Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27

BACKGROUND: DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε). RESULTS: Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function. CONCLUSIONS: The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one

Experiments and analysis advance R2100 distance sensors used for safety systems of TOMI

In order to increase safety systems reliability of TOMI harvester, it is necessary to use advance R2100 Distance sensors which can scan all kinds of targets and receive data from automatic control system. The Structure and function of R2100 Distance sensors were provided in this paper, In order to determine the best application function of the R2100, effectiveness of R2100 sensors used for TOMI robot with robotic cutting forage were tested and analyzed. For application in precision agricultural engineering automatic control safety systems, static tests were applied with a box, cylinder, cone and person as 4 target samples which were set at different points and lines with each segment at 8° angle within 11 segments, the target samples were set at 0°, 14°, 44°, 74° and 88° angles with the reference of the sensor at centre, respectively, samples represent obstacles such as tractors, telegraph pole, car, and person which were detected and received by TOMI equipped with R2100 Distance sensors. TOMI Robot equipped with R2100 sensors setting at 240, 420 and 850 mm height, respectively, were set location at about 0.2m, 0.3m, 0.5m, 1m, 1.5m, 2m, 2.5m, 3m, 3.5m and then added up to 0.5 m step up to 10 m with the reference of R2100 sensor in semicircle centre, respectively. In dynamistic testing, the target samples were set at the same method and location, and TOMI robot equipped with Advance R2100 sensors was running at speed of 0.8~1.2 m/s from 5 m to the test centre in dynamistic tests. Tests and statistical evaluate results showed that the average R2 on TOMI robot was up to 98.96% in static tests, while the average R2 is up to 98.67% in dynamistic test, and as far as TOMI robot’s safety system, 420 mm height was the best location for scanning all kinds of obstacles. The experiment results showed that the Advance R2100 was accurate sensor for application, it had been carried out on TOMI's intelligence safety systems which more practical and safety working in various fields

Design and evaluate intelligent control safety systems on the TOMI robot

Aimed to design safety systems and evaluate the behaviours about Robot grass cutting named TOMI, the false tree analysis, failure mode effects tree analysis methods were used for review and analysis about the TOMI robot, it would be liability and legislation; TOMI robot management embedded guidelines and knowledge such as Agricultural Engineering, Design and manufacture of agricultural machinery, Mechanic Theory, Mathematics, Electronics, Grass science, Computer science and several software programs; Procedure and Reliability analysis for robots TOMI safety systems are key features,the safety systems of Agricultural Robot such as TOMI should be checked in various working circumstance; With the full consideration of engineering practicability, the solutions to the safety problems of the TOMI robot are promoted, Technology Route and models about TOMI’s safety system were built, Process Management, continual improvement tools and Techniques and effects analysis were built in the new safety systems of TOMI robot. TOMI function measurements such as braking, throttle and pedal force were tested and analysed. TOMI’s Mechanical system, Hydraulics and Electrical Systems were tested for checking safety and evaluated, some sensors and laser such as Distance sensors, SICK, GPS, Dead man handle, safety red button and bumpers were built up and developed the TOMI robot’s new safety systems; To ensure the safety and reliable operation is a system engineering, it is involved to various TOMI robot design, production, operation, adjust, and management; to improve the TOMI robot reliability and reduce the failure frequency was an important way to improve the robot inherent safety; The Evaluation Criteria of Robot grass Cutting DFMEA occurrence may be suggested to use multiple complex technology knowledge and design with more experience. Application built with Microsoft Robot Development studio was run over on the www.webfarming.com. The hazard and risk analysis were detailed about the safety problems of TOMI robot and deeply studied. Development more practical and safety TOMI robot would be carried out at northwest China in the future

PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA

Consumer exposure to biocides - identification of relevant sources and evaluation of possible health effects

<p>Abstract</p> <p>Background</p> <p>Products containing biocides are used for a variety of purposes in the home environment. To assess potential health risks, data on products containing biocides were gathered by means of a market survey, exposures were estimated using a worst case scenario approach (screening), the hazard of the active components were evaluated, and a preliminary risk assessment was conducted.</p> <p>Methods</p> <p>Information on biocide-containing products was collected by on-site research, by an internet inquiry as well as research into databases and lists of active substances. Twenty active substances were selected for detailed investigation. The products containing these substances were subsequently classified by range of application; typical concentrations were derived. Potential exposures were then estimated using a worst case scenario approach according to the European Commission's Technical Guidance Document on Risk Assessment. Relevant combinations of scenarios and active substances were identified. The toxicological data for these substances were compiled in substance dossiers. For estimating risks, the margins of exposure (MOEs) were determined.</p> <p>Results</p> <p>Numerous consumer products were found to contain biocides. However, it appeared that only a limited number of biocidal active substances or groups of biocidal active substances were being used. The lowest MOEs for dermal exposure or exposure by inhalation were obtained for the following scenarios and biocides: indoor pest control using sprays, stickers or evaporators (chlorpyrifos, dichlorvos) and spraying of disinfectants as well as cleaning of surfaces with concentrates (hydrogen peroxide, formaldehyde, glutardialdehyde). The risk from aggregate exposure to individual biocides via different exposure scenarios was higher than the highest single exposure on average by a factor of three. From the 20 biocides assessed 10 had skin-sensitizing properties. The biocides isothiazolinone (mixture of 5-chloro-2-methyl-2H-isothiazolin-3-one and 2-methyl-2H-isothiazolin-3-one, CMI/MI), glutardialdehyde, formaldehyde and chloroacetamide may be present in household products in concentrations which have induced sensitization in experimental studies.</p> <p>Conclusions</p> <p>Exposure to biocides from household products may contribute to induction of sensitization in the population. The use of biocides in consumer products should be carefully evaluated. Detailed risk assessments will become available within the framework of the EU Biocides Directive.</p

Mechanism of Dinitrochlorobenzene-Induced Dermatitis in Mice: Role of Specific Antibodies in Pathogenesis

Dinitrochlorobenzene-induced contact hypersensitivity is widely considered as a cell-mediated rather than antibody-mediated immune response. At present, very little is known about the role of antigen-specific antibodies and B cells in the development of dinitrochlorobenzene-induced hypersensitivity reactions, and this is the subject of the present investigation.Data obtained from multiple lines of experiments unequivocally showed that the formation of dinitrochlorobenzene-specific Abs played an important role in the development of dinitrochlorobenzene-induced contact hypersensitivity. The appearance of dinitrochlorobenzene-induced skin dermatitis matched in timing the appearance of the circulating dinitrochlorobenzene-specific antibodies. Adoptive transfer of sera containing dinitrochlorobenzene-specific antibodies from dinitrochlorobenzene-treated mice elicited a much stronger hypersensitivity reaction than the adoptive transfer of lymphocytes from the same donors. Moreover, dinitrochlorobenzene-induced contact hypersensitivity was strongly suppressed in B cell-deficient mice with no DNCB-specific antibodies. It was also observed that treatment of animals with dinitrochlorobenzene polarized Th cells into Th2 differentiation by increasing the production of Th2 cytokines while decreasing the production of Th1 cytokines.In striking contrast to the long-held belief that dinitrochlorobenzene-induced contact hypersensitivity is a cell-mediated immune response, the results of our present study demonstrated that the production of dinitrochlorobenzene-specific antibodies by activated B cells played an indispensible role in the pathogenesis of dinitrochlorobenzene-induced CHS. These findings may provide new possibilities in the treatment of human contact hypersensitivity conditions

EEG-fMRI Based Information Theoretic Characterization of the Human Perceptual Decision System

The modern metaphor of the brain is that of a dynamic information processing device. In the current study we investigate how a core cognitive network of the human brain, the perceptual decision system, can be characterized regarding its spatiotemporal representation of task-relevant information. We capitalize on a recently developed information theoretic framework for the analysis of simultaneously acquired electroencephalography (EEG) and functional magnetic resonance imaging data (fMRI) (Ostwald et al. (2010), NeuroImage 49: 498–516). We show how this framework naturally extends from previous validations in the sensory to the cognitive domain and how it enables the economic description of neural spatiotemporal information encoding. Specifically, based on simultaneous EEG-fMRI data features from n = 13 observers performing a visual perceptual decision task, we demonstrate how the information theoretic framework is able to reproduce earlier findings on the neurobiological underpinnings of perceptual decisions from the response signal features' marginal distributions. Furthermore, using the joint EEG-fMRI feature distribution, we provide novel evidence for a highly distributed and dynamic encoding of task-relevant information in the human brain

Direction and magnitude of nicotine effects on the fMRI BOLD response are related to nicotine effects on behavioral performance

Considerable variability across individuals has been reported in both the behavioral and fMRI blood oxygen level-dependent (BOLD) response to nicotine. We aimed to investigate (1) whether there is a heterogeneous effect of nicotine on behavioral and BOLD responses across participants and (2) if heterogeneous BOLD responses are associated with behavioral performance measures. In this double-blind, placebo-controlled, cross-over study, 41 healthy participants (19 smokers)—drawn from a larger population-based sample—performed a visual oddball task after acute challenge with 1 mg nasal nicotine. fMRI data and reaction time were recorded during performance of the task. Across the entire group of subjects, we found increased activation in the anterior cingulate cortex, middle frontal gyrus, superior temporal gyrus, post-central gyrus, planum temporal and frontal pole in the nicotine condition compared with the placebo condition. However, follow-up analyses of this difference in activation between the placebo and nicotine conditions revealed that some participants showed an increase in activation while others showed a decrease in BOLD activation from the placebo to the nicotine condition. A reduction of BOLD activation from placebo to nicotine was associated with a decrease in reaction time and reaction time variability and vice versa, suggesting that it is the direction of BOLD response to nicotine which is related to task performance. We conclude that the BOLD response to nicotine is heterogeneous and that the direction of response to nicotine should be taken into account in future pharmaco-fMRI research on the central action of nicotine

Lysyl hydroxylase 3 localizes to epidermal basement membrane and Is reduced in patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

<p>Abstract</p> <p>Background</p> <p>Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.</p> <p>Results</p> <p>We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.</p> <p>Conclusions</p> <p>A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.</p