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Airway epithelial cells are exposed to environmental toxicants that result in airway injury. Naphthalene (NA) causes site-selective damage to Clara cells in mouse distal airways. N-terminally truncated recombinant human keratinocyte growth factor (ΔN23-KGF) protects against acute lung injury. The present study investigated whether or not ΔN23-KGF protects against NA-induced acute Clara cell damage by measuring airway responses specifically and in order to identify underlying molecular mechanisms.
Mice were treated with ΔN23-KGF or PBS 33 h prior to injection of 200 mg·kg body weight−1 NA. Lung function was analysed by head-out body plethysmography. Distal airways isolated by microdissection were assessed for cell permeability using ethidium homodimer-1. Immunohistochemistry of Clara cell-specific protein in conjunction with a physical dissector was used to quantify Clara cell numbers. RNA was isolated from frozen airways in order to analyse gene expression using quantitative RT-PCR.
ΔN23-KGF prevented NA-induced airflow limitation and Clara cell permeability, and resulted in twice as many Clara cells compared with PBS pre-treatment. ΔN23-KGF-pre-treated mice exhibited increased expression of proliferating cell nuclear antigen mRNA. Cytochrome P450 isoform 2F2, which converts NA into its toxic metabolite, was reduced by ∼50%.
The present results demonstrate that pre-treatment with N-terminally truncated recombinant human keratinocyte growth factor protects against naphthalene-induced injury. This suggests that N-terminally truncated recombinant human keratinocyte growth factor exerts its beneficial effect through a decrease in the expression of cytochrome P450 isoform 2F2.
Airway injury, repair and remodelling are implicated in the pathogenesis of diverse lung diseases, such as asthma, chronic obstructive pulmonary disease and lung cancer 1. Among the many agents that cause lung injury, hazardous air pollutants have chronic adverse effects on lung function 2, and are likely to contribute significantly to morbidity and mortality in humans 3. The airway epithelium is composed of a few major cell types, ciliated epithelial cells, Clara cells, neuroepithelial cells, basal cells, mucous cells and serous cells 4. It provides a selectively permeable barrier between the internal tissues and the potentially hazardous agents entering the body via the airways. In particular, the distal airways are exposed at high levels to various environmental oxidants, which may lead to epithelial injury in this specific region 5.
Naphthalene (NA), a harmful environmental toxin, can be found in ambient air, ground water and cigarette smoke and is produced by combustion 6. NA is used as feedstock in various chemical industrial processes and was suggested to pose a problem in the workplaces of various industries 7. NA, which has been found to be the most abundant polycyclic aromatic hydrocarbon in sidestream cigarette smoke 8, selectively injures nonciliated Clara cells of the conducting airways. The toxicity of NA requires metabolic activation, which is catalysed by cytochrome P450 monooxygenases 9. In addition to their general secretory role 10, mouse Clara cells are the primary cellular site of cytochrome P450 monooxygenase isoform 2F2 (CYP2F2) 11, an enzyme with both high catalytic activity for NA and great abundance in the mouse airway 12. Therefore, murine Clara cells are more susceptible to NA-induced cytotoxic injury than other types of airway epithelial cell 13.
Keratinocyte growth factor (KGF), also known as fibroblast growth factor (FGF) 7, has been identified as an important paracrine mediator of proliferation, migration and differentiation of alveolar epithelial type II cells 14. KGF binds specifically to the KGF receptor, a splice variant of FGF receptor 2 (isoform IIIb), which is expressed by epithelial cells 15. In animal studies, KGF reduces alveolar injury and mortality in rat and/or mouse models of acute lung injury to the gas exchange area 14. Knowledge is much more limited with regard to the role of KGF in acute airway epithelial injury and repair. KGF was demonstrated to be important for the detoxification of reactive oxygen species 16, and to enhance proliferation in airway cells 17. Administration of KGF was shown to protect against radiation-induced increases in airway epithelial barrier permeability 18, exert protective effects against oxidant injury 19 and limit allergen-induced alterations in airway epithelial integrity 20. Furthermore, KGF accelerated epithelial wound closure in bronchial epithelial cells cultured in vitro 21, and studies in the heterotopic tracheal transplant model revealed that KGF enhanced airway epithelial repair involving both local resident progenitor epithelial cells and the mobilisation and engraftment of circulating epithelial progenitor cells 22.
Despite the important function of KGF in the lung, the role of KGF in the airways is not well understood. Microarray analyses of human primary bronchial epithelial cells incubated with KGF in vitro revealed a decrease in the expres-sion of CYP2F1 mRNA 23. Human CYP2F1 exhibits 80% homology with mouse CYP2F2 and is also expressed in the respiratory tract 24. Therefore, it was hypothesised that pre-treatment of lungs with N-terminally truncated recombinant human KGF (ΔN23-KGF; palifermin), which is characterised by enhanced protein stability 25, protects against NA-induced acute Clara cell damage of the murine distal airways. In order to address this question, four experimental approaches were used: 1) distal airways from mice pre-treated with ΔN23-KGF or PBS were isolated by microdissection 26 and analysed for cell membrane integrity, cell proliferation and Clara cell number following NA-induced injury; 2) serial noninvasive lung function analysis was performed in unanaesthetised mice by means of head-out body plethysmography 27 in order to assess the functional relevance of NA-induced acute airway injury and the potential protective effect of ΔN23-KGF at the organ level; 3) the effect of KGF treatment on mRNA expression of CYP2F2, which catalyses the conversion of NA into its cytotoxic 1R,2S-oxide 12, was analysed in microdissected distal airways, as well as in Clara cell-enriched pulmonary epithelial cells isolated from mouse lungs; and 4) mice were therapeutically treated with ΔN23-KGF at different time-points following the injection of NA in order to test the hypothesis that the protective effect of KGF treatment was dependent upon the presence of Clara cells.
Pathogen-free male C57BL/6 mice (Harlan Winkelmann, Hanover, Germany) weighing 25–29 g and aged 8–10 weeks were used in all of the experiments. The animals were allowed free access to food and water. All of the animals were euthanised by an over-dose of sodium pentobarbital (100 mg·kg body weight−1 intraperitoneally) followed by exsanguination. All animal procedures were performed according to the guidelines of good animal experimental practice of the Philipps University of Marburg (Marburg, Germany) and approved by the local authorities for animal experiments.
Application of growth factor
Mice were anaesthetised by short-term inhalation of isoflurane (Abbot, Wiesbaden, Germany). Each mouse received a single bolus of 10 mg·kg body weight−1 ΔN23-KGF (Amgen, Thousand Oaks, CA, USA) by oropharyngeal aspiration or an equivalent volume of PBS (80 μL). This dose was found to induce the maximal proliferative response of alveolar epithelial type II cells in mice 28. Treatment was performed 33 h prior to intraperitoneal injection of NA or corn oil.
Application of NA
NA was purchased from Fisher (Aschaffenburg, Germany) and dissolved in corn oil. C57BL/6 mice were injected with 200 mg·kg body weight−1 NA or corn oil alone as vehicular control and were sacrificed 12 h later. This time-point was chosen because experiments studying the kinetics of NA-induced acute airway injury revealed that, after 12 h, Clara cells were already damaged but exfoliation was still incomplete, whereas complete exfoliation was observed 24 h after injection of NA (see online supplementary material). Distal airways were microdissected and processed for histological analysis. In order to evaluate the protective effect of ΔN23-KGF, mice were pre-treated with ΔN23-KGF or PBS. Mice were sacrificed 12 h after NA treatment and distal airways were isolated by microdissection. In order to exclude effects of circadian rhythm, all applications were performed between 08:00 and 10:00 h.
In order to evaluate whether the effect of ΔN23-KGF is primarily related to Clara cells, mice were treated with 200 mg·kg body weight−1 NA 2 or 24 h prior to oropharyngeal aspiration of 10 mg·kg body weight−1 ΔN23-KGF. Animals were sacrificed 5 days later.
Tissue preparation and microdissection
Procedures for the microdissection of defined airway generations have been described in detail previously 26. Briefly, after sacrifice by cervical dislocation, the trachea was exposed and cannulated. The lungs were removed from the thorax and inflated with 1% low melting-point agarose (Sigma-Aldrich, Munich, Germany) dissolved in 2× Waymouth's medium (Sigma-Aldrich) and incubated in Ham's F-12 nutrient mixture (Sigma-Aldrich) for 30 min on ice in order to allow the agarose to solidify. Distal airways were isolated from the left lung of each mouse.
Assessment of cell permeability
For cell permeability studies, microdissected airways were incubated with ethidium homodimer (EH)-1 (Sigma-Aldrich) in Ham's F-12 nutrient mixture for 20 min at 37°C in order to label the nuclei of any cells with permeable membranes, as described previously 29. Airways were washed three times with F-12 medium at 37°C to remove unincorporated EH-1 and fixed with 330 mOsm Karnovsky's fixative. Airways were stored in fixative in the dark until embedment into glycol methacrylate (Technovit 7100; Heraeus Kulzer, Wehrheim, Germany). Embedded airways were cut into 1-μm thick sections. A water soluble nonfluorescing mounting medium, Fluoromount-GTM (Southern Biotechnology, Birmingham, AL, USA), was used.
For imaging, an epifluorescent microscope (Olympus, Tokyo, Japan) equipped with a wide-band ultraviolet (UV) fluorescence excitation–emission filter set was used. As there is a phase shift in the wide-band UV excitation–emission filter set, the tissue autofluoresces green and EH-1-positive nuclei pinkish-white.
Airway fixation
For quantitative morphological analysis, at least four microdissected distal airways from each mouse (n = 3) were fixed in 1% phosphate-buffered formalin, orientated such that the resulting sections were normal to the longitudinal axis of the airways, and embedded in paraffin. For RNA isolation and subsequent gene expression analyses, three microdissected airways from each mouse (n = 3) were snap frozen in liquid nitrogen and stored at -80°C.
As described previously 17, cross-sections of airways 2 μm in thickness were deparaffinised in xylene and rehydrated in ethanol and PBS. Endogenous peroxidase activity was inactivated using 1% hydrogen peroxide in methanol (Roth, Karlsruhe, Germany; pH 7.2) for 30 min. Antigen retrieval was performed by microwave treatment in 3% citrate buffer (Roth; pH 6.0). After washing in PBS, the sections were incubated in PBS containing 1% bovine serum albumin (Serva, Heidelberg, Germany) for 30 min followed by incubation with a polyclonal rabbit antibody (courtesy of J. Klug, Justus Liebig University, Marburg, Germany) directed against Clara cell-specific 10-kDa protein (CC10) diluted 1:3,000 in the same solution for 1 h at 37°C. Sections were then incubated with an anti-rabbit secondary antibody diluted 1:10 for 30 min at room temperature. This was visualised using 3,3'-diaminobenzidine as chromogen according to the ABC method (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and following the manufacturer's instructions. In order to identify ciliated cells and cell proliferation, immunostaining was performed using mouse monoclonal antibodies directed against β-Tubulin-IV (Bio Genex, San Ramon, CA, USA; 1:150 dilution) and against proliferating cell nuclear antigen (PCNA; Dako Cytomation, Hamburg, Germany; 1:600 dilution), respectively. Antibodies were detected using a mouse-on-mouse immunodetection kit (Vector MOM kit; Vector Laboratories). All sections were counterstained with haematoxylin.
Quantitative–morphological analysis
The number of Clara cells was determined by design-based stereology using a physical dissector 30. Clara cell numbers were normalised to the area of the airway epithelial basal lamina. In addition, the arithmetic mean thickness of Clara cells was estimated by point and intersection counting. Four to six paraffin-embedded airways were studied per mouse. From each airway, two serial cross-sections were cut using a motorised microtome with a block-cooling device (H 355 S; Microm, Walldorf, Germany), which was calibrated for block advance. Sections 2 μm in thickness were stained for CC10 and analysed by means of a computer-assisted Olympus BX 51 light microscope, which was equipped with a CAST-Grid System (Olympus, Ballerup, Denmark).
The arithmetic mean thickness of Clara cells (τ̄CC) was calculated using the following equation:
τ̄ = 2PCC·lc/πIBL,(1)
where PCC is the sum of points that hit the Clara cells, lc is the length per point on a cycloid grid and IBL is the sum of intersections of cycloid test lines with the epithelial basal lamina.
The number of Clara cells (nCC) was calculated, using Equation 2, from their numerical density (nCC per volume of epithelium (VEPI;nVCC,EPI)), which was determined using Equation 3 and normalised to the surface area of the basal lamina (SBL) in the same reference volume, estimated using Equation 4:
nSCC,BL = nVCC,EPI/SVBL,EPI(2)
nVCC,EPI = 0.5ct/PEPI·Afr·H(3)
SVBL,EPI = πIBL/2lc·PEPI(4)
where nSCC,BL is the nCC per SBL, SVBL,EPI the SBL per VEPI, ct the number of nuclear profiles of CC10 immunoreactive cells in the look-up section of the dissector, which are not seen in the reference section, PEPI the sum of points hitting airway epithelium, Afr the area of the unbiased counting frame and H the height of the dissector (i.e. the calibrated mean block advance of 2 μm). Since counting was performed in both directions (double dissector), ct was multiplied by 0.5 in Equation 2.
RNA isolation and real-time RT-PCR
Total RNA was isolated from microdissected airways using the SV Total RNA Isolation System (Promega, Mannheim, Germany), and cDNA was synthesised using the Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time RT-PCR was performed using the QuantiTect® SYBR® Green Master Mix (Abgene, Hamburg, Germany) and BioRad ICycler (BioRad Laboratories, Munich, Germany). QuantiTect® SYBR® Green Master Mix (10 μL), 1 μL of each primer at a concentration of 50 pmol·μL−1 and 20 μL water were added to 3 μL cDNA, standard or water (negative control). Primer sequences were generated from the respective mRNA sequences obtained from the European Molecular Biology Laboratory gene bank (Heidelberg, Germany; table E1 of online supplementary material). Primers were synthesised by MWG Biotech (Ebersberg, Germany). Change in cycle threshold analysis was used to determine expression in comparison to reduced glyceraldehye-3-phosphate dehydrogenase (GAPDH). In additional, because NA caused Clara cell exfoliation and the CYP2F2 isozyme is predominantly expressed in Clara cells, CYP2F2 mRNA expression was determined relative to CC10 mRNA expression.
Effects of ΔN23-KGF on Clara cells cultured in vitro
Mouse Clara cells were isolated from C57BL/6 mice using the protocol described by Elizur et al. 31, from which a Clara cell purity of 70% was obtained. Briefly, the lung was perfused free of blood with PBS via the right ventricle, 1 mL 1% low melting-point agarose in PBS was instilled through the trachea followed by 0.5 mL 0.25% bovine pancreatic trypsin (Sigma-Aldrich). Following incubation on ice, the lungs were excised and incubated for 10 min at 37°C. The tissues were diced in medium (Dulbecco's modified Eagle medium containing 250 μg·mL−1 DNase and 2× antibiotic/antimycotic) filtered sequentially through 100- and 40-μm filters. The final pellet was resuspended in 10 mL medium. For experiments, cells were placed into 35-mm cell culture plates. Cells received fresh medium supplemented with either ΔN23-KGF (50 ng·mL−1) or an equivalent volume of PBS as control. After 1 and 4 h of in vitro culture at 37°C, the RNA Lysis buffer (SV Total RNA Isolation System) covering the cells was collected and the cells harvested for RNA isolation and subsequent real-time RT-PCR analysis as described above.
Noninvasive measurement of lung function
Immediately prior to sacrifice, mid-expiratory airflow (EF50; measured in mL·s−1), a parameter suitable for monitoring broncho-obstruction 27, was measured using head-out body plethysmography as described previously 32. Briefly, the system consisted of a glass body to which four head-out body plethysmograph chambers were attached (Forschungs-werkstätten, Hanover Medical School, Hanover, Germany). A calibrated pneumo-tachograph (PTM 378/1.2; Hugo Sachs Electronics, March-Hugstetten, Germany) and a differential pressure transducer (8T-2; Gaeltec, Dunvegan, UK) coupled to an amplifier (Gould Universal Amplifier; Gould, Dietzenbach, Germany) were attached to the top port of each plethysmograph chamber. The amplified analogue signal from the pressure transducer was digitised via an analogue-to-digital converter (DAS-16; Keithley, Germering, Germany) at a sampling frequency of 2,000 s−1. For airflow measurements, one mouse was placed into each chamber with the head protruding through a flexible neck collar so that the chamber was sealed in an airtight fashion against the main glass body. Airflow was measured during a 15-min period after the animals had resumed normal breathing. EF50 measured following treatment with ΔN23-KGF and/or NA were expressed as percentage changes from the mean baseline value of the respective control group.
Statistical analysis
Data are presented as mean±sd unless otherwise stated. One-way ANOVA was used to determine the significance of differences observed between groups if normality and equal variance corresponded to p>0.1. Otherwise, Kruskal–Wallis ANOVA on ranks was used. A p-value of <0.05 was considered significant.
Pre-treatment with ΔN23-KGF protects Clara cells against naphthalene-induced necrosis
Failure of cell membrane integrity was assessed using EH-1, a sensitive indicator of irreversible cell injury 29. EH-1-positive cells, which were identified as Clara cells based on morphological characteristics, were abundant in the distal airways of mice 12 h after injection of 200 mg·kg body weight−1 NA (fig. 1b⇓), whereas EH-1-positive cells were completely absent from control mice (fig. 1a⇓) or mice treated with ΔN23-KGF (fig. 1c⇓). Pre-treatment of lungs with ΔN23-KGF prior to injection of NA markedly reduced the appearance of EH-1-positive cells in distal airway epithelium (fig. 1d⇓).
ΔN23-KGF induces PCNA activation and airway epithelial proliferation
PCNA is a proliferation marker as its gene is expressed from early in the G1 phase to the S phase of the cell cycle, with the protein confined to the nucleus. Epithelial cells showing nuclear staining for PCNA were rarely observed in untreated airways (fig. 2a⇓). Immunoreactivity for PCNA was regularly observed in airway epithelial cells of mice 12 h after injection of NA (fig. 2b⇓). However, PCNA-positive cells were abundant in the distal airways of lungs pre-treated with ΔN23-KGF (fig. 2c⇓ and d).
Quantitative real-time RT-PCR revealed that, in mice pre-treated with ΔN23-KGF 33 h prior to injection of NA, PCNA mRNA expression was significantly increased compared with mice injected with NA that had been pre-treated with PBS (fig. 2e⇑).
Pre-treatment with ΔN23-KGF inhibits Clara cell loss
Clara cells were identified immunohistochemically via CC10, which is also known as CC16, uteroglobin or secretoglobin 33. Clara cell numbers were analysed using design-based stereology with a physical dissector approach. Quantitative morphological analysis revealed that, 12 h after injection of NA, the number of CC10-positive Clara cells per square millimeter of basement membrane (BM; 5,047±829) was reduced to 50% of the Clara cell number characteristic of the distal airways of control mice (10,157±229 cells·mm BM−2). Pre-treatment of lungs with ΔN23-KGF caused a slight increase in Clara cell number after 33 h (12,142±399 cells·mm BM−2) and significantly restricted the loss of Clara cells 12 h after NA treatment (9,404±528 cells·mm BM−2) to only 23% of that of lungs treated with ΔN23-KGF alone (fig. 3⇓).
Consistent with the physical dissector counts of Clara cell numbers, real-time RT-PCR showed significantly reduced CC10 relative to GAPDH mRNA expression in the distal airways of NA-treated mice (4.4% of PBS control), whereas the CC10 mRNA level in airways of mice pre-treated with ΔN23-KGF prior to injection of NA was approximately four-fold higher (fig. 4⇓). KGF has previously been shown to decrease CC10 mRNA expression levels in rat Clara cells 34. Consequently, pre-treatment with ΔN23-KGF alone resulted in a reduction in CC10 mRNA expression compared with PBS-treated control mice despite the increase in proliferation and Clara cell numbers.
ΔN23-KGF downregulates cytochrome P450 2F2 mRNA expression
CYP2F2 is important in the metabolism of NA since it converts NA into a cytotoxic epoxide intermediate. Administration of ΔN23-KGF with or without subsequent injection of NA resulted in significant downregulation of CYP2F2 mRNA expression (fig. 5⇓). This was corroborated by real-time RT-PCR analyses of Clara cell-enriched lung epithelial cells isolated from mouse lungs. After 4 h of incubation of isolated Clara cells with ΔN23-KGF in vitro, the level of CYP2F2 mRNA expression was significantly decreased (fig. 6⇓). Since NA resulted in a significant loss of Clara cells 12 h after injection, normalisation of CYP2F2 to GAPDH mRNA expression is problematic in that a reduction in CYP2F2 mRNA expression might simply reflect the loss of Clara cells. Therefore, CYP2F2 mRNA expression was instead normalised to CC10 mRNA expression. Since treatment with ΔN23-KGF alone reduces CC10 expression, this procedure is likely to result in underestimation rather than overestimation of the effect of ΔN23-KGF on CYP2F2.
Pre-treatment with ΔN23-KGF prevents naphthalene-induced impairment of lung function
In order to assess the functional relevance of NA-induced acute airway injury and of the effect of ΔN23-KGF, airflow was investigated by means of head-out body plethys-mography. At 33 h following administration, ΔN23-KGF or PBS alone had no significant effect on EF50 compared with EF50 recorded immediately prior to pre-treatment (0 h), 0.74±0.18 mL·s−1 in PBS treated mice and 0.78±0.43 mL·s−1 in mice treated with ΔN23-KGF. However, 12 h after injection of NA, there was a significant reduction in EF50 recorded from mice pre-treated with PBS, whereas EF50 did not change in mice pre-treated with ΔN23-KGF (fig. 7⇓).
Therapeutic effect of ΔN23-KGF after naphthalene-induced airway epithelial injury depends on the presence of Clara cells
In order to test the hypothesis that the protective effect of ΔN23-KGF is primarily related to Clara cells, treatment with ΔN23-KGF was performed 2 h after injection of NA, when Clara cells are already injured but still present, or 24 h after injection, a time-point at which Clara cells are almost completely lost from the distal airway epithelium (fig. E1 of online supplementary material). In mice treated with ΔN23-KGF 2 h after injection of NA, Clara cell numbers were significantly higher than in mice injected with NA and only slightly and insignificantly lower than in control mice injected with corn oil (fig. 8⇓). In contrast, in mice receiving ΔN23-KGF 24 h after injection of NA, Clara cell numbers were similar to those found in mice injected with NA and significantly lower than in control mice injected with corn oil.
KGF is a potent mitogen for epithelial cells and has been demonstrated to protect the lung against acute injuries of the pulmonary parenchyma in various animal models 14. However, its potential for protecting against airway epithelial injury has not been well characterised. Therefore, the aim of the present study was to investigate whether or not pre-treatment with ΔN23-KGF or palifermin, which results in enhanced KGF protein stability 25, protects the airway epithelium against NA-induced acute airway injury in vivo. Three experimental approaches were used in addressing this question. These approaches defined different aspects of the cell biology of the early injury process, the effect of treatment on pulmonary function and the effect of KGF treatment on the biology of the airway epithelium. It was found that ΔN23-KGF prevented the NA-induced loss of cell membrane integrity, increased airway epithelial cell proliferation and largely reduced the loss of Clara cells, as well as the impairment of lung function due to NA-induced acute airway injury. The decrease in CYP2F2 mRNA expression in microdissected distal airways, as well as in isolated Clara cells, indicates that ΔN23-KGF exerted its protective effects, at least in part, through downregulation of this NA-metabolising xenobiotic enzyme. The failure of ΔN23-KGF to ameliorate NA-induced injury when applied 24 h after injection of NA, i.e. at a time-point at which Clara cells are almost completely lost from the distal airway epithelium, demonstrates that the protective effect of ΔN23-KGF is primarily related to Clara cells.
The NA model of airway epithelial cell injury has been well established in the mouse 13, 35. The high susceptibility to NA cytotoxicity has been revealed to be associated with the expression of CYP2F2 in Clara cells of the distal airway epithelium of the mouse 12. Although the cytotoxic effect of NA had been extensively investigated in Swiss Webster 9, 36 and FVB mice 37–40, little was known about NA-induced injury to the distal airway epithelium of C57BL/6 mice 41, 42, this being particularly true for the early time course. In order to define the optimal time-point for the analysis of potential protective effects of ΔN23-KGF against NA-induced Clara cell injury, the kinetics of NA-induced effects on the distal airway epithelium were investigated first. The sequence of events in C57BL/6 mice following injection of 200 mg·kg body weight−1 NA was similar to that seen in Swiss Webster mice. At 3 h after injection of NA, Clara cells exhibited slight swelling and onset of vacuolisation, with exfoliation being almost complete by 24 h, which is in line with previous findings in C57BL/6 mice 41, 42. After 12 h, most of the Clara cells were swollen and contained vacuoles, but only half of them were lost by exfoliation. Therefore, this time-point was chosen for revealing the potential protective effects of ΔN23-KGF on NA-induced Clara cell injury.
Previous studies have adapted EH-1 for use on microdissected airways and demonstrated it to be a reliable probe for cells undergoing necrosis 43. Intraperitoneal injection of NA resulted in a marked increase in EH-1-permeable Clara cells by 6 h after injection in Swiss Webster mice 29, and 12 h after injection in C57BL/6 mice in the present study, indicating that NA-induced exfoliation of Clara cells was due to necrosis. Pre-treatment of C57BL/6 mice with ΔN23-KGF largely abolished the incorporation of EH-1 into Clara cell nuclei, which suggests that ΔN23-KGF protects Clara cells of the distal airways against NA-induced necrotic cell death. As a consequence, in mice pre-treated with ΔN23-KGF, only 23% of the Clara cells were lost 12 h after injection of NA, whereas 50% were lost in mice pre-treated with control vehicle, as demonstrated using design-based quantitative morphology 30. Similarly, after injection of NA in mice pre-treated with PBS, CC10 mRNA expression was reduced to <5% of that of control mice, whereas, in mice pre-treated with ΔN23-KGF, expression of CC10 mRNA was 38% of that recorded in controls treated with ΔN23-KGF alone. The reduction in CC10 mRNA expression in ΔN23-KGF-treated animals is probably a consequence of the decrease in the number of CC10 transcripts per cell, demonstrated by in situ hybridisation studies 34, rather than a result of Clara cell exfoliation. The marked loss of Clara cells following injection of NA is likely to affect the barrier function of the airway epithelium and KGF can be suspected to maintain airway epithelial integrity, which is essential to, for example, lung fluid balance, host defence, and clearance or metabolism of inhaled agents 10. This is in line with in vitro studies that have demonstrated KGF to protect monolayers of bronchial epithelial cells against loss of barrier function induced by hydrogen peroxide 19 or irradiation 18.
Head-out body plethysmography was used to assess the effects of airborne chemicals on respiratory function 44. The present study demonstrated for the first time that acute airway epithelial damage caused by NA was associated with significant airway dysfunction, indicating that the highly specific injury to Clara cells resulted in significant functional impairment at the organ level. Whether this is the result of changes in airway permeability or inflammation, a direct effect of NA on airway smooth muscle or an indirect effect due to the alterations in the airway epithelium remains to be elucidated. Pre-treatment of mice with ΔN23-KGF 33 h prior to NA-induced airway injury almost completely prevented the impairment of EF50. In previous studies, comparing noninvasive measurement of EF50 with invasive measurement of pulmonary conductance (GL) and dynamic compliance, very good agreements between EF50, GL and dynamic compliance were observed 45, 46. During airway constriction, the main changes in the tidal flow signal, recorded by head-out body plethysmography, occur during the mid-expiratory phase. Therefore, the EF50 was defined as the tidal flow at the mid-point (50%) of expiratory tidal volume 46. Consequently, EF50 is closely linked to tidal volume and expiration time. However, EF50 was clearly demonstrated to be independent of respiratory frequency 45. Only a slight and insignificant decrease in EF50 was observed 33 h after instillation of ΔN23-KGF, whereas modest-but-significant changes in lung resistance and forced expiratory airflow were seen by invasive lung function analysis 3 days after treatment with ΔN23-KGF in rats 47. These differences may relate to the fact that noninvasive assessment of lung function is less sensitive than invasive measurement of respiratory function parameters in anaesthetised and intubated animals 27, although the possibility that there may be quantitative differences between mice and rats in their response to ΔN23-KGF cannot be ruled out.
Clara cells are the preferred progenitor cell for regeneration of the distal airway epithelium following injury 9, 37, 38. Transdifferentiation of ciliated cells has been suggested to significantly contribute to epithelial repair following NA-induced injury 40. Recently, however, Rawlins et al. 42 suggested that a small population of variant Clara cells, which survive after NA injury, are most probably the progenitors of Clara cells following NA injury. Their data suggest that ciliated cells do not transdifferentiate into Clara cells. Human recombinant KGF was shown to stimulate DNA synthesis and proliferation of human bronchial epithelial cells in vitro and of rat and mouse airway epithelium in vivo. It has previously been demonstrated that the KGF-induced stimulation of distal airway epithelial proliferation was restricted to Clara cells, and peaked between days 1 and 2 following intratracheal instillation of KGF into rat lungs in vivo 17. In the present study, a similar strong induction of mouse Clara cell proliferation in response to exogenous ΔN23-KGF was observed, as evidenced by both PCNA immunohistochemistry and quantitative real-time RT-PCR for detection of PCNA mRNA expression. Consequently, Clara cell numbers were increased by ∼20% in mice 33 h after treatment with ΔN23-KGF compared with control treated mice. At 12 h after NA treatment, no significant proliferation of airway epithelium was observed in C57BL/6 mice treated with NA alone. However, proliferation of distal airway epithelium was demonstrated at later time-points, and was shown to peak at day 2 after injection of NA into Swiss Webster mice 9, 37. Notably, the induction of this endogenous repair process was recently suggested to be related to the upregulation of airway epithelial expression of KGF receptor at day 1 after NA-induced injury 48.
In rats, Northern blot analysis of whole lung homogenate revealed that administration of KGF in vivo led to a significant decrease in CC10 mRNA expression on days 2 and 3 compared with vehicle-treated control lungs and then gradually recovered to normal values 34. In the present study, treatment with ΔN23-KGF resulted in a slight, albeit insignificant, decrease in CC10 mRNA expression in the distal airways of C57BL/6 mice, which might relate to the earlier time-point examined in the present study. In rats, the decrease in CC10 mRNA levels appeared to be due to decreased expression per cell, as assessed by in situ hybridisation 34. Correspondingly, CC10, assessed by quantitative immunohistochemistry, exhibited decreased immunoreactivity per cell in distal rat airway epithelium 17. In response to intratracheal administration of exogenous KGF in vivo, a quantitative shift from Clara cells exhibiting normal strong staining for CC10 to them exhibiting weak immunoreactivity for CC10 was observed. The findings of these studies suggested that the reduction in CC10 mRNA and protein expression was not the result of a decrease in the number of CC10-expressing Clara cells but reflected decreased expression per cell. This is supported by the present findings that Clara cell numbers were increased by 20%, whereas CC10 mRNA expression was concomitantly decreased down to ∼60% in mice treated with ΔN23-KGF compared with vehicle controls.
The species-specific and region-selective cytotoxicity of NA has been directly linked to its metabolism to NA 1R,2S-oxide, which is catalysed by the xenobiotic enzyme CYP2F2 12. In adult mice, expression of CYP2F2 appears to be strictly limited to nonciliated airway epithelial cells 11. Since both ΔN23-KGF and NA had significant effects on the number of Clara cells per distal airway, as shown above, the fact that a decrease in Clara cell numbers per airway could simulate a decrease in CYP2F2 mRNA expression relative to that of GAPDH had to be taken into account. Therefore, the expression of CYP2F2 mRNA was related to that of CC10, which is highly specific for Clara cells 49. Despite the slight reduction in CC10 mRNA expression in lungs pre-treated with ΔN23-KGF, ΔN23-KGF reduced CYP2F2 mRNA expression to ≤25% of that of vehicle controls. Correspondingly, the present in vitro study of Clara cell-enriched lung epithelial cells isolated from C57BL/6 mice revealed a significant reduction in CYP2F2 mRNA expression in cells cultured for 4 h in the presence of ΔN23-KGF. Notably, a population of CC10-positive variant Clara cells, which are maintained within neuroepithelial bodies, was demonstrated to be resistant to NA-induced injury due to low CYP2F2 expression 39. This subpopulation of Clara cells with low CYP2F2 expression was suggested to be critical to the renewal of proximal airway epithelium following NA-induced injury 50. A similar NA-resistant Clara cell subpopulation was located to the bronchiolar–alveolar duct junction and exhibited a number of features characteristic of region-specific endogenous stem cells 39. These data suggest that a reduction in the expression of CYP2F2 mediated by ΔN23-KGF pre-treatment is able to significantly limit the Clara cell cytotoxicity of NA.
The failure of ΔN23-KGF to ameliorate NA-induced injury when applied 24 h after NA-injection, i.e. at a time-point at which Clara cells are almost completely lost from the distal airway epithelium 41, 42, whereas Clara cell loss was significantly reduced by treatment with ΔN23-KGF 2 h after injection of NA, demonstrates that the protective effect of ΔN23-KGF is primarily related to Clara cells. This is in line with previous findings that the proliferative effect of KGF is restricted to Clara cells in rat distal airway epithelium 17.
As reviewed recently, NA is used as feedstock in various chemical industrial processes and is suggested to pose a problem in the workplaces of various industries 6, 7. Hence it is conceivable that therapeutic application of ΔN23-KGF immediately following accidental occupational exposure to high levels of NA might help to limit the development of severe airway epithelial injury, although additional studies in humans are required before such an approach can be translated into clinics.
In summary, it has been demonstrated that pre-treatment of lungs with N-terminally truncated recombinant human keratinocyte growth factor significantly reduced the naphthalene-induced loss of Clara cells by necrosis and prevented the deterioration of respiratory function associated with airway injury. The present in vivo and in vitro findings suggest that the protective effect of N-terminally truncated recombinant human keratinocyte growth factor is mediated, at least in part, by marked downregulation of the expression of cytochrome P450 isoform 2F2, the xenobiotic enzyme responsible for the metabolism of naphthalene into its cytotoxic intermediate. The success of a therapeutic application of N-terminally truncated recombinant human keratinocyte growth factor depends upon the presence of Clara cells.
Support statement
This study was supported, in part, by a grant from the German Research Foundation (Bonn, Germany) to H. Fehrenbach (FE287/8-1). L.S. Van Winkle's role in this research was supported by grants from the National Institute of Health (Bethesda, MD, USA; ES012720) and the State of California's Tobacco-Related Diseases Program (Oakland, CA, USA; 14RT0132). The University of California (Davis, CA, USA) is a National Institute of Environmental Health Sciences Center for Environmental Health Sciences (ES05707) and supported core facilities used in sections of this study.
Statement of interest
None declared.
The authors acknowledge the help of G. John (Dept of Pulmonary Medicine, Philipps University of Marburg, Marburg, Germany) with the cell culture model and for use of laboratory equipment.
This article has supplementary material accessible from www.erj.ersjournals.com
- Received November 19, 2007.
- Accepted March 18, 2008.
- © ERS Journals Ltd


text:
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Opening Ceremony of the 2019 AIIB Annual Meeting: Around eight per cent GDP infrastructure investment key to Asia's socio-economic growth and connecting Asia to the World: AIIB President Jin Liqun
Opening the 2019 Asian Infrastructure Investment Bank Annual Meeting in Luxembourg in the presence of HRH Grand Duke of Luxembourg, Fernand Etgen, Xavier Bettel and Pierre Gramegna, AIIB President Jin Liqun set out the Bank's commitment to drive the green economy through sustainable, adaptable and innovative infrastructure investment.
Joint press conference by Prime Minister, Xavier Bettel and Prime Minister of Ireland, Leo Varadkar
Video of the joint press conference. Topics: European Union/ Brexit
National Day 2019
For the public celebration of the birthday of HRH the Grand Duke, members of the Grand Duke's family and the government will attend the festivities organised in Esch-sur-Alzette, Bourscheid and Luxembourg City on 22nd and 23rd June 2019.
National Day 2019: Official ceremony at the Luxembourg Philharmonie, Luxembourg 23 June 2019 at 10 am
Online registration for the reservation of tickets for the official ceremony on the occasion of National Day 2019 is closed. All available places have been allocated. Thank you for your interest in this event.
Funeral of HRH Grand Duke Jean
The funeral of HRH Grand Duke Jean on May 4, 2019.
Statement of Prime Minister Xavier Bettel on the occasion of the death of HRH Grand Duke Jean
Prime Minister Xavier Bettel's statement on the occasion of the death of HRH Grand Duke Jean
National VIGILNAT Exercise
On Saturday, January 12, 2019, the police and the rescue services will participate in a national anti-terrorist exercise on the site Belval and more specifically in and around the Rockhal and Esch / Belval and at the Emile Mayrisch Hospital Center (CHEM).
Animated videos on the political system in Luxembourg
In line with the governments new internet portal, launched and presented in February 2018, animated videos have been produced to put into evidence, through audiovisual elements, certain aspects of the Luxembourgish political system.
National Day of Remembrance
The National Day of Remembrance is dedicated to the memory of the efforts made by the people of Luxembourg during the Occupation from 1940 to 1945. Several ceremonies were held in Luxembourg on October 7, 2018 to mark the occasion.
NVIDIA and Luxembourg Government Announce Cooperation on Artificial Intelligence and High Performance Computing
NVIDIA and the Grand-Duchy of Luxembourg have established a cooperation to support scientists and engineers in solving society's most important challenges using artificial intelligence and high performance computing.
National Day 2018
For the public celebration of the birthday of H.R.H. the Grand Duke, members of the Grand Duke's family and the government will attend the festivities organised in Esch-sur-Alzette, Larochette and Luxembourg City on 22 and 23 June 2018.
Prime Minister Xavier Bettel in London
The 14th of March 2018, Prime minister Xavier Bettel had a meeting with his British counterpart, Theresa May, at 10 Downing Street. The two prime ministers discussed bilateral relations between the two countries, as well as European and international politics.
Working visit of Xavier Bettel to Dublin
Prime Minister Xavier Bettel traveled for a working visit to Dublin, Ireland, on March 5, 2018. Today, 5th of March 2018, the Prime Minister had a meeting with the Irish Prime Minister Leo Varadkar. The counterparts discussed about the bilateral relations between the two countries, but also about the future of the European Union and particularly Brexit.
New version of the government portal
The www.gouvernement.lu portal unveils a new design, available in four language versions: French, German, English and Luxembourgish.


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You mightn't think accounting is about adding value to your business.
We'd like to change your mind on that.
We are management accountants adding value in the following areas
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Timely information is the cornerstone of any business, now more than ever.
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It's a challenging time when there's lots to consider.And putting in place the right accounting procedures from the start is critical to any future success. At Guardian Accountants Dublin we'll talk you through all the options, and help you choose what's best for you and your business.
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For the last 20 years our accounting software package of choice has been TAS Books – it has been totally reliable, and we had a bureau version here in Guardian that we could use [...]
Many people start off in business as a sole trader, as it is cheap (€20 to register a business name), quick – you can register a business name in 2-5 days in the CRO, and its [...]
Sample Maximum Contribution Rates, as % of Salary The table assumes that the member is married with at least 10 years service at retirement. Existing pension benefits are not [...]


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Malnutrition is a global epidemic, and two organizations have gathered a group of global experts to come up with solutions in the food supply chain that can improve nutrition in emerging economies in just five years.
Can Wasting Less Food Between the Farm and Consumer Improve Nutrition?
Switching out traditional raffia baskets with plastic crates when transporting tomatoes in Nigeria can save fresh, healthy food and have multiple subsequent effects on nutrition in the region.
Opinion | Groups Urge Beef Roundtable to Send Sustainability Plan Back to Drawing Board
The beef industry's poor grazing management, unethical treatment, and environmentally unfriendly practices will rule out credible plans for sustainable beef, unless industry leaders take serious action.
Can Fixing Supply Chains Work as a Nutrition Intervention?
Global Alliance for Improved Nutrition is aligning supply chains with human need, for a better food system between farmer and consumer.


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Mother's Day Gift Ideas: Teas and Teaware that she'll love
Mother's Day is just around the corner on Sunday, May 12th
Bitter tea? Here's how to make your tea taste great
Bitter teabag tea? Change it up. It is all about quality loose leaf tea and how you brew it. You don't need to add sugar to mask bitter tea. Invest in high quality teas that taste naturally sweet and learn
Tea for Healthy Skin
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AI in Social Media
Vote on this story –>>>Read more
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The Latest on President Donald Trump (all times local): 1:28 p.m. President Donald Trump has signed an executive order thatRead more
In a post-truth world, beset by fake news, in which people segregate into information bubbles impenetrable to unfriendly ideas, constitutionalRead more
The drama, disputes and falsehoods that permeated Donald Trump's presidential campaign are now roiling his transition to the White House,Read more
CJ McCollum, Jamal Crawford and Jacob Tamme are among current and former professional athletes on social media to criticize DonaldRead more
Who remembers the short-lived site, Friendster? Six years ago I was asked to sit on a panel at a conferenceRead more
Television viewership for Monday's presidential debate is expected to be high, but you don't need a television to watch. ThereRead more
"…and the whole wide world is whistling." NONONO For years, I've joked that writing is cheaper than therapy. Come toRead more
"Goodbye yellow brick road…where the dogs of society howl." – Elton John My son Zachary had a date last yearRead more


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7th EPNS Research Meeting: 29-30 May 2020
Karolinska University Hospital, Stockholm, Sweden
Child Neurologist researchers at all stages of their career networking, collaborating and building research networks.
Draft programme:
Day 1
10:00 Arrival, registration & welcome coffee
10:45 Welcome
11:00 Plenary lecture: Petter Brodin: "The Neuro-immune Interface in Health and Disease"
11:45 Lunch
13:00 Working groups 1-4:
- Autoimmune and Infectious Diseases
- Neurometabolic
- Stroke
- Movement disorders
16:00 Break
16:30 Working groups 5-8:
- Epilepsy
- Ataxia
- Neonatal Neurology & Developmental Neurology
- Neuromuscular
19:30 Social event
Day 2
09:15 Plenary lecture: Nadia Bahi-Buisson: "How to raise EU projects"
09:45 Plenary lecture: Anna Lindstrand: "Zebrafish models of genetic neurological disorders"
10:30 Break
11:00 Selected projects /workshops
12:30 Awards with presentation from winners. Closure
13:00 Farewell lunch
8 Working Groups: please contact your working group chair if you intend to present a project; email adresses at email@example.com
|EPNS Working group
|Chair 1
|Chair 2
|Autoimmune and Infectious Diseases
|Kevin Rostasy (Germany)
|Martin Häusler (Germany)
|Maja Steinlin (Switzerland)
|Deborah Sival (Netherlands)
|Lieven Lagae (Belgium)
|Floor Jansen (Netherlands)
|Movement Disorders
|Jean-Pierre Lin (UK)
|Michel Willemsen (Netherlands)
|Neonatal Neurology & Developmental Neurology
|Regina Trollmann (Germany)
|Nadia Bahi-Buisson (France)
|Barbara Plecko (Austria)
|Dimitrios Zafeiriou (Greece)
|Thomas Sejersen (Sweden)
|Guenther Bernert (Austria)
|Paediatric Stroke
|Finbar O´Callaghan (UK)
|Lucia Gerstl (Germany)
10 Bursaries will be granted upon individual application: the application process for bursaries will be added to the website nearer the time.
EPNS Research Prizes: Two prizes of 2,500 Euros per person awarded to young neurologists who present outstanding, innovative ideas in research in Paediatric Neurology.


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Join hands with parteners for 10 years and share CEES benefits
China Energy and Environment Summit (brief as CEES), jointly sponsored by National Development and Reform Commission (NDRC), National Energy Administration (NEA) , Ministry of Science and Technology( MOST), Ministry of Environmental Protection(MEP), University of International Business and Economics, China Center for Urban Development, NDRC, Center for International Economic Exchanges, NDRC,State Information Center and Xiamen University has been successfully held for 9 consecutive sessions since 2008(see Attachment 3 ). In 2009, the State Council approved the Summit as the Sub-Forum (The Largest one) of the Global Think-Tank Summit. And former Premier WEN Jiabao, Vice-Premier LI Keqiang (current Premier) of the State Council met with our honourable guests both domestic and international, and delivered speeches
In this 10th-year anniversary, CEES is honored by having had the presence of more than 1000 distinguished guests from home and abroad, which included leaders and officials from the State Council of China, CPPCC, NDRC, NEA, MEP, MOST and state-owned energy companies; the former President of India, leaders from international organizations, the Fortune Global 500 and multinationals; Nobel laureates, academicians of the Chinese Academy of Sciences, Chinese Academy of Engineering and counsellors from the State Council, and has made a lot of new achievements including a series of green/low-carbon/energy-efficient/environmental/ highly- efficient innovations, inspiring advice, classic cases and systematic theory of Green Development. All the observations and research have been included in the two blue books of CEES published by China Economic Publishing House. One is Green Development Global Dream, the other is Theory of Green Development for Ash Haze Dissipation, which have been highly regarded by People's Daily and was translated into 8 languages by Central Compilation and Translation Bureau. Some important achievements have been adopted by the Central Committee of Communist Party of China (CCCPC)and the Central People's Government of PRC, etc. and have had a widespread effect throughout the whole society at home and abroad. CEES has been providing suggestions and advices to the authorities of energy, environment and climate change; promoting green development all across the globe and serving as the guiding and supervising principle for the green campaigns in various industries and regions, the application of green technologies; promoting international technological and economic cooperation on energy, environment and climate change.
We launched a large number of world-class innovative technologies and excellent haze-eliminating products. Many organizations with international influence have become advocators of green development and our partners, including China's national energy and environment competent ministries. We have made leapfrog progress towards our commitment 'more than one hundred million audiences' by gathering tens of thousands of media and institutions to spread ideas of green development and ecological civilization construction over the past 9 years.
In order to enforce decisions and arrangements of green development and ecological civilization construction of CCCPC, The 10th CEES (2017) will be jointly hosted by China Energy and Environmental Research Center, China Center for Urban Development, NDRC. etc. It will be held in Beijing(the Great Hall of the People and/or Xiyuan Hotel) , Mid to late September, 2017. Topics and issues will include Green Development for ash haze dissipation——Green Technology, environmental protection, electricity industry deregulation, renewable energy , efficiency , low carbon , energy saving.
The Summit will invite leaders and experts from NDRC, NEA, MOST, MEP, the Board of Supervisors of Key Large State-Owned Enterprises of the State Council, Ministry of Education ; leaders from international organizations, the Fortune Global 500 and multinationals, foreign ambassadors to China; Nobel laureates, academicians of the Chinese Academy of Sciences , Chinese Academy of Engineering and counsellors from the State Council, to deliver speeches or hold group discussions.
We will consolidate the political, industrial, academic community , media and the audience at home and abroad , and high-end resources and strength of overseas Chinese we have gathered for the past 10 years. We will conduct close cooperation with other countries and international organizations, and organize sub-forums, special project pairing, the 10th anniversary serial activities, domestic and overseas investigation activities of green technology with our new and old partners. We will publish the third blue book and conduct cloud seminars and exchanges. Hundreds of central and frontline media including People's Daily, Xinhua News Agency will cover the forum. The event will be co-hosted by Consulting Center of CEES in Beijing and other organizations which will host the sub-forum or series of activities.
1. Industry cooperation : Continue to host a series of sub-CEES on Green Technology and Environmental Governance ; Promote automobile enterprise and product propagation, cooperation on space solar energy, science, technology and education, green building cultural. The cooperation in areas such as Green energy technology and industry between China and USA, China and Europe, China and Austria will also be addressed
2. International Cooperation: set up an international electricity industry deregulation forum, which will review 20 years deregulation experiences in the world, together with 2 years of experiences in China, foster international power sector reformation, discuss the cooperation of power markets (Asia Pacific, North America, Europe and China) development, exchange experiences among nations such as U.S.A., China, Canada, Germany, France, United Kingdom, North Europe, Australia, Mexico, Philippines, Brazil, Russia, and Singapore. There will be discussions about the interactions between energy storage and power grid operations, renewable energy and green dispatch, demand side response, retail markets, and the revolution of energy industry.
3. Regional cooperation: Promote green environmental protection cooperation in Shenzhen , Guandong and some counties/cities, Fujian; Dalian cooperation in aspects of development and utilization of clean energy; regional cooperation concerning ecological civilization planning in Sichuan province.
We welcome more talents with funds, technologies, and a good knowledge of management, market and economy to join us so as to expand industrial and regional cooperation at home and abroad(see Attachment 1 ). We will systematically sum up and show the green results in the past decade and work together to push the 10th CEES to a higher level in this autumn. We will make breakthrough in key areas such as the innovation and integration of green technology, green ideas and green capital, green transformation of energy, restructuring of the electric power industry as well as the control of key issues on green development and haze eliminating. At the national and local level, we will promote the process of green economy and energy, green energy revolution, green institutional system, green culture and technology, evaluate green standards and the application of new green technology and clean the "haze" from the source in order to set out on a green path with Chinese characteristics, promote social and economic sustainable development for global well-beings.
Finally, we are looking forward to meeting you in Beijing, China.
For more details, please contact us via:
Ms. LIU Fang(CEES Vice -SG & Executive chairman of Sub-CEES), Mr. LI Yonggan
etc.( head of one Sub-CEES) , Ms. Quan Yujie, Ms.Xie, Ms.Zhang, Ms.Lian, Mr.Zhu etc.
Beijing; CCUD -NDRC, NO.5 East Sanlihe Rd, Xicheng District, Beijing, China
Please accept the assurances of my highest consideration.
Yours sincerely,
Attachment 1
WAN Gang ,Vice Chairman of the Chinese People's Political Consultative Conference
(CPPCC) and Minister of Science and Technology
ZHU Zhiwen, Deputy Minister of Education; The Hon.Chinese Speakers of West Coast of the
Straits Summit, CEES 2011
ZHANG Xiaoying, President and chief editor, The Economic Daily
ZHANG Guobao, Member of Standing Committee of CPPCC,P.R.C.; Former Vice Chairman of
the National Development and Reform Commission (NDRC) ; Former Chairman of
National Energy Administration (NEA)
ZHANG Hongtao, Counselor of the State Council ; Chief Engineer of the Ministry of Land
and Resuorces, P.R.C
ZHANG Jing-an, Chairman of the Association of China's High-tech Industrial Development
Zone; Former Vice Minister of Science and Technology
JI Xiaonan, Chairman of the Supervisory Board of the State Council for Key Large State- Owned Enterprises
FU Chengyu , Former General Manager of China National Offshore Oil Corporation
XIE Zhenhua , Chinese minister for climate change negotiations; Deputy Director of Population
Resources and Environment Committee, the CPPCC National Committee; original deputy
director of the NDRC
WEI Jianguo , Vice chairman of China Center for International Economic Exchanges; Former
Vice Minister of Commerce Ministry
※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※ ※
WANG Siqiang, Director-General of Department of Energy Conservation and Technological
Equipment; National Energy Administration (NEA)
WANG Xiji , Academician of Chinese Academy of Science; a distinguished precursor of Chinese
A-bomb, H-bomb, artificial satellite; Consultant of Chinese Academy of Space Technology
WANG Dong, Director and Researcher of Environmental planning institute, the Ministry of
Environmental Protection.
WANG Xiaoming, Director and Researcher of Industry Research Department of Development
Research Center of the State Council
MAO Zengyu, Vice President of China Economic Publishing House
LU Jichuan, Prof.& Deputy Dean of the Research Institute of Chinese Academy of
Management Science
SHI Lishan , Deputy Director-general of Nuclear Power Department , Deputy Director-
general, Department of New Energy and Renewable Energy, NEA
LV Wenbin , Deputy Director-General of Department of Resource Conservation and
Environment Protection, NDRC
QIAO Runling, Vice Chairman of joint CEES, Prof. & Deputy director, China Center for
Urban Development, NDRC;
LIU Fang, Prof., Vice -SG of CEES ; Executive chairman of Sub-Forum , China Energy and Environment Summit; Vice Chairman of Beijing Yishui Environment Investment Co. Ltd.
ZHUANG Rui, Prof. and Vice-Dean of Research Institute of International Economics,
University of International Business and Economics
LI Ming , Prof. & Vice Dean of Chinese Academy of Space Technology(the fifth branch)
(WANG Li, Prof.& Deputy chief researcher, HOU Xinbin, Prof. of Qian Xuesen Laboratory
of Launch Vehicle Technology, China Academy of Space Technology, the China Aerospace
Science and Technology Corp.)
LI kai, Deputy director of the State Information Center
LI Tie , Prof. & chairman of the council, China Center for Urban Development, NDRC;
LI Jun-Feng , Director of National Center for Climate Change strategy and international
Cooperation, NDRC
LI Nuyun, Deputy director of Climate office, State Forestry Administration
HE Bingguang, Director of Major Project Inspector 's Office , NDRC; Former Director-General
Of Department of Resource Conservation and Environment Protection , NDRC
ZHANG Shouying, Prof & Director of Theory Dept., People's Daily
ZHANG Jianhua , Governor of Beijing Rural Commercial Bank, Former Director of China
People's Bank Research Bureau
ZHANG Zhenlong, Vice- SG of China Energy and Environment Summit; Professor of China
Energy and Environment Research Center
CHEN Quan-shi, Professor of Tsinghua university; Director of Electric vehicles sub-committee,
China Auto Standard Committee
LIN Guijun, Prof. & Vice President, University of International Business and Economics
XU Dingming, Counselor of the State Council ; Vice Director General of the State Energy
Advisory Board ,P.R.C
LIN Zhiqin, Director of China Energy and Environment Research Center, Executive Vice-
President & SG of China Energy and Environment Summit, Professor of University of
International Business and Economics.
LIN Yaxian, Diapatched Diretor of Central Huijin Investment LTD, Diretor of China Orient
Asset Management Co., LTD
ZHOU Xiaoxin, Academician of Chinese Academy of Sciences, Honorary President of China
Electric Power Research Institute
ZHAO Yinong, Vice- SG of China Energy and Environment Summit, Professor of China
Energy and Environment Research Center
ZHAO Zhongxiu, Director of International Institute of Low Carbon Economy; Prof. and Vice
President, University of International Business and Economics
JIA Chengzao, Academician of Chinese Academy of Sciences; former deputy President of
China National Petroleum Co.; Member of the CPPC National Committee
GAO Shixian, deputy director of Energy Research Institute of NDRC
GUO Jiaofeng, Deputy Director & Researcher, Resource and Environmental Policy Research
Institution of DRC (Development Research Center of the State Council)
SANG Baichuan, Prof. and Dean of Research Institute of International Economics, University
of International Business and Economics
HUANG Xiaozeng, deputy director of Pollution control department, the Chinese Ministry of
Environmental Protection
GE Chang-Chun, Academician of Chinese Academy of Science; Prof. of Beijing Science and
Technology University
JIANG Qingzhe, Prof. & Secretary-General of the Party Committee of UIBE; the Vice-Standing
President of China Petroleum Enterprises Association
JIANG Liping, Vice President & Senior Engineer of the State Grid Energy Research Institute
XIE Ji , Deputy Director , Department of Climate Change, NDRC, P.R.C
A Leader from Department of Electricity Power, National Energy Administration
A Leader from Bureau of Quality and Safety Supervision for Agro-products, Ministry of
Agriculture of the People's Republic of China
David Mohler , VP & Chief Technology Officer Duke Energy
Irene Giner-Reichl, Chairman of the Global Forum on Sustainable Energy; Vice-chairman of
the Global Network for Renewable Energy in 21st Century; the Austrian ambassador
plenipotentiary in People's Republic of China and Mongolia.
Feng Hsu, Chairman, SSP Committee, National Space Society; Former Sr. Staff & Technical
Manager of NASA & Lockheed Matin USA
LANG Lixin, Vice President of LEXUS China
LI Yonggan , Independent Market Monitor of Texas Electricity Market, experienced in Power
Market design, operations and monitoring
LIU Dongsheng, Energy Market Principal Analyst, ALG Energy Limited, Australia
Mark Hopkins, President of US National Space Society
Steven Chu, Nobel Prize winner in physics; the United States Former-Secretary of Energy.
S.Julio Friedmann, Carbon Management Program Leader at the US Department of Energy
S.Ming SUNG, Asia-Pacific chief Representative of Clean Air Task Force Organization
Sergio C. Trindade , A former United Nations Assistant Secretary-General for Science and
Technology; Nobel Prize Co-Laureate
ZHAI YongPing, Technical General Counsel(Energy) , Sustainable Development & Climate
Chang Department , Asian Development Bank
In areas such as international power sector reformation and electricity market development, there will be distinguished speakers from more than 300 experts and entrepreneurs, who are mainly from countries such as U.S.A. (Southwest, California, Texas, New York, New England, PJM, MISO etc), China (Guangdong, Yunnan etc), Canada, Germany, France, United Kingdom, Nord Pool, Australia, Philippines, Brazil, Russia, Singapore, and Denmark. While the following eight subjects will be discussed, more organizations and experts with new ideas and concepts are welcomed. On the basis of the following eight topics, we welcome Chinese and overseas institutions and experts with new ideas to further join the partner team(please fill in Attachment 2 Receipt form of the 2nd round VIP of the 10th CEES):
1. The value of energy storage in power market and how will it interact friendly with power grid operations?
2. How will traditional energy and renewable compete in the same market?
3. How can market design minimize policy distortion and foster the sustained development of electricity industry?
4. Risks facing load serving entities and mitigation measures
5. Risks of power market development and mitigation measures
6. The sequence of power market development and detailed adjustment
7. The pros and cons of aggressive and progressive power market developments
8. The pros and cons of zonal markets and nodal markets
Attachment 2-1
The 10th (2017) China Energy and Environment Summit
CEES Guests Receipt
2. Speech and Communication: The Hon. speaker ( ); speaker ( ); academic
discussion and exchange of papers ( abstract of paper attached)
3. Participants or Media Reports: senior VIP ( ); VIP ( ); reporter ( )
(Multiple Choice shall be permitted)
(no more than 150 words)
Yonggan etc.( head of one Sub-CEES) , Ms. Quan Yujie, Ms.Xie, Ms.Zhang, Ms.Lian, Mr.Zhu etc.
District, Beijing; CCUD/NDRC , NO.5 East Sanlihe Rd, Xicheng District,
Attachment 3


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History Making Assembly of ITF Leaders
Greenland Taekwon-Do which made history by celebrating its 40 year anniversary on October 12, also hosted a history making assembly of ITF leaders. Meanwhile, young competitors in the Anniversary tournament proudly display their medals. Some of the natural beauty of…Read More
Kukkiwon Director Election Results
In the first round of voting, Grand Master Choi Young Ryul(candidate 1) received 29 votes, Grand Master Oh No Kyun(candidate 3) received 28 votes and Grand Master Kim Hyun Sung(candidate 2) received 4 votes. In the second round Grand Master Choi received 31 votes and Grand Master Oh received 30 votes, with 1 vote invalid. Grand…Read More
Greenland Taekwon-Do Invites Taekwondo Times to 40th Anniversary
TaeKwonDo Times is very grateful to have been invited to Geenland Taekwon-Do's 40th anniversary celebration on October 12. Woo Jin Jung and Dr. George Vitale will be representing our publication. Over 40 years 172,000 students at 11 dojangs have trained under Grand Master Leong Wai Meng's guidance. Grand Master Leong was featured in our Hall…Read More
Greenland TKD Federation 40th Anniversary
1979-2019, Geenlands's Taekwondo Federation is celebrating its 40th birthday on Oct 11 and 12. Congratulations Grand Master Leong.Read More
Taiwan Sports Association Cuts Funding to Taipei Taekwondo Association
The Sports Administration is set to suspend funding for the Chinese Taipei Taekwondo Association (CTTA) for one year after an investigation found that the association's senior management had misappropriated its funds. The agency launched an investigation into the association's finances after receiving a complaint from a member of the board of supervisors that accused CTTA…Read More
Taiwan Strong at World Taekwondo Poomsae Championships
Outstanding start last week for China Taipei at the World Poomsae Championships; topping the medals table with 12 gold on day one. The event at the International Expo Center, in the Chinese city of Wuxi, features 286 athletes from 10 countries. The World Team Championships began last weekend at the same venue, the first time…Read More
Cultural Treasures
We are soliciting applications for "Cultural Treasures". Pioneering individuals who have contributed to TaeKwonDo, by the donation of their time and resources to charities, special needs students or other philanthropic activities. If you know someone that embodies these characteristics please email a resume to firstname.lastname@example.com with Cultural Treasure in the subject line. Individuals selected for…Read More
Why was General Choi Exiled from South Korea? The Truth Revealed.
Ambassador Choi Hong-Hi Two-star Major-General (army serial #10044) This is the second part of the story of General Choi's Tae Kwon Do. The first part ended with his locating the ITF headquarters in Vienna, Austria, which allowed him to disseminate his martial art around the world. POLITICAL INFLUENCES Advantages & Disadvantages General Choi was…Read More
Tribute to General Choi by: Grandmaster Bobby Kim
Tribute to General Choi By Grandmaster Bobby Kim I met General Choi Hong-Hi in Aurora, Colorado, when he visited my dojang, Kim's Taekwondo Institute, during the early nineteen seventies. At that time, he was already regarded as the "father of Tae Kwon Do," but he still had much important work ahead of him. Before his…Read More
My Father: The Daughter of TaeKwon-Do Founder General Choi Hong Hi
My Father By Meeyun Choi My father would have been 100 years old this November(2018). As if he could avoid death by being constantly on the move, he was already planning the next trip as soon as he came home. Perhaps he was the happiest when he was teaching, discussing, demonstrating Tae Kwon Do in…Read More


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Books ordered may be returned for a full refund if they are not as described. Delivery is guaranteed - or your money back.
Order online or by phoneAUS 1800 067 877 NZL 0800 771 773 INT +61 2 4861 7666
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London: Peter Davies, 1951. This is the first of a series of 7 books of fictionalized autobiography similar to the Little House stories by Laura Ingalls Wilder. The dj (price unclipped) is badly chipped on spine tips, lightly frayed on edges, short splits on the spine. Bright green cloth with red vignette & lettering on spine, bottom of spine a bit bumped, very light page edge foxing, small name and address stamp on front flyleaf, vg. 8vo, 229pp. Illustrated by EDWARD SHENTON.. First UK. Very Good/Good. Illus. by Edward Shenton.


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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Our mission is to help people manage the risks of everyday life, recover from the unexpected and realize their dreams.
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* Contact me to Get to a Better State
* Get all the discounts you deserve
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The State Farm mission is to help people manage the risks of everyday life, recover from the unexpected, and realize their dreams. We are people who make it our business to be like a good neighbor; who built a premier company by selling and...
State Farm products include auto insurance, home owner insurance, Commercial insurance, small business insurance, life insurance, health insurance, long-term care, annuities, recreational vehicle insurance, trailers, farm & ranch insurance, mutual funds and college savings plans.
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Kevin and team have drastically changed over the years. I haven't ever had the most pleasant experience at his offices due to bad attitudes from some of his staff members; however Kevin always seemed to patch things up and was understanding of his customers. As of recent, myself and my entire family chose to leave state farm. Instead of looking into situations, Kevin chose to live in a bed of excuses after failing us time and time again. The same issues I ran into, extended family members faced. These were very simple things too like bad attitudes, never getting calls back about information or MORE QUOTES (MORE MONEY), or even a simple address change. We began to feel like when we called they would just ignore the call and never return your voicemails. Ultimately, it took a call from his corporate office to get a hold of him via transfer (from his corporate). At this point I was looking elsewhere for insurance and leaving his company. On top of this once again a bed of lies and excuses... After I thought I was going to his office to inform him I was leaving his agency and had attempted to contact other SF agents, I was informed by Mr. Coan "the good agents never let you transfer, we dont work like that and things like this could always be resolved in house". Any intelligent business would understand that watching out for your in house profits doesnt globally assist you in the long run if you have a dissatisfied customer especially in a world of social networking. Mr. Coan
Insurance Automobile
Our mission is to help people manage the risks of everyday life, recover from the unexpected and realize their dreams.


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The Kennel Club Dog Photographer of the Year 2015
The Competition: The UK-based Kennel Club has announced the Kennel Club Dog Photographer of the Year 2015. The competition is now open for entries.
Categories: Dogs at play, Dogs at work, Portrait, I love dogs because… (Under 18's only), Puppies, Assistance dogs and Dogs charity, Men's best friends and Oldies.
Prize: The overall winner's photograph will be exhibited in the Kennel Club Art Gallery in Mayfair London. Also, the winner will be provided 1 SmugMug business account and a two day course hosted by professional dog photographer Andy Biggar. In addition, the overall winner will receive an original oil painting of their winning image by internationally awarded artist, Sara Abbott.
Winning entries of other categories will also be displayed in the Kennel Club Art Gallery in Mayfair London.
Entry Fees: Free participation for photographers world over
Website: https://www.thekennelclub.org.uk
Deadline: 13 March 2016Tags: Kennel Club Art Gallery, Kennel Club Dog Photographer of the Year 2015, Mayfair London, Professional dog photographer Andy Biggar


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Cabernet Sauvignon is the most famous grape variety in Bordeaux, used at the heart of all the wines of the Grand Cru châteaux of the Médoc but wherever it is planted in the world it continues to show its class. Make no mistake this is still a very young wine with at least 5 years ahead of it. Punchy Cabernet expression, medium bodied palate of plush, mid-weight firmness. Drink now to 2025+.
Product Detail
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Microsoft underestimated the iPhone , unlike Google who took serious actions, and so didn't make the necessary changes for their phone to become competitive after the market was redefined. See why some people love iPhones.
People had strong reasons to buy both Android phones and iphones but there was no compelling reason to own a windows phone. See why Microsoft acquisition for Nokia failed.
Some of the important Apps people wanted to use such as Snapchat and many others weren't available on windows phone. See why people like Snapchat.
On a Quora thread it was suggested that Microsoft had a hard time competing with Apple because windows phone was among the many things the company does while Apple was laser focused on very few products.
Android is backed by a wide open source community while the iPhone is backed by so many hardcore fans. The windows Phone didn't have a strong enough community to convince developers to develop Apps for it.
Microsoft had designed the windows phone to make many of its desktop Apps , such as excel, work perfectly on it forgetting that the majority of smart phone users have very different needs than desktop users. See why Microsoft bought Nokia.
The windows phone had the goal of supporting the windows operating system which showed signs of slowing down.
The windows Brand wasn't strong enough to compete with other Brands such as Android and IOS in the smartphone market. See the importance of branding.
Microsoft Lumia had bad naming conventions that confused the consumers. Phones were called 640, 930 and 535. Such number prevented Consumers from knowing what each phone does easily. See also why Gopro got popular.
As Microsoft failed to make enough profits or to gain big market share through its Nokia acquisition it had to write down the amount and change its strategy.
Thank you so much for helping out!


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Mobile devices and high technology.
Vseweb.com — webmaster, website, web
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Not so long ago on our site you can get acquainted with a very famous trading system no less than the famous trader bill Williams Profitunity. To work under this system, the author has created several indicators, and one of them has a very intriguing title — a Wonderful Oscillator bill Williams (Awesome Oscillator, AO). And as you may have guessed, is exactly what we will examine today.
the Characteristics of the indicator
Platform: Any
Currency pairs: any
Timeframe: any
trading Time: around the clock
Recommended brokers: Alpari, RoboForex, Amarkets
Despite the intriguing title, this oscillator is not very different from the well-known MACD. Principles of work with display in General the same. In fact, the indicator helps to determine the driving force of the market by subtracting the values of the slow moving average with the period 34, built at the median price, from the fast moving average with a period of 5. The histogram consists of columns of two colors — red and green. If the column is red, it is below the previous one, if the green is higher. Ranges the histogram of the AO oscillator around a zero line and has no overbought and oversold.
the AO Indicator is a standard set of terminal MT4 and does not have any settings except the color. To add it to the chart trading tool — open the menu "Insert/Indicators/bill Williams 'indicators/Awesome Oscillator".
the periods of the moving averages hard-coded in the code of the indicator. However, for experimentation you can download a set of custom indicators prepared by our undisputed king of indicators Pavel888 on the forum in this section. There are many variants of the indicator AO, including setting periods.
the indicator
the Median price (Median Price) is the quotient of the sum of the maximum prices and minimum prices for two. Sometimes to calculate also used Typical price (Typical Price), which is obtained by dividing the maximum, minimum price and closing price for the three.
the Method of calculation of the AO indicator is very simple – it suffices to calculate the difference between the moving averages of median prices with periods of 34 and 5:
As you can see, two moving averages have the same shape of the indicator. Thus, the calculation formula will look like the following:
AO = SMA (Median Price, 34) — SMA (Median Price, 5)
SMA — simple moving average,
, Median Price — median price.
the Signals generated by the indicator Awesome Oscillator
Despite the fact that AO belongs to the category of oscillators, and developed as one of the filtering tools in the strategy Profitunity, this indicator also provides and independent signals.
crossing the zero line or the signal "Zero cross"
The basic signal generated by the indicator is Awesome, is a Zero Line Cross. The signal to buy is generated when the histogram crosses the zero line upwards. The first column is below the zero line, and the second above the zero line. Selling signals the opposite.
the Two pikes Signal
Signal "Twin Peaks" (or "Two peaks") for the purchase occurs if there is a second local minimum is higher than the previous, and these local minimums are below zero. For sale need to find the two falling peak. If there is a third local minimum or higher than the second maximum below the second for the sales, it is regarded as a signal amplifying:
this is actually a pretty weak signal, so how can it be four consecutive lower high, as in the figure above.
the Signal "Saucer"
the Signal "Saucer" (or "Saucer") for the purchase is formed when the Awesome Oscillator is above zero line and the histogram has reversed its direction from downward to upward, that is, of the three columns of the histogram have any first color, the second is red and below the first, the third above the second column and green. This signal is sometimes called a "Saucer", and it looks as follows:
Is the only signal to buy that comes when the histogram is above the zero line. It is also a pretty weak signal, because after the first peak/trough movement may be too short-term in nature, and it is possible to earn not enough. It is best to take signals "saucer" when the first peak or trough is located near the zero line and the indicator has plenty of room to fall, as in the case of sales in the above picture.
Remember that the occurrence of the signal "Saucer" for buying the histogram should be above zero line and the sales – below. In fact, this model describes the entrance to the continuation of the trend after a small pullback, so on the chart to enter on this signal you should see the uptrend, and the indicator ideally should consistently rise from the opposite zone. Here's a good example for purchases:
by the Way, this option sell signal is called the "Inverted saucer".
Awesome Oscillator is an interesting and efficient tool, able to give good entry signals. However, as with any other indicator and Awesome Oscillator should you use in the trading system in conjunction with other indicators. Remember — indicators that I am not mistaken, does not exist. Follow money management and do not uprate risks on transactions. Follow the rules of your trading system that have been properly tested on historical data.
the signals from the Awesome Oscillator is quite unambiguous and easy to interpret, so their use in their trading systems can be recommended to beginners. It should be remembered that the AO catches the short pulses, and should adjust the holding time according to the system based on it, to 5-10 candles, no more.
forum Topic
best regards, Dmitry aka Silentspec
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Pelvic Wedge Cushion
Special Price £19.03 Regular Price £22.39
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Pelvic Wedge Cushion
The charcoal tweed covered cushion has been designed to assist those who spend long periods sat down.
The 11° angle is accepted as producing the optimum sitting position and correct posture and should help in reducing pain in the lower back and legs.
This cushion can also be used in a car seat as an infill.
- Size: 36x36cm (14x14")
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Extreme Locks Ltd > Auto-locksmith > Waterloo
If your looking for the service of a local auto locksmith in Waterloo for lost car key replacements, car key repairs and vehicle unlocking then we can usually have an auto locksmith with you 24 hours a day 365 days a year within 60 minutes of your call.
If your car keys have been lost or stolen and you need them replacing at short notice rather than waiting weeks for the manufacturers then you can give us a call and we will respond the same day. We can delete old car keys when they have been stolen and then replace them with brand new car keys to start and run your vehicle.
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text: Category: Off-the-grid living Live Green In The Coastal Range! Have you been dreaming of living in the country and reducing your carbon footprint? Check out this beautiful 84 acre ranch with a roomy 2800 sq ft 4 bedrooom/3 bedroom… Written by Sue Kretzu September 18, 2017 No Comments About our blog Our agents write often to give you the latest insights on owning a home or property in the local area. Recent Posts Affordable Conveniently Located Santa Margarita Mobile Home Escape the City and Find Your Bliss Cattlemen's Western Art Show and Sale Remodeled Quality 2 Bed 1 Bath Lake Nacimiento Archives June 2018 May 2018 April 2018 March 2018 February 2018 January 2018 December 2017 November 2017 October 2017 September 2017 August 2017 July 2017 June 2017 May 2017 April 2017


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This Adding Vectors Graphically And Component Method has 1275 x 1651 pixel resolution with jpeg format. Adding Vectors In Magnitude And Direction Form, Adding Vectors Calculator, Adding Vectors By Components, Add Vectors Magnitude And Direction Calculator, Magnitude Of A Vector, Adding Vectors Algebraically, Vector Addition Formula, Magnitude Of A Vector Formula, Adding Vectors By Components, Magnitude Of A Vector, Vector Addition Formula was related topic with this Adding Vectors Graphically And Component Method. You can download the Adding Vectors Graphically And Component Method picture by right click your mouse and save from your browser.
In adding the east-west components of all the individual vectors one must consider that an eastward component and a westward component would add together as a positive and a negative. Some students prefer to think of this as subtraction as opposed to addition. In actuality it really is addition - the addition of vectors with opposite direction. Similarly a northward and a southward This Adding Vectors Graphically And Component Method has 1275 x 1651 pixel resolution with jpeg format. How To Add Vectors With Angles Vector Addition Problems Adding Vectors Calculator How To Find The Resultant Vector Of 3 Vectors Adding Vectors Graphically Vector Addition Formula Adding Vectors Algebraically Adding Vectors With This geometric procedure for adding vectors works in general. For any two vectors u and v in the plane the sum of the vectors is graphically given as in the following figure Figure % The sum of the vectors u and v in the plane. The geometric procedure is valid for 3-dimensional vectors as well
Vector Basics - Drawing Vectors Vector Addition This graphic discusses the basic notion of a vector and how to add vectors together graphically as well as what it means graphically to multiply a vector


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Engine Failure Analysis
Engine failures result from a complex set of conditions, effects, and situations. To understand why engines fail and remedy those failures, one must understand how engine components are designed and manufactured, how they function, and how they interact with other engine components. To this end, this book examines how engine components are designed and how they function, along with their physical and technical properties. Translated from a popular German reference work, this English edition sheds light on determining engine failure and remedies. The authors present a selection of engine failures, investigate and evaluate why they failed, and provide guidance on how to prevent such failures. A large range of possible engine failures is presented in a comprehensive, readily understandable manner, free of manufacturer bias.