Iterative image transformations for an automatic screening of cervical smears.

The new generation of image analysis systems permits the use of iterative image transformations. It is now possible to construct algorithms where the elementary steps are not arithmetic operations but image transformations. This will be illustrated by two examples. In the first, the absorption image of Feulgen Stained nuclei is processed by contrast algorithms in order to detect suspect cells. In the second, free lying cells are separated from overlapping cells and other artefacts by the use of skeletonization procedures.     

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.     

Drug screening for influenza neuraminidase inhibitors

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC50 was from 0.1 μmol/L to 3μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The resuit indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Sequence scanning chicken cosmids: a methodology for genome screening

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.     

The border effect in surname structure: an Italian-Slovenian case study

Population surname structure in northeastern Italy municipalities, in which small Slovenian-speaking groups are present, are compared with neighboring populations of both Italian and Slovenian nationality. In the early 20th century those municipalities were part of the Italian territory and underwent a massive Italianization process that led to the mutation of the original Slovenian surnames into a derived Italian form. In order to track surname relationships among the examined communities, two different similarity analyses were performed: the first using the surnames as they are recorded in the Italian and Slovenian official telephone directories, and the second restoring the Italianized Slovenian surnames to their original form and then recalculating the populations' similarity. Both comparisons evidence a separation that depends on nationality, which is less marked after restoring surnames to their original form. Geographical elements, older historical events, and socioeconomic traits allow interpretation of these results.     

The ferret: an animal model to study influenza virus

There has been much critical influenza research conducted in a little-known laboratory animal--the ferret. The authors review some of these findings, discuss the reasons the ferret often becomes a model for influenza infection, and compare the ferret with other animal models.     

Psychological consequences for parents of false negative results on prenatal screening for Down's syndrome: retrospective interview study

ObjectiveTo determine the psychological consequences for parents of children with Down''s syndrome of having received a false negative result on prenatal screening.DesignComparison of adjustment of parents who received a false negative result with that of parents not offered a test and those who declined a test.SettingParents were interviewed in their own homes.ParticipantsParents of 179 children with Down''s syndrome (mean age 4 (range 2-6) years).ResultsOverall, regardless of screening history, parents adjusted well to having a child with Down''s syndrome. Compared with mothers who declined a test, mothers in the false negative group had higher parenting stress (mean score 81.2 v 71.8, P=0.016, 95% confidence interval for the difference 1.8 to 17.0) and more negative attitudes towards their children (124.9 v 134.2, P=0.009, −16.2 to −2.4). Fathers in the false negative group had higher parenting stress test scores (77.8 v 70.0, P=0.046, 1.5 to 14.2) than fathers not offered a test. Mothers in the false negative group were more likely to blame others for the outcome than mothers who had not been offered the test (28% v 13%, P=0.032, 3% to 27%). Mothers and fathers in the false negative group were more likely to blame others for this outcome than parents who had declined a test (mothers 28% v 0%, P=0.001, 19% to 37%; fathers 27% v 0%, P=0.004, 17% to 38%). Blaming others was associated with poorer adjustment for mothers and fathers.ConclusionsA false negative result on prenatal screening seems to have a small adverse effect on parental adjustment evident two to six years after the birth of an affected child.     

Structure of an influenza neuraminidase-diabody complex by electron cryomicroscopy and image analysis

The structure of a complex between a bivalent diabody and its antigen, influenza neuraminidase, has been determined by electron cryomicroscopy of single particles and image analysis. A three-dimensional reconstruction has been interpreted in terms of high-resolution X-ray models of the component proteins. The complex consists of two neuraminidase tetramers cross-linked by four diabodies with 422 point symmetry. The structure and symmetry of the complex is determined uniquely by packing constraints consistent with the maximum possible number of diabody cross-links. Diabodies may provide a useful approach to the structure determination of small proteins by incorporating the proteins into large symmetric complexes followed by single-particle electron microscopy.     

The scanning model for translation: an update

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.     

Increase of sensitivity of sputum cytology using high-resolution image cytometry: field study results

Lung cancer remains the leading cause of cancer deaths in the developed world. There is no widely accepted method to screen for this cancer. The most commonly used method remains conventional sputum cytology, but this method is hampered by low sensitivity. We tested the hypothesis that sensitivity of sputum cytology for early lung cancer can be greatly improved by using image analysis of sputum cells, at a modest reduction of specificity.The study was double-blinded and used sputum samples from subjects with well-characterized clinical diagnoses. There were 177 cancers, 98 dysplasias, and 558 normals. The study samples were separated into two independent sets: training set and test set. Sputum samples were collected prospectively from subjects with a high probability of having lung cancer. Seven institutions from five countries participated in the study. All subjects had complete clinical diagnoses which included, as a minimum, negative chest x-rays for all negative cancers, while all cancers had confirmed tissue pathology. Samples were prepared according to the Saccomanno method. For conventional cytology, slides were stained using Papanicolaou stain. For image analysis, slides were stained using a DNA-specific (Feulgen-Thionin) stain. An automated, high-resolution image cytometer was used for measurements.At 90% specificity, sensitivity of 60% can be achieved for adenocarcinoma, compared to only 14% sensitivity of conventional cytology (at 99% specificity). Similarly, 45% sensitivity at 90% specificity can be reached for stages 0 and I lung cancer, compared to only 14% (at 99% specificity) using conventional cytology.Cytometry combined with conventional cytology shows an increase in sensitivity to early-stage cancer and to adenocarcinomas compared to conventional cytology alone. While the results are encouraging, the sensitivity to detect early lung cancer should be further improved to 70-80% at 90-95% specificity before this test can be considered for screening of high-risk individuals for lung cancer. Cytometry (Clin. Cytometry) 50:168-176, 2002.     

Genome scanning for linkage: an overview.

Several different methods for linkage analysis are shown to arise from a single likelihood function L for the observed allele-sharing data at multiple markers in a chromosomal region. These include classical parametric lod score methods, nonparametric or "model-free" affected pedigree-member (APM) methods, and the Gaussian process method. Setting the methods in the context of the likelihood function L clarifies their underlying assumptions. A test statistic derived from L, the efficient score statistic, is introduced. It is asymptotically equivalent to the lod score, but it can be easier to compute when the penetrances and frequencies of alleles of the trait gene are not known. APM test statistics and the Gaussian lod score are shown to be special cases of efficient score statistics. This unified framework facilitates exploration of a range of models for the effects of a putative trait-predisposing gene, and it facilitates sensitivity analyses to examine the consequences of model misspecification.     

Preparing for an influenza pandemic: ethical issues

In the near future, experts predict, an influenza pandemic will likely spread throughout the world. Many countries have been creating a contingency plan in order to mitigate the severe health and social consequences of such an event. Examination of the pandemic plans of Canada, the United Kingdom and the United States, from an ethical perspective, raises several concerns. One: scarcity of human and material resources is assumed to be severe. Plans focus on prioritization but do not identify resources that would be optimally required to reduce deaths and other serious consequences. Hence, these plans do not facilitate a truly informed choice at the political level where decisions have to be made on how much to invest now in order to reduce scarcity when a pandemic occurs. Two: mass vaccination is considered to be the most important instrument for reducing the impact of infection, yet pandemic plans do not provide concrete estimates of the benefits and burdens of vaccination to assure everyone that the balance is highly favorable. Three: pandemic plans make extraordinary demands on health care workers, yet professional organizations and unions may not have been involved in the plans' formulation and they have not been assured that authorities will aim to protect and support health care workers in a way that corresponds to the demands made on them. Four: all sectors of society and all individuals will be affected by a pandemic and everyone's collaboration will be required. Yet, it appears that the various populations have been inadequately informed by occasional media reports. Hence, it is essential that plans are developed and communication programs implemented that will not only inform but also create an atmosphere of mutual trust and solidarity; qualities that at the time of a pandemic will be much needed.     

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.     

A Computer-based image analysis system for biocide screening

A computer-based image analysis system that measures the behavioral response of the marine diatom Amphora coffeaeformis to potentially toxic challenges is described. At any one time the system is capable of measuring in a population of about 80 cells, changes in the number motile, their speed, direction, and if they are turning, their angular velocity and acceleration. Chemical compounds that interfere with the generation of energy, protein or glycoprotein synthesis, calcium homeostasis and cytoskeletal activity, change the motile behavior of the cell. A pre-requisite of motility is that the cells be attached to a substratum. It is proposed that the system, which requires only a 1-2 minute data collection period per replicate, could be used to screen for potential antifouling activity in chemical agents, and it is speculated that it could be used also for measuring the physicochemical interaction between attached motile organisms and their substratum, as well as in environment toxicology measurements.     

Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: a differential scanning calorimetry study

Peanut agglutinin is a homotetrameric legume lectin. The crystal structure of peanut agglutinin shows that the four subunits associate in an unusual manner, giving rise to open quaternary structure [Banarjee, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 227-231]. The thermal unfolding of peanut agglutinin has been characterized by differential scanning calorimetry and gel filtration to elucidate its thermal stability and its mode of oligomerization. The unfolding process is reversible and could be described by a three-state model with two transitions occurring at around 331 and 336 K. For the tetramer, the ratio of DeltaHc/DeltaHv for the first transition is close to 4 and for the second transition is close to 0.25, suggesting that 4 and 0.25 cooperative unit(s) of the tetramer are involved in the first and second transitions, respectively. The agreement between the model-independent DeltaHv(S) determined from the values of the temperatures of the peak maximum (Tp1) with the protein concentration with the values of DeltaHv obtained from the fit of the data to the transition confirms that the first peak is associated with the dissociation of peanut agglutinin tetramers (A4) to "folded" monomers (4A), whereas the second peak describes the unfolding (4U) of these monomers. The overall process for the thermal unfolding of peanut agglutinin could therefore be summarized as A4 <==> 4A <==> 4U. Gel filtration studies confirm this process, as peanut agglutinin elutes as a tetramer up to 50 degrees C, and at and above 56 degrees C (Tm of first transition), it elutes at a position commensurate with that of the folded monomer of peanut agglutinin. The unfolding behavior of peanut agglutinin in the presence of saturating amounts of carbohydrate ligands is similar to that observed for the unligated form. The temperature of maximal stability of the peanut agglutinin tetramer at pH 7.4 is calculated to be around 33 degrees C with a maximal free energy of stabilization of 8.70 kcal/mol. The results demonstrate that unfolding of peanut agglutinin goes through two distinct phases with folded monomer being the intermediate.     

Age is an independent risk factor for left atrial dysfunction: results from an observational study

Introduction. The degenerative changes of myocardial tissue are thought to influence left atrial (LA) function. Changes of left atrial function are generally due to changes in left ventricle (LV) compliance. But valvular dysfunction and hypertension as comorbidity cannot be ignored. Women have a different clinical profile compared with men concerning the risk of heart failure. We investigated the influence of increasing age and gender corrected for comorbidity, on left atrial function. Methods. Using an open access echocardiography database, supplemented with additional LA function measurements, we defined three different LA function parameters. Odds ratios (OR) were calculated to reproduce the relation between age, gender and LA function. The association between age, gender and LA function was estimated, and corrected for comorbid conditions as valve disease, high blood pressure and LV dysfunction, using logistic regression. Results. Higher age was positively correlated with increased LA volume, decreased ejection fraction and increased LA kinetic energy. Age per decade increase, corrected for comorbidity, resulted in an increased risk of LA dysfunction (OR between 1.5 and 1.9). Gender had little influence on LA function parameters except for LA maximal volume. Men had a significantly larger LA maximal volume compared with women. Conclusions. In this open access echocardiography database, increasing age was correlated with LA dysfunction. Age per decade increase, corrected for comorbid conditions such as mitral and aortic valve disease, hypertension and heart failure, is an independent risk factor for LA dysfunction. The gender influence on LA dysfunction seems to be limited. (Neth Heart J 2010;18:243-7.)     

Thermal precipitation fluorescence assay for protein stability screening

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies.