they may have undergone endocytosis subsequent to binding to the endogenous peptide

A comparative analysis of preprocessing techniques of cardiac event series for the study of heart rhythm variability using simulated signals

In the present study, using noise-free simulated signals, we performed a comparative examination of several preprocessing techniques that are used to transform the cardiac event series in a regularly sampled time series, appropriate for spectral analysis of heart rhythm variability (HRV). First, a group of noise-free simulated point event series, which represents a time series of heartbeats, was generated by an integral pulse frequency modulation model. In order to evaluate the performance of the preprocessing methods, the differences between the spectra of the preprocessed simulated signals and the true spectrum (spectrum of the model input modulating signals) were surveyed by visual analysis and by contrasting merit indices. It is desired that estimated spectra match the true spectrum as close as possible, showing a minimum of harmonic components and other artifacts. The merit indices proposed to quantify these mismatches were the leakage rate, defined as a measure of leakage components (located outside some narrow windows centered at frequencies of model input modulating signals) with respect to the whole spectral components, and the numbers of leakage components with amplitudes greater than 1%, 5% and 10% of the total spectral components. Our data, obtained from a noise-free simulation, indicate that the utilization of heart rate values instead of heart period values in the derivation of signals representative of heart rhythm results in more accurate spectra. Furthermore, our data support the efficiency of the widely used preprocessing technique based on the convolution of inverse interval function values with a rectangular window, and suggest the preprocessing technique based on a cubic polynomial interpolation of inverse interval function values and succeeding spectral analysis as another efficient and fast method for the analysis of HRV signals

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

Measuring higher order optical aberrations of the human eye: techniques and applications

In the present paper we discuss the development of "wave-front", an instrument for determining the lower and higher optical aberrations of the human eye. We also discuss the advantages that such instrumentation and techniques might bring to the ophthalmology professional of the 21st century. By shining a small light spot on the retina of subjects and observing the light that is reflected back from within the eye, we are able to quantitatively determine the amount of lower order aberrations (astigmatism, myopia, hyperopia) and higher order aberrations (coma, spherical aberration, etc.). We have measured artificial eyes with calibrated ametropia ranging from +5 to -5 D, with and without 2 D astigmatism with axis at 45º and 90º. We used a device known as the Hartmann-Shack (HS) sensor, originally developed for measuring the optical aberrations of optical instruments and general refracting surfaces in astronomical telescopes. The HS sensor sends information to a computer software for decomposition of wave-front aberrations into a set of Zernike polynomials. These polynomials have special mathematical properties and are more suitable in this case than the traditional Seidel polynomials. We have demonstrated that this technique is more precise than conventional autorefraction, with a root mean square error (RMSE) of less than 0.1 µm for a 4-mm diameter pupil. In terms of dioptric power this represents an RMSE error of less than 0.04 D and 5º for the axis. This precision is sufficient for customized corneal ablations, among other applications.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

Influência da adição de íons Ca++ e Mg++ na dureza de géis de concentrado proteíco de soro de leite bovino

Inúmeros estudos vêm sendo realizados com o objetivo de compreender o comportamento das proteínas do Concentrado Protéico de Soro (CPS). A capacidade destas proteínas em formar géis estáveis a temperaturas entre 70ºC e 90ºC, é uma propriedade funcional importante para a confecção de vários produtos alimentícios, tais como: produtos de padaria, cárneos, texturizados e lácteos. A concentração protéica, pH, composição iônica e temperatura podem ser controladas para obter um gel com as características desejadas. Neste trabalho foi investigado o efeito dos íons metálicos bivalentes Ca++ e Mg++, na propriedade de dureza de géis de CPS induzidos termicamente. Dispersões protéicas a 6; 7 e 8% de proteína do CPS a pH 6,3 com 0; 7,5; 15; 30 e 75mm de CaCl2 ou MgCl2 foram aquecidas a 75ºC por 45 min, resfriadas a 4ºC por 12 horas, e os géis avaliados em um texturômetro TA-XT2. Os géis formados com maiores concentrações do sal adicionado obtiveram maiores valores de dureza na faixa estudada. Nas concentrações de 15 a 75mM os valores de dureza dos géis com a adição de CaCl2 foram significativamente maiores (p<0,05) do que com adição de MgCl2. Na concentração de 8% de proteínas do CPS ambos os tratamentos atingem o maior valor de dureza (p<0,05) a 30mM do sal adicionado. O cálcio iônico mostrou ter um importante papel na formação do gel de proteínas do soro.

Perfil de textura e capacidade de retenção de água de géis ácidos de concentrado protéico de soro de leite

A capacidade dos concentrados protéicos de soro de leite (CPS) de formar géis é importante propriedade funcional. O objetivo deste estudo foi avaliar a influência das variáveis concentração de proteína, pH, temperatura e tempo de desnaturação, nos intervalos de 8 a 12%; 4,0 a 5,2; 81 a 89ºC e 15 a 27 minutos, respectivamente, no perfil de textura e capacidade de retenção de água de géis ácidos de CPS. O perfil de textura foi determinado em texturômetro TAXT2 e a capacidade de retenção de água avaliada através da umidade espremível dos géis. O delineamento estatístico foi um planejamento fatorial 2(4) completo. Os géis de CPS apresentaram os maiores valores de firmeza, coesividade, elasticidade e capacidade de retenção de água, de maneira geral, nas maiores faixas de concentração protéica, tempo e temperatura de desnaturação. Com relação a variável pH, géis formados em pH 4,0 apresentaram-se mais elásticos e com maior capacidade de retenção de água, enquanto que os géis formados em pH 4,9 a 5,2 mostraram-se mais firmes e coesos.

Perfil de textura e umidade espremível de géis do albume de ovos recobertos com soro de leite

A gelatinização é uma importante propriedade funcional das proteínas alimentares, devido ao seu grande potencial de uso nos alimentos estruturados. As proteínas da clara do ovo de galinha têm sido extensivamente usadas como ingredientes em alimentos processados. O objetivo deste trabalho foi avaliar as mudanças no pH, no perfil de textura e na umidade espremível de géis de clara de ovos de galinha com e sem cobertura de concentrado protéico de soro de leite, armazenados a 25ºC, por 3, 7, 10, 14, 21 e 28 dias. A dureza do gel do albume de ovos sem cobertura foi maior do que a de ovos recobertos, durante todos os períodos de armazenamento. Não houve efeito do tempo de armazenamento na dureza dos géis dos ovos sem cobertura. Em ovos cobertos, a regressão linear explicou 60% do comportamento da dureza em relação ao período de armazenamento. No caso da elasticidade, não houve interação entre período de armazenamento e a cobertura. Houve diferença entre as médias dentro de cada período, mas não durante o armazenamento. A maior elasticidade foi dos géis de ovos sem cobertura, comparados com os géis de ovos recobertos. O índice de coesividade e a mastigabilidade de géis de ovos sem cobertura foi maior que o de géis de ovos recobertos, em todos os períodos de armazenamento. A percentagem de umidade espremível (UE) de géis de clara de ovos recobertos foi maior do que a de ovos sem cobertura em todo o período de estocagem.

Estudo reológico da gelificação induzida pelo calor de proteínas do soro do leite e dos géis resultantes sob condições variadas de pH

Medidas reólogicas sob cisalhamento oscilatório foram realizadas em reômetro de tensão e deformação controladas com suspensões de concentrado de proteínas do soro do leite (WPC) a 10% (m/m) em água e a diferentes condições de pH (pH 4,0, 4,6 e 7,0). O processo de gelificação induzida pelo calor foi investigado, assim como as propriedades viscoelásticas dos géis formados a 80°C e daqueles formados após o decréscimo da temperatura a 20°C. Foi verificado que, em presença de teores significativos de sais, procedentes do próprio soro, a concentração usada nos experimentos foi suficiente para a formação de géis macroscópicos, e que o pH exerce papel importante na formação e na natureza estrutural dos géis.

Estabilidade de géis de amido de milho normal, ceroso e com alto teor de amilose adicionados de gomas guar e xantana durante os processos de congelamento e descongelamento

O objetivo do presente trabalho foi estudar os efeitos das gomas guar e xantana sobre a estabilidade dos géis de amido de milho normal, ceroso e com alto teor de amilose submetidos aos processos de congelamento e descongelamento. Os géis desses amidos, com concentração total de sólidos de 10% e adicionados das gomas (0,15; 0,50; 0,85 e 1%), foram submetidos a 5 ciclos de congelamento (20 horas a -18 °C) e descongelamento (4 horas a 25 °C), com exceção dos géis com alto teor de amilose, que foram submetidos a apenas 1 ciclo, devido à perda da estrutura de gel. A determinação da sinérese (porcentagem de água liberada) foi realizada pela diferença entre a massa inicial e a massa final das amostras. O gel de amido de milho normal liberou 74,45% de água, sendo que a adição de 1% da goma xantana reduziu significativamente a sinérese para 66,43%. A adição de 0,85 e 1% da goma xantana também reduziu a sinérese dos géis de amido ceroso. O menor teor de sinérese foi obtido com a utilização de 1% de goma xantana ao gel de amido de milho com alto teor de amilose, evidenciando a ação crioprotetora desta goma.

Evaluation of the quality of pet foods using fast techniques and official methods

This paper was designed to evaluate the rancidity of 18 pet food samples using the Diamed FATS kits and official AOCS methods for the quantification of free fatty acids, peroxide value and concentrations of malonaldehyde and alkenal in the lipid extracted. Although expiration dates have passed, the samples presented good quality evidencing little oxidative rancidity. The results of this study suggest that the Brazilian pet food market is replete with products of excellent quality due to the competitiveness of this market sector.

Optimization of image analysis techniques for quality assessment of whole-wheat breads made with fat replacer

The cellular structure of healthy food products, with added dietary fiber and low in calories, is an important factor that contributes to the assessment of quality, which can be quantified by image analysis of visual texture. This study seeks to compare image analysis techniques (binarization using Otsu’s method and the default ImageJ algorithm, a variation of the iterative intermeans method) for quantification of differences in the crumb structure of breads made with different percentages of whole-wheat flour and fat replacer, and discuss the behavior of the parameters number of cells, mean cell area, cell density, and circularity using response surface methodology. Comparative analysis of the results achieved with the Otsu and default ImageJ algorithms showed a significant difference between the studied parameters. The Otsu method demonstrated the crumb structure of the analyzed breads more reliably than the default ImageJ algorithm, and is thus the most suitable in terms of structural representation of the crumb texture.

Several techniques for the preparation of flour from carcasses of the Pantanal alligator (Caiman crocodilus yacare)

AbstractFlour prepared via current assay methodologies from the carcasses of the Pantanal alligator (Caiman crocodilus yacare) was analyzed for its chemical composition, minerals, fatty acids, and sensorial profile. Carcasses of the Pantanal alligator, originating from the Coocrijapan scientific zoo, Cáceres MT Brazil, were utilized. The carcasses were cooked for 60 minutes in a pressure cooker with water containing 2% salt and 5% chimichurri. The cooked carcasses were then ground and the mass was used for the manufacture of flour via three techniques: non-smoked, hot-smoked, and liquid-smoked. After each technique, the carcasses were dehydrated at 60ºC for 3h and were ground. Alligator flour was then produced. The moisture of liquid-smoked flour (10.97%) was higher than that of non-smoked flour (3.78%) and hot-smoked flour (4.43%). The flours provided high protein (57.11% - 58.27%) and ash (23.45 – 26.42%) rates, and were predominantly calcium (6.77% - 7.69%), phosphorus (3.67% - 4.05%), and iron (73.13 – 273.73 ppm/100 mg). Smoked-flour had a better acceptance rate by tasters when compared to non-smoked flour. Results show that flours produced from alligator carcasses had high protein, ash, and mineral rates and a reasonable acceptability by most tasters.

Effects of different packaging techniques on the microbiological and physicochemical properties of coated pumpkin slices

Abstract In this study the effects of zein film coating along with benzoic acid on the quality of sliced pumpkin samples, which were packaged with different techniques were investigated. The samples were allocated into different groups and were treated with different processes. Following processing, the samples were stored at +4 °C for twenty days. Physicochemical and microbiological analyses were carried out on the samples once every five days during the storage period. According to color analysis, the L* value was observed to have significantly decreased in the processed and packaged samples in comparison with the control group. Besides, a* and b* values increased in all groups. It was determined that zein film alone did not exhibit the expected effectiveness against moisture loss in the samples. According to the results of microbiological analysis, a final decrease at approximately 1.00 log level was determined in total count of mesophilic aerobic bacteria (TMAB) in the group which was vacuum packaged in PVDC with zein coating when compared with the initial TMAB. Furthermore, no molding occurred in zein-coated group on the last day of the storage period, while massive mold growth was noted in the group which was packaged without any pretreatment procedure.