SUBJECTIVE METHODS FOR ASSESSMENT OF DRIVER DROWSINESS

The paper deals with the issue of fatigue and sleepiness behind the wheel, which for a long time has been of vital importance for the research in the area of driver-car interaction safety. Numerous experiments on car simulators with diverse measurements to observe human behavior have been performed at the laboratories of the faculty of the authors. The paper provides analysis and an overview and assessment of the subjective (self-rating and observer rating) methods for observation of driver behavior and the detection of critical behavior in sleep deprived drivers using the developed subjective rating scales

MODEL CAR TRANSPORT SYSTEM - MODERN ITS EDUCATION TOOL

The model car transport system is a laboratory intended for a practical development in the area of the motor traffic. It is also an important education tool for students’ hands-on training, enabling students to test the results of their own studies. The main part of the model car transportation network is a model in a ratio 1:87 (HO), based on component units of FALLER Car system, e.g. cars, traffic lights, carriage way, parking spaces, stop sections, branch-off junctions, sensors and control sections. The model enables to simulate real traffic situations. It includes a motor traffic in a city, in a small village, on a carriageway between a city and a village including a railway crossing. The traffic infrastructure includes different kinds of intersections, such as T-junctions, a classic four-way crossroad and four-way traffic circle, with and without traffic lights control. Another important part of the model is a segment of a highway which includes an elevated crossing with highway approaches and exits

Assessment of external interface of autonomous vehicles

The paper deals with the problem of a communication interface between autonomous vehicles (AV) and pedestrians. The introduced methodology for assessing new and existing e-HMI (external HMI) contributes to traffic safety in cities. The methodology is implemented in a pilot experiment with a scenario designed in virtual reality (VR). The simulated scene represents an urban zebra crossing with an approaching autonomous vehicle. The projection is implemented with the help of a head-up display – a headset with a built-in eye tracker. The suggested methodology analyses the pedestrian’s decision making based on the visual cues – the signals displayed on the autonomous vehicle. Furthermore, the decision making is correlated to subjects’ eye behaviour, based on gaze-direction data. The method presented in this paper contributes to the safety of a vehicle-pedestrian communication of autonomous vehicles and is a part of a research that shall further contribute to the design and assessment of external communication interfaces of AV in general

Deficit of the budget: essence, reasons and management

Стаття присвячена проблемі дефіциту державного бюджету в умовах формування соціально спрямованої ринкової економіки. У дослідженні обґрунтовано економічну сутність та виявлено основні фактори існування негативного сальдо бюджету. Визначено умови використання дефіциту державного бюджету як інструменту впливу на економічні процеси. Обґрунтовано, що бюджетний дефіцит є складним економічним явищем, має вплив на весь спектр економічних відносин в Україні й може розглядатися у контексті гарантування економічної безпеки держави. Визначено сутнісні характеристики дефіциту бюджету у таких аспектах: за причинами виникнення, типами, формами прояву, зв’язком із державним боргом, термінами дії. Метою дослідження є поглиблення теоретичних засад виникнення дефіциту бюджету та розробка рекомендацій щодо підвищення ефективності управління ним. Основним завданням наукової статті є дослідження економічної суті та виявлення основних причин дефіциту бюджету.Статья посвящена проблеме дефицита государственного бюджета в условиях формирования социально направленной рыночной экономики. В исследовании обоснованно экономическую сущность и обнаружены основные факторы существования негативного сальдо бюджета. Определенно условия использования дефицита государственного бюджета как инструмента влияния на экономические процессы. Обоснованно, что бюджетный дефицит является сложным экономическим явлением, имеет влияние на весь спектр экономических отношений в Украине и может рассматриваться в контексте гарантирования экономической безопасности государства. Определенно сущностные характеристики дефицита бюджета в таких аспектах: за причинами возникновения, типами, формами проявления, связью, с государственным долгом, сроками действия. Цель исследования – углубление теоретических принципов формирования дефицита бюджета и разработка рекомендаций относительно повышения эффективности управления им. Основным заданием научной статьи является исследование экономической сути и выявление основных причин дефицита бюджета.The article is devoted to the problem of deficit of the state budget in the conditions of forming the socially directed market economy. The economic essence and the basic factors of existence of negative balance of the budget have been interpreted and identified. The conditions of the use of deficit of the state budget as the instrument of influence on economic processes have been determined. The purpose lies in principles of forming the deficit of budget and developing recommendations in relation to the increasing management efficiency. The basic task of the scientific article is to investigate the economic essence and identify the principal reasons for deficit of budget

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

<p>Abstract</p> <p>Background</p> <p>Plasmid-less, engineered <it>Bacillus </it>strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid.</p> <p>Results</p> <p>A plasmid-less <it>B. subtilis </it>JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the <it>mpr </it>gene from <it>B. amyloliquefaciens</it>) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an <it>mpr^B.amy</it>cassette in which the GSP gene was placed between the promoter of the <it>B. amyloliquefaciens rplU-rpmA </it>genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the <it>mpr^B.amy</it>cassette was carried on a multi-copy plasmid.</p> <p>Conclusion</p> <p>A novel strategy for ectopically integrating a cassette into multiple random locations in the <it>B. subtilis </it>chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells.</p

Use of the λ Red-recombineering method for genetic engineering of Pantoea ananatis

<p>Abstract</p> <p>Background</p> <p><it>Pantoea ananatis</it>, a member of the <it>Enterobacteriacea </it>family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of <it>Pantoea ananatis </it>have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in <it>E. coli </it>and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.</p> <p>Results</p> <p>In this study, the expression of λ Red genes in <it>P. ananatis </it>was found to be highly toxic. A screening was performed to select mutants of <it>P. ananatis </it>that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ <it>gam</it>, <it>bet</it>, and <it>exo </it>genes. Using this strain, procedures for fast introduction of multiple rearrangements to the <it>Pantoea ananatis </it>genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated.</p> <p>Conclusion</p> <p>The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected <it>P. ananatis </it>recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different <it>P. ananatis </it>strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.</p

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

<p>Abstract</p> <p>Background</p> <p>The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on <it>in vitro </it>and <it>in vivo </it>recombination processes have been developed.</p> <p>Results</p> <p>Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant <it>E. coli </it>strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-<it>attB </it>sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-<it>attP</it>-site, and second, the λ system is used for excision of inserted vector part, including the plasmid <it>ori</it>-replication and the marker, flanked by λ-<it>attL/R</it>-sites.</p> <p>Conclusion</p> <p>The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.</p

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

<p>Abstract</p> <p>Background</p> <p>RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.</p> <p>Results</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed <it>in vitro </it>linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, <it>repB</it>, the plasmid loci <it>oriT</it>, <it>mobC </it>and <it>mobA </it>were substituted by the DNA fragment containing P_{<it>lac</it>UV5}→<it>lacI</it>. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after <it>lacI </it>elimination. High stability of both constructed plasmids has been demonstrated in <it>Escherichia coli </it>and <it>Pantoea ananatis</it>. Design of RSFmob allows easy substitution of P_{<it>lac</it>UV5}by any desirable promoter for construction of novel derivatives with changed copy number or host range.</p> <p>Conclusion</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in <it>E. coli </it>and <it>P. ananatis </it>have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.</p

The complete genome sequence of Pantoea ananatis AJ13355, an organism with great biotechnological potential

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds