Expanding application of digital pathology in Japan - from education,telepathology to autodiagnosis

Background

Digital pathology, i.e., applications of digital information technologies to pathology practice, has been expanding in the recent decades and the mode of pathology diagnostic practice is changing with enhanced precision. In the present study the changing processes of digital pathology in Japan were investigated and trends to future were discussed.

Methods

The changing status of digital pathology was investigated through reviewing the records of annual meetings of the Japanese Research Society of Telepathology and Pathology Informatics (JRST-PI) and of the Japanese pathology related medical and informatics journals. The results of the Japanese questionnaire survey conducted in 2008-2009 on telepathology and virtual slide were also reviewed. In addition effectiveness of an experimental automatic pathology diagnostic aid system using computer artificial intelligence was investigated by checking its rate of correct diagnosis for given prostate carcinoma digital images.

Results

Telepathology played a central role in the development of digital pathology in Japan. Both macroscopic and microscopic pathology digital images were routinely generated and used for diagnostic purposes in major hospitals. Virtual slide (VS) digital images were used first for education then for conference, consultation and also gradually for routine diagnosis and telepathology. The experimental automatic diagnostic aid system achieved the rate of correct diagnosis around 95% for prostate carcinoma and its use for automatic mapping of cancerous areas in a given tissue image was successful.

Conclusions

Advance in the digital information technologies gave revolutionary impacts on pathology education, conference, consultation, diagnosis, telepathology and also on pathology diagnostic procedures in Japan. The future will be bright for pathologists by the advanced digital pathology but we should pay attention to make the technologies and their effects under our control.

     

Dynamic active telepathology over National Health Laboratory service network, South Africa: feasibility study using Nikon Coolscope

Telepathology recently entered a new era with the introduction of digital microscopes combined with Internet technology. The microscope allows viewing real time of whole slide (macro) as well as different chosen fields in four different magnifications. Three Nikon Coolscope were installed in NHLS laboratories in Mthatha, East London and Port Elizabeth. All these microscopes are connected to NHLS server allowing real time viewing of the full slide at any time of the day using Internet browser. Viewing is possible from any PC connected to NHLS Intranet. The challenge was to be able to view slides from other than NHLS computers due to NHLS IT Department network security measures. This was solved by installing NHLS Virtual Private Network server. About 60 cases were viewed by pathologists in Cape Town (Stellenbosh University) and Pretoria (MEDUNSA). All users assessed the system as a helpful tool allowing easy access to cases needing consultation or second opinion. The quality of images was very good. Our experience with Nikon Coolscope is positive. It is an excellent tool for remote small histopathology departments lacking specialists in such areas as dermatopathology, oncology, and haematopathology. Further studies are needed especially in the scope of full utilization of the microscopes installed and impact on laboratory services.     

Telepathology in emerging countries pilot project between Italy and Egypt

Pathological examination includes gross & microscopic examinations at different magnification. Through the steps of examination, we obtain many images that can be used for telepathology. Telepathology is the practice of pathology at a distance, viewing images on a monitor rather than directly through a light microscope. It can be used for primary diagnosis, second opinion, quality assurance and distance learning. Telepathology is classified into Static, Dynamic, Hybrid and Whole Slide Imaging (WSI). We have a successful experience in Egypt in applying the static & dynamic techniques in a pilot project between the Italian Hospital in Cairo (NPO) and the Civico Hospital in Palermo. This project began in 2003 and continued till now. From the second year 2004, Ospedale S. Giovanni e Paolo Hospital in Venice, Charing Cross Hospital in London and the University of Pittsburgh Medical Center Health System (UPMC) in the USA participated actively in our project. During the past five years we consulted on many problematic pathological cases with these different specialized pathological centers in Italy, UK & USA. In addition to the highly specialized scientific value of consulting on the cases and exchanging knowledge, we saved a lot of time and money and succeeded in providing our patients with a better medical service.We are now in the process of establishing a Digital Telepathology Center (DTC) in the pathology department, Cairo University, using the latest technique of telepathology which is Whole Slide Imaging (WSI). We believe that it will help us to improve and extend diagnosis for our difficult pathological cases and will facilitate increased E-learning opportunities for staff and students both in Egypt and in the longer term in the wider Eastern Mediterranean.     

Directionlet transform based sharpening and enhancement of mammographic X-ray images

Due to difficulty in detecting the low contrast and noisy nature of X-ray mammography images, they have to be enhanced to obtain a clear and good view. Though Sharpening Technique (ST) is used to enhance the contrast, it introduces noise in the enhancement process, and they do not include anisotropic features. This paper proposes a ST, which uses multiscale linear and anisotropic geometrical features obtained from directionlet transform (DT). The newly formulated method that combines multidirectional geometrical information has various tunable parameters and improved noise control by means of multiscale features. The DT that uses skewed and elongated directional basis functions not only captures the point singularities, but also links them into linear structure. The performance of the proposed DT ST is compared with non-linear unsharp masking (NLUSM). While the DT and LoG based sharpened images are given to the input of standard AHE, their performance is improved. Enhancement Measure and structural similarity measure are used to analyze the performance of the proposed method. Though the images are enhanced, the quality of the image is not degraded. As a specific application, the enhanced images are used to detect the microcalcification and spiculated masses in mammograms.     

Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin α_vβ₃-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin α_vβ₃, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin α_vβ₃ compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment.     

Metabolic pathways of the fossil dinosaur bones. Part IV. Modes of linkage between osteocytes and a variety of nexuses of osteocytes processes

Examinations were carried out on the fossil dinosaur bones 80 million years old. Samples for examination were prepared with specially elaborated methods. The light, transmission electron, and sanning electron microscopic images showed the spatial distribution of osteocytes with the system of processes as well as by thin and short ones. The axial processes, retaining the same diameter along their entire course, usually connected the polar parts of the osteocytes which frequently lay considerable distance apart. Such connections were of two kinds. In one case the link was attained by catoplasmic continuity. In the other, the processes belonging to one osteocyte came into contact with definite spot of plasmalemma of another osteocyte by means of a terminal, club-shaped widening, the so called contact body. This spot looked like a hollow or a conical protuberance. In this type of contact there were always tiny apertures between the contacting elements. Moreover, three types of contact, especially between thin processes, were distinguished, i.e. "end to end", "side to side", and "end to side". It seems that direct connections achieved through cytoplasmic continuity are utilized in rapid exchange, which may be indicated by the thickness of processes and the lack of a membranous barrier. The remaining, indirect links (with participation of membranes) though varied in the type of contact seem to fulfil identical functions.     

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.     

Recent developments and present status of telepathology.

Telepathology which is the diagnostic work of a pathologist at a distance has been developed to routine application within the last ten years. It can be classified in relation to application, technical solutions, or performance conditions. Diagnostic pathology performance distinguishes primary diagnosis (for example, frozen section statement) from secondary diagnosis (for example, expert consultation) and quality assurance (diagnostic accuracy, continuous education and training). Applications comprise (a) frozen section service; (b) expert consultations; (c) remote control measurements; and (d) education and training. The technical solutions distinguish active (remote control, live imaging) systems from passive (conventional microscope handling, static imaging), and the performance systems with interactive (on-line, live imaging) use from those with passive (offline, static imaging) practice. Intra-operative frozen section service is mainly performed with remote control systems; whereas expert consultations and education/training are commonly based upon Internet connections with static imaging in an off-line mode. The image quality, transfer rates, and screen resolution of active and passive telepathology systems are sufficient for an additional or primary judgment of histological slides and cytological smears. From the technical point of view, remote control telepathology requires a fast transfer and at least near on-line judgement of images, i.e., image acquisition, transfer and presentation can be considered one performance function. Thus, image size, line transfer rate and screen resolution define the practicability of the system. In expert consultation, the pixel resolution of images and natural color presentation are the main factors for diagnostic support, whereas the line transfer rate is of minor importance. These conditions define the technical compartments, especially size and resolution of camera and screen. The performance of commercially available systems has reached a high quality standard. Pathologists can be trained in a short time and use the systems in a routine manner. Several telepathology systems have been implemented in large Institutes of Pathology which serve for frozen section diagnosis in small hospitals located in the local area. In contrast, expert consultation is mainly performed with international connections. There is a remarkable increase of expert consultations by telepathology according to the experiences of the Armed Forces Institute of Pathology or the Department of Pathology, Thoraxklinik, Heidelberg. In expansion of these experiences, a "globalization" of telepathology can be expected. Telepathology can be used to shrink the period necessary for final diagnosis by request for diagnostic assistance to colleagues working in appropriate related time zones. Telepathology is, therefore, not a substitute of conventional diagnostic procedures but a real improvement in the world of pathology.     

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.     

Basic aspects of and recent developments in telepathology in Europe, with specific emphasis on quality assurance

Telepathology is the diagnostic work of a pathologist from a distance and includes all specific fields of diagnostic pathology, such as frozen section services, expert consultation, cytometric and histometric measurement, and continuous education. For about 15 years experience has been collected at several universities in the United States and Europe based upon analog telephone lines (9.2 kbaud), digitized lines (ISDN, 64 kbaud), broad band connections (1.5 Mbaud) and the World Wide Web (28 kbaud). Potential use can be expected in the application of telepresentation, remote slide preparation, remote central diagnostics and telediscussion. The transfer of still images is well developed; that of live images is used in only a few institutions for frozen section services. The image quality and spatial resolution as well as the transfer speed are sufficient for expert consultations, morphometric measurements, quality assurance and education. All applications focus on discontinuous work flow. Although the European Community focuses on user needs and standardization aspects of telepathology by sponsoring a widespread telepathology project (Europath), implementation of telepathology into routine application in the continuous work flow has still to be developed. The technical equipment has still to be adjusted to the labor flow charts in routine pathologic diagnostic procedures. Telepathology seems to be the appropriate technique to offer both improvement in diagnostic quality and inclusion of the "control institution" into diagnostic responsibility.     

Visualizing 3D data obtained from microscopy on the Internet

The Internet is a powerful communication medium increasingly exploited by business and science alike, especially in structural biology and bioinformatics. The traditional presentation of static two-dimensional images of real-world objects on the limited medium of paper can now be shown interactively in three dimensions. Many facets of this new capability have already been developed, particularly in the form of VRML (virtual reality modeling language), but there is a need to extend this capability for visualizing scientific data. Here we introduce a real-time isosurfacing node for VRML, based on the marching cube approach, allowing interactive isosurfacing. A second node does three-dimensional (3D) texture-based volume-rendering for a variety of representations. The use of computers in the microscopic and structural biosciences is extensive, and many scientific file formats exist. To overcome the problem of accessing such data from VRML and other tools, we implemented extensions to SGI's IFL (image format library). IFL is a file format abstraction layer defining communication between a program and a data file. These technologies are developed in support of the BioImage project, aiming to establish a database prototype for multidimensional microscopic data with the ability to view the data within a 3D interactive environment.     

Postural Control in Dual-Task Situations: Does Whole-Body Fatigue Matter?

Postural control is important to cope with demands of everyday life. It has been shown that both attentional demand (i.e., cognitive processing) and fatigue affect postural control in young adults. However, their combined effect is still unresolved. Therefore, we investigated the effects of fatigue on single- (ST) and dual-task (DT) postural control. Twenty young subjects (age: 23.7 ± 2.7) performed an all-out incremental treadmill protocol. After each completed stage, one-legged-stance performance on a force platform under ST (i.e., one-legged-stance only) and DT conditions (i.e., one-legged-stance while subtracting serial 3s) was registered. On a second test day, subjects conducted the same balance tasks for the control condition (i.e., non-fatigued). Results showed that heart rate, lactate, and ventilation increased following fatigue (all p < 0.001; d = 4.2–21). Postural sway and sway velocity increased during DT compared to ST (all p < 0.001; d = 1.9–2.0) and fatigued compared to non-fatigued condition (all p < 0.001; d = 3.3–4.2). In addition, postural control deteriorated with each completed stage during the treadmill protocol (all p < 0.01; d = 1.9–3.3). The addition of an attention-demanding interference task did not further impede one-legged-stance performance. Although both additional attentional demand and physical fatigue affected postural control in healthy young adults, there was no evidence for an overadditive effect (i.e., fatigue-related performance decrements in postural control were similar under ST and DT conditions). Thus, attentional resources were sufficient to cope with the DT situations in the fatigue condition of this experiment.     

Virtual plankton: A novel approach to the investigation of aquatic predator‐prey interactions

Small‐scale zooplankton swimming behaviors can affect aquatic predator‐prey interactions. Difficulties in controlling prey swimming behavior however, have restricted the ability to test hypotheses relating differences in small‐scale swimming behavior to frequency of predation by fish. We report here a Virtual Plankton (VP) system that circumvents this problem by allowing the observation of fish “preying"on computer‐generated prey images whose size, shape, color and swimming behavior can be precisely controlled. Two experiments were performed in which bluegill sunfish (Lepomis macrochirus) were given a choice of either two VP images, one of which moved twice as fast as the other, or six VP, one of which moved either faster (1.25 x, 1.5 x or 2 x ) or slower (0.5 x) than the other five. Current predator‐prey models based on encounter probabilities and prey visibility predict that moving faster increases predation risk and conversely, moving slower decreases predation risk. In agreement with existing predator‐prey models, in both experiments, fish chose faster moving VP significantly more often than their slower moving neighbors. Contrary to the predictions of existing models, in the second experiment with six VP, the rate at which fish chose a prey image moving half as fast as the five surrounding images did not differ significantly from the rate predicted by chance(l/6). These results suggest that current fish‐zooplankton predation models would benefit by the incorporation of small‐scale swimming behavior and assessments of its influence on overall prey visibility.     

Colocalization of β1,4galactosyltransferase with mannose 6-phosphate receptor in monensin-induced TGN-derived structures

Previously, we demonstrated that beta 1,4galactosyltransferase (gal-T1) reversibly segregates from alpha 2,6sialyltransferase (ST6Gal) to swollen vesicles after monensin treatment of the cells. To further explore this phenomenon, we investigated the response to monensin of various Golgi proteins. Within 30 min of monensin treatment, gal-T1 moved from the Golgi apparatus, as defined by localization of giantin, to swollen vesicles whereas ST6Gal, alpha 2,3(N)sialyltransferase, mannosidase II, and N-acetylgalactosaminyltransferase 2 remained associated with the Golgi apparatus. Stably transfected CHO cells exhibited a similar phenomenon of monensin-induced displacement of recombinant gal-T1 to swollen vesicles while recombinant ST6Gal remained colocalized with endogenously expressed giantin. Gal-T1 and the cation-insensitive mannose 6-phosphate receptor colocalized in swollen vesicles as observed at both light and electron microscopic levels. When monensin was replaced by chloroquine, gal-T1 remained arrested in swollen vesicles. Brefeldin A treatment known to cause relocation of Golgi-associated gal-T1 to the endoplasmic reticulum had no effect on gal-T1 trapped in swollen vesicles. This evidence suggests that monensin blocks gal-T1 trafficking in post-Golgi structures and argues against swelling of gal-T1-containing trans Golgi cisternae as previously assumed.     

Virtual slides in peer reviewed,open access medical publication

Background

Application of virtual slides (VS), the digitalization of complete glass slides, is in its infancy to be implemented in routine diagnostic surgical pathology and to issues that are related to tissue-based diagnosis, such as education and scientific publication.

Approach

Electronic publication in Pathology offers new features of scientific communication in pathology that cannot be obtained by conventional paper based journals. Most of these features are based upon completely open or partly directed interaction between the reader and the system that distributes the article. One of these interactions can be applied to microscopic images allowing the reader to navigate and magnify the presented images. VS and interactive Virtual Microscopy (VM) are a tool to increase the scientific value of microscopic images.

Technology and Performance

The open access journal Diagnostic Pathology http://www.diagnosticpathology.org has existed for about five years. It is a peer reviewed journal that publishes all types of scientific contributions, including original scientific work, case reports and review articles. In addition to digitized still images the authors of appropriate articles are requested to submit the underlying glass slides to an institution (DiagnomX.eu, and Leica.com) for digitalization and documentation. The images are stored in a separate image data bank which is adequately linked to the article. The normal review process is not involved. Both processes (peer review and VS acquisition) are performed contemporaneously in order to minimize a potential publication delay. VS are not provided with a DOI index (digital object identifier). The first articles that include VS were published in March 2011.

Results and Perspectives

Several logistic constraints had to be overcome until the first articles including VS could be published. Step by step an automated acquisition and distribution system had to be implemented to the corresponding article. The acceptance of VS by the reader is high as well as by the authors. Of specific value are the increased confidence to and reputation of authors as well as the presented information to the reader. Additional associated functions such as access to author-owned related image collections, reader-controlled automated image measurements and image transformations are in preparation.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1232133347629819.

     

When GIS zooms in: spatio-genetic maps of multipaternity in<Emphasis Type="Italic"> Armadillidium vulgare</Emphasis>

Geographic information system (GIS) tools are designed to illustrate, analyse and integrate geographic or spatial data, usually on a macroscopic scale. By contrast, genetic tools focus on a microscopic scale. Because in reality, landscapes have no predefined scale, our original study aims to develop a new approach, combining both cartographic and genetic approaches to explore microscopic landscapes. For this, we focused on Armadillidium vulgare, a terrestrial isopod model in which evolutionary pressures imposed by terrestrial life have led to the development of internal fertilisation and, consequently, to associated physiological changes. Among these, the emergence of internal receptacles, found in many taxa ranging from mammals to arthropods, allowed females to store sperm from several partners, enabling multipaternity. Among arthropods, terrestrial isopods like the polygynandrous A. vulgare present a female structure, the marsupium, in which fertilised eggs migrate and develop into mancae (larval stage). To test our innovative combined approach, we proposed different males to four independent females, and at the end of incubation in the marsupium, we mapped (using GIS methods) and genotyped (using 12 microsatellite markers) all the incubated mancae. This methodology permitted to obtain spatio-genetic maps describing heterozygosity and spatial distribution of mancae and of multipaternity within the marsupial landscape. We discussed the interest of this kind of multidisciplinary approach which could improve in this case our understanding of sexual selection mechanisms in this terrestrial crustacean. Beyond the interesting model-focused insights, the main challenge of this study was the transfer of GIS techniques to a microscopic scale and our results appear so as pioneers rendering GIS tools available for studies involving imagery whatever their study scale.     

Acquired immunity and allergy of cells cultured in vitro

It is in the reinoculated cell line FL that the sensitivity of cells under the action of a small dose of diphtheria toxin (DT) changes to the toxin itself as well as to the Coxsackie enterovirus. (B-5). After surviving the infection and getting again "healthy", the cells have acquired simultaneously an elevated sensibility to DT and reduced susceptibility to B-5 transmissible even in long passages. These acquired properties are a transient phenomenon, after a certain number of passages the culture returns to initial stage with all properties inherent in the initial line of cells: with regard to DT in 10 months, to coxsackie virus in 7 months. The stage of elevated sensibility to DT and reduced susceptibility to B-5, however, may be not only renewed but also considerably prolonged and even strengthened by repeated contact of cells with a small dose of DT. The small dose of DT is the first delusion of the toxin not provoking any visible morphological changes in the monolayer. The experimental data obtained render it possible to presume that application of reinoculated cell cultures offers a promising method for studying manifestations of laws governing the third factor of immunity and allergy at the level of cells.     

Means, Advantages and Limits of Merging Biology with Technology

The natural world spent billions of years in solution-finding during evolution, which could benefit Technology. How do we put that in a nutshell? Biological systems are more complex than the most complex current technology. Any given functiofi and effect are simultaneously coordinated and linked with others at many levels of biological organisation-from cell organelle to organism, to population and ecosystem. Technology does not have tools to deal with the complexity and “goalintendedness“ of living systems. But limits for interaction exist on both sides-Biological science itself is also too empirical and not mature enough to provide a solid base for correlating living with technical systems. Moving towards a synthesis, where engineers can utilize the vast amount of available biological data, we suggest using a tool called “Theory of Inventive Problem Solving“ (TRIZ) and clarifying some important methodological issues, which have not previously been recognised in bionic engineering: 1) Requirement for more appropriate definitions of “system“, “effect“, “function“, “law“ and “rule“. 2) Requirement for understanding or even measuring the degree of contradiction or analogy between functions in biological and artificial and/or non-living engineering system-there is no simple direct correlation between what engineers find useful and what biology does.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).