Auswahl der wissenschaftlichen Literatur zum Thema „Molecular documentation“

PURPOSE The Ewing tumor (ET) family of peripheral primitive neuroectodermal tumors (pPNETs) are primitive small round-cell tumors (SRCTs) of the bone and soft tissue that occur predominantly in children and adolescents. However, pPNETs only rarely enter the differential diagnosis of bone and soft tissue SRCTs in adults. Recently, gene fusions between the EWS gene and different members of the ETS transcription factor family have been shown to occur in virtually all pPNETs and thus constitute a pathognomonic marker for this tumor subclass. The aim of the present study was to document EWS/ETS fusion gene expression in suspected pPNETs of adults as objective evidence for the existence of this tumor family in older patients. PATIENTS AND METHODS The three contributing molecular diagnostic laboratories retrospectively compiled a cohort of all SRCT cases in which EWS/ETS gene fusions had been shown by molecular analysis. This cohort was surveyed for cases that occurred in patients aged 40 years or older, which were then analyzed for their clinical and pathologic features. RESULTS Nine patients between 40 and 65 years of age were found to have tumors positive for EWS/ETS gene fusions. Standard histopathologic and clinical features of these cases, other than age, were similar to those of childhood pPNETs. Patients were initiated on appropriate therapy after molecular analysis confirmed the diagnosis of pPNET. CONCLUSION Identification of an EWS/ETS gene fusion is useful in providing objective evidence of the diagnosis of pPNET in patients over the age of 40 years. This diagnosis should be considered in adults who present with bone and soft tissue SRCTs and appropriate biopsy specimens should be collected for molecular analysis at the time of diagnosis.

A common laboratory method involves gel electrophoresis followed by photographic documentation of the results, a procedure which is performed worldwide by students and experienced scientists alike. Proprietary Gel Documentation Systems are convenient and useful for documentation of electrophoresis results, but the systems can be prohibitively expensive to purchase and repair, they contain features that are not necessary for everyday documentation, and some users may not find the systems intuitive to operate. We describe our gel documentation setup that meets the everyday needs for documentation in our lab. The setup is inexpensive, modular, user friendly, and increases sustainability through extending the working life of obsolete cell phones, iPads, or other electronic devices containing a camera. More importantly, the setup completely shields users from potentially damaging ultraviolet radiation.

Este trabajo de tesis doctoral se centra en el uso de ontologías de dominio y su refinamiento enfocado a la recuperación de la información. El dominio seleccionado ha sido el de la Biomedicina, que dispone de una extensa colección de resúmenes en la base de datos Medline y recursos que facilitan la creación de ontologías muy extensas, tales como MeSH o UMLS. En este trabajo se ha desarrollado también un modelo de formulación de consulta que permite relacionar un modelo de documento con una ontología dentro de los modelos de lenguaje. Además hemos desarrollado un algoritmo que permite mejorar la ontología para la tarea de recuperación de la información a partir de recursos no estructurados. Los resultados muestran que el refinamiento de las ontologías aplicado a la recuperación de la información mejora el rendimiento, identificando automáticamente información no presente en la ontología. Además hemos comprobado que el tipo de contenido relevante para las consultas depende de propiedades relacionadas con el tipo de consulta y la colección de documentos. Los resultados están acordes con resultados existentes en el campo de la recuperación de la información.

Osteosarcoma is the most common type of malignancy of bone and generally occurs among adolescent and young adults. Like the osteoblast cells of normal bone, osteosarcoma also forms the bone matrix, but the osteoid is not as strong as that of normal bones. Osteosarcoma is characterized by the production of weak or immature bones by the malignant cells. As the diagnosis of osteosarcoma is extremely poor, it suggests a critical need to develop some promising anti-osteosarcoma drugs to improve disease outcome. Many anti-cancer compounds induce apoptotic cell suicide via some potent cellular, molecular and biochemical markers. The cancer cell lines provide a valuable model system to study an extensive variety of cancer characteristics in the cell biology, genetics and chemotherapy or the impact of therapeutic molecules. The methods presented in this chapter describe the experimental technique used to culture the osteosarcoma cells for the documentation of DNA fragmentation and Caspase-3 activation associated with apoptosis.

Abstract The documentation of vertebrate diversity traditionally involved the collection of specimens for further study. Properly curated collections constitute treasure troves for biologists interested in systematics, biogeography, and evolutionary biology. The collection of specimens and their preservation was justified on the grounds that our understanding of nature depended upon it. This is no longer always the case, and in situations involving rare or threatened species it may be completely unjustified. Collections that were well-intended at the time can seem appalling in today’s conservation-minded world. Four examples illustrate the conflicts between the need to conduct scientific research and the need to conserve the subjects of that research: The 1906 California Academy of Sciences expedition to the Galapagos dis¬ covered that the Pinzon island race of giant tortoises was, in fact, not extinct and accordingly collected the remaining 86 for study (Thornton, 1971).

Abstract The 'revolution' that began in the 1980s with the application of mitochondrial DNA (mtDNA) analyses to address questions of molecular evolutionary and population biology has now largely become the paradigm for such work (1). Studies ranging in scope from purely systematic problems to identification and recognition of species to documentation of genetic changes in populations that have occurred over a very short period of time, are based on both indirect or direct estimates of nucleic acid variation. While progress continues to be made in analysing coding regions of nuclear DNA, the sequences retained in organellar genomes are still the most widely studied by evolutionary biologists. Restriction site or fragment analyses of mtDNA and direct sequencing of specific regions of the mitochondrial genome following amplification by the polymerase chain reaction (PCR) are currently the methods of choice for the majority of population level studies. The reasons for the increasingly wide spread application of mtDNA markers in studies of animal populations are many; however, there are also a few noteworthy drawbacks (reviewed in ref. 1). Figure 1 shows the typical vertebrate gene order of mtDNA, with birds having a single rearrangement involving ND 6 (a subunit of NAD dehydrogenase) and tRNAGlu not found in other vertebrates examined to date. Other minor rearrangements have been reported, and gene order can provide important synapomorphies (shared derived characters) for deciphering some relationships.

CovidGraph, developed by the HealthECCO community, is a platform designed to foster research and data exploration to fight COVID-19. It is built on a graph database and encompasses data sources from different biomedical data domains including publications, clinical trials, patents, case statistics, molecular data and systems biology models. The tool provides multiple interfaces for data exploration and thus serves as a single point of entry for data driven COVID-19 research. Availability and Implementation: CovidGraph is available from the project website: https://healthecco.org/covidgraph/. The source code and documentation are provided on GitHub: https://github.com/covidgraph.

Abstract After several decades of very active research in comparative animal physiology, an outside observer currently might get the impression that the situation has changed radically. A recent documentation of the success of comparative animal physiology is, for example, given in the book Biochemical Adaptation: Mechanism and Process in Physiological Evolution by Peter Hochachka and George Somero (2002). With the success of molecular biological techniques, however, sayings like “Everything or everyone goes molecular” are frequently encountered in discussions on future perspectives of scientific work in biological sciences. Departments and institutes are being renamed, the “evolutionary” aspect is replacing the “comparative” angle. More and more referees and thus journals and funding agencies ask for the mechanistic (“molecular”) part of the story, and disapprove of the descriptive (“physiological”) part, irrespective of the fact that the story “Factor A increases during hypoxia, activates transcription factor B, and this results in the expression of protein C” is as descriptive as the reported cardiovascular modifications encountered on hypoxic exposure. Is this the end of comparative physiology?

Abstract There are several reviews that cover the biological questions that have been addressed with fluorescent techniques in plants (1-3) and there is extensive on line documentation on the properties of the fluorescent probes themselves (e.g. Molecular Probes, . This chapter focuses on optimization of the systems used for live cell imaging, the methods used to mount plant specimens and load them with dyes, a compendium of dyes that have proved useful in plant tissues, and a series of protocols to convert qualitative observations to quantitative measurements. The hazards associated with these dyes are, in the main, not known, so all should be treated as potentially harmful.

At this point, we hope to have demonstrated that the algebraic approach provides a viable method for the quantitative description of molecular vibrotational spectra. Chapters 4 (triatomic molecules, both linear and bent) and 5 (linear tetratomic molecules) and Appendix C provide extensive documentation for the quantitative applications, while Chapter 6 shows that larger molecules can also be treated. Throughout, but most particularly in Chapter 7, we have sought to forge a link with the more familiar geometrical approach. It is precisely our requirement that even in zeroth order the Hamiltonian with which we start describes an anharmonic motion, which makes this link not trivial. The advantage of our approach in providing, even in zeroth order, high overtone spectra that are typically more accurate than 10 cm−1, should not be overlooked. Yet much remains to be done. In this chapter we look to the future: Where and why do we think that the algebraic approach will prove particularly advantageous? Of course, what we really hope for is to be surprised by unexpected new developments and applications. Here, however, is where we are certain that some of the future progress will be made, with special reference to the spectroscopy of higher-energy states of molecules. One area of spectroscopy where the Hamiltonian in matrix form is the route of choice is that of large polyatomics, particularly so when in an electronically excited state (see Note 3 of the Introduction). Such states are isoenergetic with very high vibrational overtones of the ground electronic states so that a fully geometrical approach is impractical. Even at lower energies, the exceedingly high density of vibrational states strongly favors an alternative approach, and the use of model Hamiltonian matrices is not uncommon. Such model matrices are introduced in order to account for the regularities that often survive in the observed spectrum. One such striking feature is often referred to as a “clump” (Hamilton, Kinsey, and Field, 1986). Consider a pure vibrational progression of states, as can be observed in stimulated emission spectroscopy.

A new edition of the HITRAN molecular spectroscopic database and the high-temperature analog (HITEMP) have recently been released.1,2 They are included in a compilation called HAWKS (HITRAN Atmospheric Workstation). HAWKS represents more fully a “matter” database. Besides an updated HITRAN high-resolution molecular database of about one million transitions, there are files of aerosol indices of refraction, UV line-by-line and cross-section parameters, supplemental files of gases such as ionic species and ozone parameters suitable for atmospheric non-local thermodynamic equilibrium conditions, extensive IR cross-sections now at different pressures and temperatures, and molecular parameters suitable for modeling high-temperature radiance. In addition there is a moderate-resolution band-model code, MODTRAN3. There is also vastly improved software handling of the data in both WINDOWS and UNIX platforms, such as more sophisticated selection filters, plotting capabilities, pointers to significant references, and documentation.

We are approaching single molecule detection in fluid solution. Our current limit with dissolved dye molecules is equivalent to the detection of 8 rhodamine-6G fluorophores on a single species. Documentation of this limit is given by the detection of microspheres containing the simulated equivalent of 25 rhodamine-6G fluorophores.

Symbiotic bacteria have been shown to influence host reproduction and defense against biotic and abiotic stressors, and this relates to possible development of a symbiont-based control strategy. This project was based on the hypothesis that symbionts have a significant impact on Culicoides fitness and vector competence for animal viruses. The original objectives in our proposal were: 1. Molecular identification and localization of the newly-discovered symbiotic bacteria within C. imicola and C. schultzei in Israel and C. sonorensis in California. 2. Determination of the prevalence of symbiotic bacteria within different vector Culicoides populations. 3. Documentation of specific symbiont effects on vector reproduction and defense: 3a) test for cytoplasmic incompatibility in Cardinium-infected species; 3b) experimentally evaluate the role of the symbiont on infection or parasitism by key Culicoides natural enemies (iridescent virus and mermithid nematode). 4. Testing the role(s) of the symbionts in possible protection against infection of vector Culicoides by BTV. According to preliminary findings and difficulties in performing experimental procedures performed in other insect symbiosis systems where insect host cultures are easily maintained, we modified the last two objectives as follows: Obj. 3, we tested how symbionts affected general fitness of Israeli Culicoides species, and thoroughly described and evaluated the correlation between American Culicoides and their bacterial communities in the field. We also tried alternative methods to test symbiont-Culicoides interactions and launched studies to characterize low-temperature stress tolerances of the main US vector, which may be related to symbionts. Obj. 4, we tested the correlation between EHDV (instead of BTV) aquisition and Cardinium infection. Culicoides-bornearboviral diseases are emerging or re-emerging worldwide, causing direct and indirect economic losses as well as reduction in animal welfare. One novel strategy to reduce insects’ vectorial capacity is by manipulating specific symbionts to affect vector fitness or performance of the disease agent within. Little was known on the bacterial tenants occupying various Culicoides species, and thus, this project was initiated with the above aims. During this project, we were able to describe the symbiont Cardinium and whole bacterial communities in Israeli and American Culicoides species respectively. We showed that Cardinium infection prevalence is determined by land surface temperature, and this may be important to the larval stage. We also showed no patent significant effect of Cardinium on adult fitness parameters. We showed that the bacterial community in C. sonorensis varies significantly with the host’s developmental stage, but it varies little across multiple wastewater pond environments. This may indicate some specific biological interactions and allowed us to describe a “core microbiome” for C. sonorensis. The final set of analyses that include habitat sample is currently done, in order to separate the more intimately-associated bacteria from those inhabiting the gut contents or cuticle surface (which also could be important). We were also able to carefully study other biological aspects of Culicoides and were able to discriminate two species in C. schultzei group in Israel, and to investigate low temperature tolerances of C. sonorensis that may be related to symbionts. Scientific implications include the establishment of bacterial identification and interactions in Culicoides (our work is cited in other bacteria-Culicoides studies), the development molecular identification of C. schultzei group, and the detailed description of the microbiome of the immature and matched adult stages of C. sonorensis. Agricultural implications include understanding of intrinsic factors that govern Culicoides biology and population regulation, which may be relevant for vector control or reduction in pathogen transmission. Being able to precisely identify Culicoides species is central to understanding Culicoides borne disease epidemiology.

The Genetic Technology (Precision Breeding) Act (England) aims to develop a science-based process for the regulation and authorisation of precision bred organisms (PBOs). PBOs are created by genetic technologies but exhibit changes which could have occurred through traditional processes. This current review, commissioned by the Food Standards Agency (FSA), aims to clarify existing terminologies, explore viable methods for the detection, identification, and quantification of products of precision breeding techniques, address and identify potential solutions to the analytical challenges presented, and provide recommendations for working towards an infrastructure to support detection of precision bred products in the future. The review includes a summary of the terminology in relation to analytical approaches for detection of precision bred products. A harmonised set of terminology contributes towards promoting further understanding of the common terms used in genome editing. A review of the current state of the art of potential methods for the detection, identification and quantification of precision bred products in the UK, has been provided. Parallels are drawn with the evolution of synergistic analytical approaches for the detection of Genetically Modified Organisms (GMOs), where molecular biology techniques are used to detect DNA sequence changes in an organism’s genome. The scope and limitations of targeted and untargeted methods are summarised. Current scientific opinion supports that modern molecular biology techniques (i.e., quantitative real-time Polymerase Chain Reaction (qPCR), digital PCR (dPCR) and Next Generation Sequencing (NGS)) have the technical capability to detect small alterations in an organism’s genome, given specific prerequisites of a priori information on the DNA sequence of interest and of the associated flanking regions. These techniques also provide the best infra-structure for developing potential approaches for detection of PBOs. Should sufficient information be known regarding a sequence alteration and confidence can be attributed to this being specific to a PBO line, then detection, identification and quantification can potentially be achieved. Genome editing and new mutagenesis techniques are umbrella terms, incorporating a plethora of approaches with diverse modes of action and resultant mutational changes. Generalisations regarding techniques and methods for detection for all PBO products are not appropriate, and each genome edited product may have to be assessed on a case-by-case basis. The application of modern molecular biology techniques, in isolation and by targeting just a single alteration, are unlikely to provide unequivocal evidence to the source of that variation, be that as a result of precision breeding or as a result of traditional processes. In specific instances, detection and identification may be technically possible, if enough additional information is available in order to prove that a DNA sequence or sequences are unique to a specific genome edited line (e.g., following certain types of Site-Directed Nucelase-3 (SDN-3) based approaches). The scope, gaps, and limitations associated with traceability of PBO products were examined, to identify current and future challenges. Alongside these, recommendations were made to provide the infrastructure for working towards a toolkit for the design, development and implementation of analytical methods for detection of PBO products. Recognition is given that fully effective methods for PBO detection have yet to be realised, so these recommendations have been made as a tool for progressing the current state-of-the-art for research into such methods. Recommendations for the following five main challenges were identified. Firstly, PBOs submitted for authorisation should be assessed on a case-by-case basis in terms of the extent, type and number of genetic changes, to make an informed decision on the likelihood of a molecular biology method being developed for unequivocal identification of that specific PBO. The second recommendation is that a specialist review be conducted, potentially informed by UK and EU governmental departments, to monitor those PBOs destined for the authorisation process, and actively assess the extent of the genetic variability and mutations, to make an informed decision on the type and complexity of detection methods that need to be developed. This could be further informed as part of the authorisation process and augmented via a publicly available register or database. Thirdly, further specialist research and development, allied with laboratory-based evidence, is required to evaluate the potential of using a weight of evidence approach for the design and development of detection methods for PBOs. This concept centres on using other indicators, aside from the single mutation of interest, to increase the likelihood of providing a unique signature or footprint. This includes consideration of the genetic background, flanking regions, off-target mutations, potential CRISPR/Cas activity, feasibility of heritable epigenetic and epitranscriptomic changes, as well as supplementary material from supplier, origin, pedigree and other documentation. Fourthly, additional work is recommended, evaluating the extent/type/nature of the genetic changes, and assessing the feasibility of applying threshold limits associated with these genetic changes to make any distinction on how they may have occurred. Such a probabilistic approach, supported with bioinformatics, to determine the likelihood of particular changes occurring through genome editing or traditional processes, could facilitate rapid classification and pragmatic labelling of products and organisms containing specific mutations more readily. Finally, several scientific publications on detection of genome edited products have been based on theoretical principles. It is recommended to further qualify these using evidenced based practical experimental work in the laboratory environment. Additional challenges and recommendations regarding the design, development and implementation of potential detection methods were also identified. Modern molecular biology-based techniques, inclusive of qPCR, dPCR, and NGS, in combination with appropriate bioinformatics pipelines, continue to offer the best analytical potential for developing methods for detecting PBOs. dPCR and NGS may offer the best technical potential, but qPCR remains the most practicable option as it is embedded in most analytical laboratories. Traditional screening approaches, similar to those for conventional transgenic GMOs, cannot easily be used for PBOs due to the deficit in common control elements incorporated into the host genome. However, some limited screening may be appropriate for PBOs as part of a triage system, should a priori information be known regarding the sequences of interest. The current deficit of suitable methods to detect and identify PBOs precludes accurate PBO quantification. Development of suitable reference materials to aid in the traceability of PBOs remains an issue, particularly for those PBOs which house on- and off-target mutations which can segregate. Off-target mutations may provide an additional tool to augment methods for detection, but unless these exhibit complete genetic linkage to the sequence of interest, these can also segregate out in resulting generations. Further research should be conducted regarding the likelihood of multiple mutations segregating out in a PBO, to help inform the development of appropriate PBO reference materials, as well as the potential of using off-target mutations as an additional tool for PBO traceability. Whilst recognising the technical challenges of developing and maintaining pan-genomic databases, this report recommends that the UK continues to consider development of such a resource, either as a UK centric version, or ideally through engagement in parallel EU and international activities to better achieve harmonisation and shared responsibilities. Such databases would be an invaluable resource in the design of reliable detection methods, as well as for confirming that a mutation is as a result of genome editing. PBOs and their products show great potential within the agri-food sector, necessitating a science-based analytical framework to support UK legislation, business and consumers. Differentiating between PBOs generated through genome editing compared to organisms which exhibit the same mutational change through traditional processes remains analytically challenging, but a broad set of diagnostic technologies (e.g., qPCR, NGS, dPCR) coupled with pan-genomic databases and bioinformatics approaches may help contribute to filling this analytical gap, and support the safety, transparency, proportionality, traceability and consumer confidence associated with the UK food chain.