A recombineering pipeline for functional genomics applied to Caenorhabditis elegans

Genome sequencing and annotation projects define the complete sets of RNA and protein components for living systems. They also present the challenge to generate functional information for thousands of previously uncharacterized genes. Protein tagging with fluorescent or affinity tags provides a generic way to describe protein expression and localization patterns and protein-protein interactions. The genome wide application of this approach in Saccharomyces cerevisiae has resulted in a comprehensive picture of the core proteome of a simple, well-studied model system. Extending these studies to more complex, multicellular model organisms, would allow us to place protein function onto a 4 dimensional space-time map, and will improve our understanding of the complex processes of development and differentiation. This will require efficient protein tagging methods and new high performance tags. Here we present a generic protein tagging approach for the model nematode Caenorhabditis elegans. The method is based on recombination mediated DNA engineering of genomic BAC clones into tagged transgenes for integrative transformation. C.elegans offers unique advantages for function discovery through protein tagging: compact and a well annotated genome, combined with a simple and well-understood anatomy and pattern of development. However, the methods for protein tagging in C.elegans have so far been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. In contrast, our approach combines the advantages of authentic regulation with a new application of recombineering, which is simple, fast and efficient. For the first time we apply liquid culture cloning for multiple recombineering steps. This is particularly important when high throughput applications are considered, as it offers significant advantages in scale up and automation. We show that the BAC derived transgenes can be used for stable, integrative transformation in C. elegans. We show that the tagged transgene can take over the function of its endogenous counterpart. Using florescent reporter, we reproduce known and document new expression patterns. The second part of the thesis describes a project that we undertook to develop improved double affinity cassettes for protein purification. We evaluated the performance of 5 new double tag combinations in vitro and in mammalian culture cells. All of the new cassettes performed well and present a valuable tool for protein interaction studies in higher model systems

MEDICINE IN CRISIS OR IN FRONT OF NEW OPPORTUNITIES

The author presents classification of medical sciences from a holistic perspective. Modern classification based on structural functionalism shows a lack of consistency. Holistic vision, based on role determinism, considers function as a role of the organ in biological variables determination. Biological variables plays an organizing role in the overall functioning of the body and determines its physiological identity being healthy or sick. Holistic vision could be successfully applied in medical theory, diagnosis and education

THE NECESSITY OF KNOWLEDGE, SERVING THE NEEDS OF THE BIO-ECONOMY IN BULGARIA

The main challenge of the bioeconomy is to achieve economic growth, increase the competitiveness of the economy, balanced production and consumption of products, conservation of natural resources, improve the marketing of farms. The aims of this article is to analyze the knowledge needs for the bioeconomy in Bulgaria. We first present the socio-economic benefits of the bioeconomy, with a focus on agriculture as a leading sector, and then identify the basic needs. A three-step approach is applied to cover all stakeholders directly involved in bioeconomic knowledge acquisition: Education and awareness; Dialogue and consult; Co-creation of new knowledge based on cooperation and training. The results imply: identifying the knowledge and technological knowledge needs required for regional specialized bioeconomies; clarification of the knowledge delivery mechanism; knowledge transfer networks; assessment of knowledge in the bioeconomics. The need for bioeconomic knowledge would support a economic growth to more resource efficient use. Increasing the knowledge base and understanding of specific areas of the bio-economy will be based on obtaining more data, generating better information and systematic analysis

TNF and PGE2 in human monocyte-derived macrophages infected with Chlamydia trachomatis

In this study levels of prostaglandin E2 (PGE2), tumour necrosis factor (TNF) and interleukin-1 (IL-1) alpha in medium from monocyte derived macrophages (MdM) infected with Chlamydia trachomatis (L2/434/Bu or K biovars). TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM

To a holistic classification in medical theory and education

ABSTRACT Introduction: Nowadays teaching medicine is based only on structural functionalism. Thus the conventional organization of the content of functional medical sciences like physiology, pathophysiology and internal medicine is sometimes ridiculous and difficult for learning and practicing

Mortality in the Bedouin Population and Proximity to a Regional Industrial Complex

Background: The study was initiated by public concern about exposure to an industrial park (IP) emission. The study examined whether mortality in the Bedouin population in the southern part of Israel is associated with the residential distance to the IP.Material and Methods: Ecological study during 1995–2001 included the entire Bedouin population. Mortality data was obtained from the Central Bureau of Statistics. As an indirect measurement of exposure we used residential distance to the IP (with 20 km radius as a cut-of-point) based on residents’ complaints about odor related to the IP. Differences in mortality rates by distance were assessed by the Mantel-Haenszel relative risk (M-H RR) within the 95% CI. The country Arab population served as a reference for calculation of the age-adjusted standardized mortality ratio (SMR).Results: Increased mortality rates due to symptoms/ill-defined conditions and non-external causes were observed in the Bedouin population of both sexes, residing up to 20 km from the IP, compared to those living in more remote areas. Corresponding M-H RR (plus 95% CI) were 1.66 (1.17–2.36), 1.24 (1.06–1.44) in females, and 1.55 (1.15–2.10), 1.32 (1.15–1.52) in males.Conclusions: The study results suggest an association between residential proximity to the regional IP and increased mortality rates in the Negev Bedouin population. These findings have been accepted by the authorities as an issue for community health protection

Major congenital malformations and residential proximity to a regional industrial park including a national toxic waste site: An ecological study

BACKGROUND: Public concern about exposure to emissions from the regional industrial park (IP), including 17 chemical plants and the national industrial toxic waste site, initiated this study of the possible association between major congenital malformations (MCM) and residence near the IP in Israel's Southern District. METHODS: The study was conducted during the period 1995–2000 and included 63,850 deliveries. Data on deliveries and MCM detected at births were obtained from the regional medical center, and stratified by ethnicity and type of locality. As exposure indicator we used distance categories (proximal and distant) and predominant wind direction from the IP. Distance stratification was based on the geographical distribution of the localities and complaints about the odor related to IP emissions. Based on these complaints, localities up to 20 km from the IP were considered proximal to the IP. RESULTS: Average rates of MCM were 5.0% and 4.1% for Bedouin and Jewish newborns, respectively. The rate of MCM for Bedouin from proximal localities was significantly greater compared with distant localities (5.6% vs. 4.8%; RR = 1.17 with 95% CI: 1.04–1.29). In the proximal Bedouin permanent localities, the MCM rate reached 8.2 %, which was significantly higher than in distant areas (RR = 1.63, 95% CI: 1.39–1.80). Significant risk increase of central nervous system MCM was found in these localities, compared to distant ones (RR = 2.27, 95% CI: 1.44–3.60). Among newborns from the traditional tribal settlements, proximity to the IP was associated with increased rates of the following MCM: 1) all combined, 2) those associated with chromosomal abnormalities, and 3) those defined as "others unclassified MCM." Comparison of autosomal recessive disease rates by proximity to the IP in Bedouin newborns indicates that the observed increased risk of MCM is not explained by consanguineous marriages. The rates of MCM in the Jewish population were similar among "exposed" and "unexposed" inhabitants. CONCLUSION: Residential proximity to the IP is associated with increased rates of MCM among Arab-Beduin but not in Jewish populations. These observations indicate the need for public health protection of a vulnerable society in transition, although the relative importance of chemical exposure and health care utilization requires further study

Framework for assessing the governance pillar of agrarian sustainability

In many other countries, there are no comprehensive systems for assessment of the governance sustainability of agriculture and its importance for the overall agrarian development. This study tries to fill the gap and suggests a holistic framework for understanding and assessing the governance sustainability. The newly elaborated approach is “tested” in a large-scale study for assessing the governance sustainability of Bulgarian agriculture. The study has proved that it is important to include the “missing” Governance Pillar in the assessment of the Integral sustainability of agriculture and sustainability of agro-systems of various type. Assessment of the Governance sustainability of Bulgarian agriculture indicates that the Overall Governance Sustainability is at a “Good” but close to the “Satisfactory” level. There is a considerable differentiation in the level of Integral Governance sustainability of different agro-systems in the country. Results on the integral agrarian sustainability assessment based on micro (farm) and macro (statistical, etc.) data show some discrepancies which have to be taken into consideration, while assessment indicators, methods and data sources improved

Как да измерим управленческата устойчивост на българското селско стопанство?

A need to include “the fourth” Governance pillar in the concept for understanding and the assessment system of (overall and) agrarian sustainability is increasingly justified in academic literature and finds place in the frameworks of government, international, private, etc. organizations. Nevertheless, still there is no general consensus on: whether and how to include the governance as a new pillar of agrarian sustainability; how to define the governance sustainability; what are the relations between the governance sustainability of a farming enterprise and agriculture; what are the critical factors of governance sustainability; how to formulate, select, measure and integrate diverse sustainability indicators; and how to properly evaluate the level of governance sustainability, etc. In Bulgaria, like in many other countries, practically there are no comprehensive assessments of the governance sustainability of agriculture and its importance for the overall agrarian development. This study tries to fill the gap and suggests a holistic framework for understanding and assessing the governance sustainability of Bulgarian agriculture. The newly elaborated approach is “tested” in assessment of the governance sustainability of country’s agriculture The study has proved that it is important to include the “missing” Governance Pillar in the assessment of the Integral sustainability of agriculture. Governance sustainability of Bulgarian agriculture indicates that the Overall Governance Sustainability is at a “Good” but very close to the “Satisfactory” level. Besides, results on the integral agrarian sustainability assessment based on micro (farm) and macro (statistical, etc.) data show some discrepancies which have to be taken into consideration in the analysis and interpretation, while assessment indicators, methods and data sources further improved. Having in mind the importance of holistic assessments of this kind for improving the agrarian sustainability in general, and the Governance sustainability of agriculture in particular, they are to be expended and their precision and representation increased. The later requires improvement of the precision through enlargement of surveyed farms and stakeholders, and incorporating more “objective” data from surveys, statistics, expertise of professionals in the area, etc. Since the elaboration of an effective framework for Governance sustainability assessment is far from complete our and other emerging suggestions have to be further discussed, experimented, improved and adapted to the specific conditions of evaluating agricultural system and needs of decision-makers at different levels