Cell block as an adjunct to conventional Papanicolaou smear for diagnosis of cervical cancer in resource-limited settings

OBJECTIVE: To assess the utility of indigenously prepared cell blocks (CBs) as an adjunct to a conventional smear test in providing a reliable diagnosis of clinically suspicious cervical cancer in resource-limited settings. METHODS: Eighty-six clinically suspicious cervical cancer cases underwent a conventional smear test, CB preparation from residual cellular samples and biopsies at the same sitting. Correlations were performed between these modalities in order to derive the sensitivity and specificity of the CB technique to diagnose cervical cancer. OBSERVATION & RESULTS: Out of 86 clinically suspicious cervical cancers, 72 (83.7%), 70 (81.4%) and 67 (77.9%) cases were diagnosed as malignant on tissue biopsies, CBs and smears respectively. CB-biopsy agreement in the diagnosis of malignancy was feasible in 87.5% of the cases while CB-Pap smear agreement was feasible in 92.5% of the cases. Sensitivity and specificity of CB preparation to diagnose malignancy was 92.5% and 100%, respectively, when the smear was taken as the reference test (excluding the unsatisfactory smears). When biopsy was taken as the gold standard, the sensitivity and specificity of CBs were 87.5% and 100% respectively (excluding the unsatisfactory biopsies). In 8/19 cases where the smear diagnoses were either unsatisfactory or atypical squamous cells/atypical glandular cells, CBs picked up malignant lesions. CONCLUSION: CBs prepared from the residual cellular sample of conventional cervical scrapes augment the sensitivity of the smear test. When used as an adjunct to the smear, CBs aid in providing a reliable diagnosis of cervical cancer in the majority of the clinically suspected cases and thus the biopsy load can be reduced significantly in resource-poor settings.     

Tannic Acid as an Electron Microscope Tracer for Permeable Cell Membranes

To recognize damaged cells in preparations for transmission electron microscopy, high molecular weight (1700 MW) tannic acid (1-4%) has been added to glutaraldehyde fixing solutions. During fixation, the tannic acid penetrates only those cells whose plasma membranes were previously damaged. It enhances the electron density of the injured cells, which become clearly distinguishable from the undamaged ones. As a tracer tannic acid shows great advantages over either lanthanum hydroxide, ruthenium red, or horseradish peroxidase. It diffuses evenly throughout the tissue block and is not removed by preparative steps. Furthermore, it is also a good tracer at the light microscope level.     

Confined-pyrolysis as an experimental method for hydrothermal organic synthesis

A closed pyrolysis system has been developed as a tool for studying the reactions of organic compounds under extreme hydrothermal conditions. Small high pressure stainless steel vessels in which the ratio of sediment or sample to water has been adjusted to eliminate the headspace at peak experimental conditions confines the organic components to the bulk solid matrix and eliminates the partitioning of the organic compounds away from the inorganic components during the experiment. Confined pyrolysis experiments were performed to simulate thermally driven catagenetic changes in sedimentary organic matter using a solids to water ratio of 3.4 to 1. The extent of alteration was measured by monitoring the steroid and triterpenoid biomarkers and polycyclic aromatic hydrocarbon distributions. These pyrolysis experiments duplicated the hydrothermal transformations observed in nature. Molecular probe experiments using alkadienes, alkenes and alkanes in H₂O and D₂O elucidated the isomerization and hydrogenation reactions of aliphatic compounds and the competing oxidative reactions occurring under hydrothermal conditions. This confined pyrolysis technique is being applied to test experiments on organic synthesis of relevance to chemical evolution for the origin of life.     

A heat polyethylene glycol procedure as an alternative method for plasmafractionation

Polyethylenglycol 4000 was used in a combined process of fractionation at normal temperature for preparing gammaglobulin and albumin in a heat denaturation process. Albumin can be gained from human plasma with a high yield and purity by using a heat procedure at the presence of polyethylglycol 4000 and sodium octanate at 75 degrees C. The concentration of polyethylglycol 4000 varies during this heat procedures from 3-10% W/V.     

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.     

‘Collar propagation’ as an alternative propagation method for rhizomatous miscanthus

The demand for perennial nonfood crops, such as miscanthus, is increasing steadily, as fossil resources are replaced by biomass. However, as the establishment of miscanthus is very expensive, its cultivation area in Europe is still small. The most common propagation method for miscanthus is via rhizomes, the harvesting of which is very labour‐intensive. Seed propagation is promising, but not suitable for sterile genotypes. In this study, a new vegetative propagation method, ‘collar propagation’, was tested in field and controlled environment studies. Collars are built at the junction between rhizome and stem. They can be harvested in a less destructive way than rhizomes by pulling out the stems from winter‐dormant miscanthus plants. One genotype of each of the species M. sacchariflorus, M. × giganteus, M. sinensis in combination with three fragment types (collars, rhizomes, collars + rhizomes) were tested for establishment success and plant performance. The performance (e.g. dry matter yield) of collar‐propagated plants was either better than or not significantly different from rhizome‐propagated plants. Pregrown plantlets transplanted into the field showed no significant differences in establishment success between the fragments within a genotype. When directly planted into the field however, the fragment ‘rhizome+collar’ had a significantly better establishment success than the other two. The winter survival rate of the fragment ‘rhizome+collar’ was 70% for M. sacchariflorus and 75% for M. × giganteus. Emergence success from collar‐derived plants was not affected by harvest date (harvested monthly from November to February). This study showed that miscanthus propagation via collars is feasible and a promising alternative to rhizome propagation, as the multiplication rate of collars is comparable to that of rhizome propagation. Collar propagation is the more suitable method for the tested genotypes of the species M. sachariflorus and M. × giganteus, but not for M. sinensis genotypes, which may be better propagated by seeds.     

Liquid- and Air-Filled Catheters without Balloon as an Alternative to the Air-Filled Balloon Catheter for Measurement of Esophageal Pressure

Background

Measuring esophageal pressure (P_es) using an air-filled balloon catheter (BC) is the common approach to estimate pleural pressure and related parameters. However, P_es is not routinely measured in mechanically ventilated patients, partly due to technical and practical limitations and difficulties. This study aimed at comparing the conventional BC with two alternative methods for P_es measurement, liquid-filled and air-filled catheters without balloon (LFC and AFC), during mechanical ventilation with and without spontaneous breathing activity. Seven female juvenile pigs (32–42 kg) were anesthetized, orotracheally intubated, and a bundle of an AFC, LFC, and BC was inserted in the esophagus. Controlled and assisted mechanical ventilation were applied with positive end-expiratory pressures of 5 and 15 cmH₂O, and driving pressures of 10 and 20 cmH₂O, in supine and lateral decubitus.

Main Results

Cardiogenic noise in BC tracings was much larger (up to 25% of total power of P_es signal) than in AFC and LFC (<3%). Lung and chest wall elastance, pressure-time product, inspiratory work of breathing, inspiratory change and end-expiratory value of transpulmonary pressure were estimated. The three catheters allowed detecting similar changes in these parameters between different ventilation settings. However, a non-negligible and significant bias between estimates from BC and those from AFC and LFC was observed in several instances.

Conclusions

In anesthetized and mechanically ventilated pigs, the three catheters are equivalent when the aim is to detect changes in P_es and related parameters between different conditions, but possibly not when the absolute value of the estimated parameters is of paramount importance. Due to a better signal-to-noise ratio, and considering its practical advantages in terms of easier calibration and simpler acquisition setup, LFC may prove interesting for clinical use.     

Development of an experiment-split method for benchmarking the generalization of a PTM site predictor: Lysine methylome as an example

Many computational classifiers have been developed to predict different types of post-translational modification sites. Their performances are measured using cross-validation or independent test, in which experimental data from different sources are mixed and randomly split into training and test sets. However, the self-reported performances of most classifiers based on this measure are generally higher than their performances in the application of new experimental data. It suggests that the cross-validation method overestimates the generalization ability of a classifier. Here, we proposed a generalization estimate method, dubbed experiment-split test, where the experimental sources for the training set are different from those for the test set that simulate the data derived from a new experiment. We took the prediction of lysine methylome (Kme) as an example and developed a deep learning-based Kme site predictor (called DeepKme) with outstanding performance. We assessed the experiment-split test by comparing it with the cross-validation method. We found that the performance measured using the experiment-split test is lower than that measured in terms of cross-validation. As the test data of the experiment-split method were derived from an independent experimental source, this method could reflect the generalization of the predictor. Therefore, we believe that the experiment-split method can be applied to benchmark the practical performance of a given PTM model. DeepKme is free accessible via https://github.com/guoyangzou/DeepKme.     

Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method

We describe a simple and effective method for inhibition of endogenous peroxidase activity in the immunoperoxidase technique. Specimens are pre-treated with a mixture of azide and hydrogen peroxide, which is then followed by an indirect immunoperoxidase procedure. Comparison studies showed no significant loss of antigenicity or morphological details by this pre-treatment. The method is most useful for evaluating cell-specific antigens on specimens that have abundant endogenous peroxidase activity, such as blood and bone marrow.     

Neonatal thyroid-stimulating hormone screening as an indirect method for the assessment of iodine deficiency in Estonia

According to neonatal thyroid screening the incidence of congenital hypothyroidism in Estonia is 1:2,860. Transient hyperthyrotropinemia with a raised thyroid-stimulating hormone level of 5 microU/ml occurred in 17.7% of infants and was not associated with low birth weight, small birth length, low gestational age or congenital anomalies. Based on WHO criteria (WHO/UNICEF, 1994) it corresponds to mild iodine deficiency in Estonia (3% or less is in iodine-sufficient areas). This is in agreement with the previously reported median urinary iodine content of 65 microg/l in children. The frequency of infants with TSH >5 microU/ml was 16.4, 21 and 17. 2% in three regions (north, central and south) of Estonia, respectively, indicating mild to moderate iodine deficiency. These findings show the possibility of using the results of newborn screening for congenital hypothyroidism to assess the severity of iodine deficiency in Estonia. The introduction of universal iodine prophylaxis is recommended.     

A novel method for detection and counting of single bacteria in a wide field using an ultra-high-sensitivity TV camera without a microscope

We describe a novel method for enumeration of bacteria, based on the principle that small, light emitting particles on a flat surface can be easily and rapidly detected and counted using an ultra-high-sensitivity TV camera. To test this method, we obtained TV images of individual cells of a luminous bacterium on a membrane filter without the use of a microscope. The positions of the luminous points in the TV images were almost the same as the positions of the bacterial colonies after growth. Our results show that the single cells can be efficiently detected and counted by our method if they emit light or can be stimulated to emit light.     

Three dimensional printing as an effective method of producing anatomically accurate models for studies in thermal ecology

Hollow copper models painted to match the reflectance of the animal subject are standard in thermal ecology research. While the copper electroplating process results in accurate models, it is relatively time consuming, uses caustic chemicals, and the models are often anatomically imprecise. Although the decreasing cost of 3D printing can potentially allow the reproduction of highly accurate models, the thermal performance of 3D printed models has not been evaluated. We compared the cost, accuracy, and performance of both copper and 3D printed lizard models and found that the performance of the models were statistically identical in both open and closed habitats. We also find that 3D models are more standard, lighter, durable, and inexpensive, than the copper electroformed models.     

Oil-stop method as an alternative to filtration for transport studies on plasma membrane vesicles. A preliminary report

The oil-stop procedure applied in transport studies on whole cells was adapted for isolated plasma membrane vesicles. The advantages of the proposed technique with respect to the commonly used filtration method are the reduction of the period of the termination step, the increase in the allowed membrane content of the samples and the absence of the leakage of substrates from the intravesicular space.     

Synthesis of carboxyacyl derivatives of phosphatidylethanolamine and use as an efficient method for conjugation of protein to liposomes

Carboxyacyl derivatives of phosphatidylethanolamine with different chain length were synthesized. These compounds were generally prepared by conversion of an appropriate dicarboxylic acid to its anhydride with dicyclohexylcarbodiimide, then reaction with phosphatidylethanolamine (PE) and triethylamine, followed by acidification. These derivatives, when incorporated into liposomes, were highly efficient in conjugating protein to liposomes. Liposomes with PE amide derivative incorporated were activated with water-soluble carbodiimide, and subsequently reacted with protein. The protein to lipid coupling efficiency was shown to be dependent on the chain length of the derivative, and the optimum coupling efficiency was achieved with PE amide of 1,12-dodecanedicarboxylic acid. Up to 60% covalent coupling efficiency of mouse IgG to liposomes was demonstrated with little non-covalent binding. This method will be of great importance in the liposome-targeting field.     

Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells

The influence of pH on the adhesion of two Lactobacillus strains to Caco-2 human intestinal cells was investigated. One strain, Lactobacillus johnsonii La1, was adherent at any pH between 4 and 7. The other one, L. acidophilus La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for L. johnsonii La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the L. johnsonii La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of L. johnsonii La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by L. johnsonii La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of L. johnsonii La1 to human Caco-2 cells involves LTA.