Stable isotopes were determined in each sample by continuous-flow isotope mass spectrometer (Isoprime100, Isoprime Limited, Cheadle Hulme, UK) coupled with an elemental analyzer (Elementar vario MICRO CUBE, Elementar, Hanau, D). Each fragment (2.0–2.5 mg dry-weight) was analyzed individually. Isotopic ratios were expressed in ‘δ’ units as the relative difference (in parts per thousand)

between the sample and conventional standards (atmospheric N2 (Air) for 15N; PD-belemnite [PDB] carbonate for 13C) in accordance with the formula δR   (‰) = [(R  sample − R  standard)/R  standard] ∗∗ 103 ( Ponsard and Arditi, 2000), where R is the heavy-to-light isotope ratio of the element (R = 13C/12C or 15N/14N). Bleomycin datasheet Results were monitored with reference to an internal standard calibrated to International Atomic Energy Agency reference materials (Caffeine: IAEA-CH6). Differences in δ15N values between T0 and T1 and among sites, and in isotopic enrichment between bathymetries and among sites, were tested by

t-test or Wilcoxon rank-sum test. The assumption of homogeneity of variances was checked using Cochran’s C-test, and data transformations were used where necessary. Spearman Statistical significance was evaluated at α = 0.05. The δ15N values of the macroalgae after 48 h of exposure were analyzed for spatial autocorrelation by Moran’s test with uniform spatial weights ( Cliff and Ord, 1981) and SP600125 mw by distance-based nearest neighbour. Spatial analyses were performed using R software 2.15.2 (geoR and spdep package). In the reference area (Circeo), the initial N and C isotopic signatures (mean ± S.D.) of macroalgae were δ15N = 5.96 ± 0.54‰ and δ13C = −22.53‰ ± 2.04‰ in U. lactuca, and δ15N = 7.69 ± 0.39‰ and δ13C = −19.73‰ ± 2.43‰ in C. amentacea. After 48 h (T1), δ15N and δ13C values were not significantly different in U. lactuca Methane monooxygenase ( Table 1; t-test, n.s.) whereas δ13C was greater in C. amentacea (t-test, p-value < 0.001). In the Gulf of

Gaeta, the isotopic signature of U. lactuca was δ15N = 5.71 ± 1.25‰ and δ13C = −22.26 ± 2.23‰ at start (T0) and δ15N = 8.15 ± 1.03‰ and δ13C = −22.42 ± 2.10‰ at T1. There was thus a statistically significant increase in δ15N ( Fig. 2 and Table 1; t-test, p < 0.001) and no significant change in δ13C (t-test, n.s.). The high 15N enrichment of U. lactuca was also evident in comparison with the reference area (Circeo) ( Fig. 2; t-test, p < 0.001). During 48 h of submersion in the Gulf, the coefficient of variation of N15 among replicate fronds of U. lactuca fell considerably, from 12.37% at T0 to 1.85% at T1 in the Vendicio area, from 23.73% to 6.76% in Formia, from 26.40% to 16.00% in Scauri, and from 14.70% to 6.03% in Garigliano. C. amentacea, which had higher starting values, was much less enriched in δ15N than U. lactuca after 48 h in the Gulf ( Table 1; Fig. 2).

Anthropometric measurements were performed in duplicate and included body weight, height, BMI (current weight in kilograms divided by square meters), waist circumference (measured at the midpoint between the lower rib margin and the iliac crest, perpendicularly to the long axis of the body, with the subject standing balanced on both feet, spread approximately 20 cm apart, with both arms hanging freely) [9], [22] and [23], hip circumference

(widest circumference over the buttocks) [24], and waist-to-hip ratio. Obesity was defined as BMI of at least 30 kg/m2 [25]. Hirsutism was defined as a modified Ferriman-Gallwey score of 8 or higher [26], [27] and [28]. Blood pressure was measured after a rest period of 10 minutes, with the subject in the Selleckchem Epigenetic inhibitor supine position [29]. Hormonal and metabolic evaluation was performed between days 2 and 10 of the menstrual cycle or on any day if the patient was amenorrheic. After a 12-hour overnight fast, blood samples were drawn from an antecubital

vein for determination of plasma cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides at baseline, and glucose Obeticholic Acid order and insulin before and 2 hours after the ingestion of a 75-g oral glucose load. Impaired glucose tolerance was determined by glucose levels between 140 and 200 mg/dL, as defined by the World Health Organization [30]. Blood samples were also drawn for measurement of sex hormone–binding globulin (SHBG) and total testosterone (TT). All samples

were obtained between 8:00 am and the 10:00 am. Free androgen index was estimated by dividing TT (in nanomoles per liter) by SHBG (in nanomoles per liter) and multiplying by 100. Homeostasis model assessment (HOMA) index was calculated by multiplying insulin (in micro–international units per milliliter) by glucose (in millimoles per liter) and dividing this product by 22.5 [31]. The cutoff point to define insulin resistance was arbitrarily defined as a HOMA index of at least 3.8 [23]. Total cholesterol, HDL-cholesterol, triglycerides, and glucose were determined by colorimetric-enzymatic methods using the Bayer 1650 Advia System (Mannheim, Germany). Non–HDL-cholesterol levels were calculated by subtracting HDL-cholesterol from total cholesterol values. Low-density lipoprotein (LDL) cholesterol was estimated indirectly using the following formula: LDL = total cholesterol − HDL − triglycerides/5. Hormonal measurements were performed using commercially available kits, as previously described [23], [32] and [33]. Serum luteinizing hormone (LH) was measured by a specific immunometric assay (Diagnostic Products Corporation–DPC, Los Angeles, CA) with sensitivity of 0.05 mIU/mL and intra- and interassay coefficients of variation (CVs) of 3.6% and 6.7%, respectively. Total testosterone levels were measured by radioimmunoassay (ICN, Costa Mesa, CA) with an intra- and interassay CVs of 10% and 11.6%, respectively.

, 2010). In addition, Cyanothece sp. ATCC 51142 and C. watsonii WH 8501 might Selleckchem DAPT use circadian fluctuations in DNA topology and chromosomal compaction as a mechanism to control global gene expression like it was shown for S. elongatus ( Mori and Johnson, 2001, Pennebaker et al., 2010, Vijayan

et al., 2009 and Woelfle et al., 2007). Other works pursue a comprehensive study of transcriptional activity in Cyanobacteria — an approach absolutely necessary to understand the temporal choreography of gene expression and cellular metabolism at the global level. The fact that marine Cyanobacteria have a tight schedule for cellular processes to take place has been confirmed by gene expression analyses for several species like Cyanothece sp. ATCC 51142, C. watsonii WH 8501, and Prochlorococcus marinus MED4 (hereafter MED4), where the transcripts of 20–80% of all genes in the genome oscillate tightly linked to diurnal cycles ( Shi et al., 2010, Stöckel et al., 2008 and Zinser et al., p38 MAPK inhibitor 2009). A genome-wide transcript analysis in Cyanothece sp. ATCC 51142 showed that about 10% of all genes oscillate in a true circadian fashion ( Toepel et al., 2008). Using the same species but an indirect approach because no free-running conditions were tested, a DNA microarray study revealed that diurnal changes in even 20–30% of transcripts of all genes are regulated in anticipation of biological

activities at day and night, respectively (e.g. photosynthesis and nitrogen fixation). This strongly suggests a circadian clock behind these changes ( Stöckel et al., 2008). Charting the proteome it was found that only less than 10% of the proteins exhibit circadian rhythms ( Stöckel et al., 2011). This discrepancy is also seen in MED4 ( Waldbauer et al., 2012) and illustrates

that not only transcriptional but also post-transcriptional mechanisms might be working, which schedule the cellular activities. Even marine microbial populations including cyanobacterial species display cross-specific, synchronous, tightly regulated, temporally variable patterns of gene expression suggesting that multi-species metabolic and biogeochemical processes are well coordinated ( Ottesen et al., 2013). Prochlorococcus, the smallest known oxygenic phototroph and important primary producer in the ocean ( Goericke and Welschmeyer, 1993 and Partensky Tyrosine-protein kinase BLK et al., 1999), represents a genus with a reduced number of kai genes: All strains harbor kaiB and kaiC genes, but have no (full-length) kaiA present ( Dvornyk et al., 2003). This lack of kaiA is the result of a stepwise deletion that occurred about 500–400 Ma ago in the course of genome streamlining ( Axmann et al., 2009, Baca et al., 2010 and Holtzendorff et al., 2008). For natural populations of Prochlorococcus and/or laboratory cultures, grown under light–dark cycles, diel variations of gene expression ( Bruyant and Babin, 2005, Garczarek et al., 2001, Holtzendorff et al., 2001, Holtzendorff et al., 2002, Holtzendorff et al.

, 1970), and two of catalases were most similar to hydroperoxidase I (catalase, katG), Manganese containing catalase, which was an important antioxidant enzyme that catalyzes decomposition and disproportionation check details of hydrogen peroxide, respectively ( Chelikani et al., 2004),

forming dioxygen and water. The other catalase was most similar to hydroperoxidase II (catalase, katE), which were haem-containing enzymes that use hydrogen peroxide as the electron acceptor to catalyze a number of oxidative reactions ( Nelson et al., 1994). Moreover, FS-N4 genome contained genes coding for proteins regulating the responses to hydrogen peroxide (H2O2) and superoxide (O2−), including alkyl hydroperoxide reductase subunits (ahpC and ahpF), glutaredoxin I (grxA), glutathione reductase (gorA), Fur repressor, Zinc uptake regulation protein ZUR, and peptide Linsitinib mouse methionine sulfoxide reductase (gsrA). Alkyl hydroperoxide reductase (Ahp), KatG and KatE were the most important proteins in the process of scavenging hydrogen peroxide in vivo (Seaver and Imlay, 2001). The thiol-based peroxidase Ahp consisted of two subunits, AhpC and AhpF, it transfered electrons from NADH

to H2O2 and reduced H2O2 to water. Fur repressor and Zinc uptake regulation protein ZUR were both involved in the PerR regulon, which was known to be highly induced by oxidative stress caused by hydrogen peroxide or paraquat. According to the annotation results of RAST, the genes related to the oxidative-stress-inducible activities were compared with those of Halomonas zhejiangensis, the results showed that the related genes were almost the same, except a phytochrome-like gene. As the definite enhancement by phytochrome of the catalase level was demonstrated in mustard ( Drumm and Schopfer,

1974) and the induction of the Cat3 expression is probably regulated by a very low fluence phytochrome response ( Polidoros and Scandalios, 1997), the phytochrome-like gene might be an important gene for strain FS-N4 to survive in the high-hydrogen-peroxide environment and produce high-catalase-activity Coproporphyrinogen III oxidase extract. It needed more works to reveal it. The following are the supplementary data related to this article. Fig. S1.   Circular representation of the chromosome of Halomonas sp. FS-N4, displaying relevant genome features. From outside to center; Genes on forward strand (tRNAs brown, rRNAs light purple), Genes on reverse strand (tRNAs brown, rRNAs light purple), GC content and GC skew. We would like to thank all brothers and sisters of the lab of extremophiles, Zhejiang University, PR China, for help in experiment skill. We also thank Qi-Lan Wang, Lu-Feng Li for help in gene annotations. This work was financially supported by the National Natural Science Foundation of China (grant no. 31170001).

He stayed at ILTS until his obligatory retirement in 1983. Upon his retirement, he received a title of emeritus professor from Hokkaido University. At ILTS, Sakai-sensei explored and developed a new direction of the research on “plant cold hardiness.” He studied physiological mechanisms of cold acclimation, cold hardiness and freezing avoidance in check details a wide variety of plants ranging from herbaceous plants to woody plants, from many regions of the world—tropical to sub-arctic. In 1960, Sakai-sensei published a scientifically outstanding and academically very interesting paper in the journal Nature (“Survival of the twig of woody plants at −196 °C”,

vol. 185, pp. 393–394). This paper demonstrated for the first time CAL-101 molecular weight the amazing abilities of plant organs/tissues to survive at an extremely low temperature, opening up a new research field: studies to understand the plants’ ability and mechanisms to keep them alive at freezing temperatures. Whilst without the recognition of many people (perhaps including Sakai-sensei himself), the paper in Nature revealed for the first time a strategy that allowed plant cells to survive at extremely low temperatures—the phenomenon of “vitrification”, another area Sakai-sensei pioneered in his career. He spent the last years of his tenure at ILTS measuring cold hardiness of thousands

of plant species collected from all over the world, focusing on the evolutionary aspects of wintering strategies in plants. Altogether, he published a number of papers in prestigious plant science journals, including Plant Physiology, Plant and Cell Physiology, Plant, Cell and Environment and Ecology, learn more as well as a few papers in Nature. Sakai-sensei indeed made many great achievements in his career at ILTS

in Hokkaido University. His enthusiasm and curiosity in plant science, however, did not stop him from continuing to pursue his research even after his official retirement from ILTS. In the time when only a very few retired professors continued their research without funding or support for projects, Sakai-sensei continued his research and published over 50 articles/books during his “retirement”. He devoted himself to the development of cryopreservation methods using vitrification for long-term preservation of plant genetic resources and endangered wild species. During the course of his research career, Sakai-sensei and his colleagues successfully developed a plant vitrification solution (PVS2), the most widely used solution for plant cryopreservation to date (“Cryopreservation of nucellar cells of navel orange [Citrus sinensis Osb. var. brasiliensis Tanaka] by vitrification”, Plant Cell Reports 9: 30–33, 1990, 300+ citations).

This effect was not significant for the lexical stimuli. As found previously, there was a late positivity for high tone-inducing suffixes incorrectly preceded by a low stem tone. It had the same onset (400 ms) as in Roll et al. (2010), but its duration was shorter, probably due to noise introduced by the interfering hand movement, which occurred on average around 700 ms after suffix onset. The positivity was only found in PI3K inhibitor the blocks involving the semantic task. This was also the only task yielding a corresponding interaction between stem

tone and suffix in the response times. This would suggest that the positivity does indeed show some kind of reprocessing of the uncued high tone-inducing suffix, i.e. a P600-like effect. Seventeen right-handed native speakers of Central Swedish, age 23.5 years, SD=3.8, 9 women, participated in the study. All were undergraduate students at Lund University. Thirty different words per condition, 360 in total, were presented in 6 blocks in pseudorandomized order with SOA jittered between 4 and 8 s. Stimulus nouns containing two syllables with voiceless stops

AZD6244 ic50 at the boundary between stem and suffix were chosen for ease of splicing. Two male Central Swedish speakers recorded the words in an anechoic chamber. The words were pronounced in isolation without any focal prominence. Test-words were cut between stem and suffix in order to create mismatching stem–suffix combinations, and the intensity was normalized

over stems and suffixes separately. The stem/suffix fragments were then spliced to obtain words with match and mismatch between stem tone and suffix. Stems were on average 631 ms long for high tones, SD=91, and 648 ms long for low tones, SD=85. High tone-inducing suffixes (plural) were 835 ms, SD=57, and low tone-inducing suffixes (singular definite) were 715 ms, SD=49. The high tone was 10.9 semitones (st), SD=0.7, and fell to 3.5 st, SD=2.4 during 388 ms, SD=117. The corresponding low tone was 3.2 st, SD=0.71 st, falling to 2.2 st, SD=1.1, with a duration of 406 ms from the lowest to the highest point, SD=154. Response times in the semantic task were measured from suffix onset, i.e. the unique disambiguation point where the also test words could be identified as being either singular or plural. Reaction times in the boundary tasks were measured from word offset, i.e. the word boundary. A 129-channel HydroCel Geodesic Sensor Net from Electrical Geodesics Incorporated (EGI) recorded the EEG at a sampling rate of 250 Hz. Band-pass filter with cutoff frequencies 0.01–70 Hz was used online, and a 0.1–30 Hz filter was applied offline. Impedances were kept below 50 kΩ (manufacturer’s recommendation, high impedance amplifiers). CZ was used as online reference, and average re-referencing was computed offline.

The sequence associated contextual (meta)data are MIxS (Yilmaz et al., 2011) compliant. This study was supported by the German Federal Ministry of Education and Research (BMBF) as part of the Microbial Interactions in Marine Systems (MIMAS) project (Grant No. 03F0480A). “
“Rhodopirellula is a member of the ubiquitous bacterial phylum Planctomycetes. Members of the Planctomycetes are abundant in particulate fractions of marine ecosystems and considered as important players in the global carbon and nitrogen cycles. They convert substantial amounts of organic material, such as “marine snow” (aggregates of zooplankton, phytoplankton and protists), into carbon dioxide. Their importance in

marine systems was recently discovered and documented in several Bleomycin publications ( Glöckner et al., 2003, Winkelmann and Harder, 2009 and Winkelmann et al., 2010). A collection of 70 Rhodopirellula strains obtained

from different European seas revealed 13 distinct operational taxonomic units (OTUs). These were defined by taxonomic studies with a combination of 16S ribosomal DNA (rDNA) sequence comparisons, DNA–DNA-hybridization (DDH) and a novel multi-locus sequence analysis (MLSA) approach that employed primers in putatively conserved regions of nine housekeeping genes ( Winkelmann et al., 2010). First evidence for a limited habitat spectrum of these sessile bacteria was detected by annotation and genome comparison of the strains. Here we report the permanent draft genome sequence of the Rhodopirellula sallentina strain SM41 (= DSM 24067 = JCM17618) which was isolated DNA ligase from a mixed sediment water selleck products sample originating from San Cataldo, Italy (40.3861 N 18.3055 E) ( Winkelmann and Harder, 2009). The genomic DNA was isolated using the FastDNA SpinKit for Soil (MP Biomedicals, Germany), randomly sheared into fragments (“shot gun sequencing”) and transferred into 96 well plates with 24 wells that were assigned

to each strain. Sequencing was performed with the Roche 454 Titanium pyrosequencing technology. The assembly was done with Newbler v. 2.3. Gene prediction was carried out by using a combination of the Metagene (Noguchi et al., 2006) and Glimmer3 (Delcher et al., 2007) software packages. Ribosomal RNA genes were detected by using the RNAmmer 1.2 software (Lagesen et al., 2007) and transfer RNAs by tRNAscan-SE (Lowe and Eddy, 1997). Batch cluster analysis was performed by using the GenDB (version 2.2) system (Meyer et al., 2003). Annotation and data mining were done with the tool JCoast, version 1.7 (Richter et al., 2008) seeking for each coding region observation from similarity searches against several sequence databases (NCBI-nr, Swiss-Prot, Kegg-Genes, genomesDB) (Richter et al., 2008) and to the protein family database InterPro (Mulder et al., 2005). Predicted protein coding sequences were automatically annotated by the software tool MicHanThi (Quast, 2006).

The FD is dependant on the external moments developed by gravity and inertia at each of the joints and the internal moments required to be produced by the muscles crossing that joint in order to counteract the external moment generated

during a functional task (Samuel, Rowe, Hood, & Nicol, 2011). Conventionally, the loading on the muscle group has been evaluated by comparing the peak external moment in a functional task with the maximum muscle strength. However this method is flawed because the peak external moment may occur at a joint angle different to the position of maximal selleck inhibitor muscle strength and muscle strength is highly dependent on joint angle (Samuel & Rowe, 2009). Hence, in this study we defined “FD” as the muscle moment required at a particular joint angle during a functional task, divided by the maximum isometric muscle strength available at the joint

angle (expressed as a percentage) (Rowe, Samuel, & Hood, 2005). Therefore, the aim of the present study was to characterize the level of FD placed on the hip and knee joints during gait, CR, CSt and SA and SD in older adults. Ethical approval was obtained from the Ethics Committee of the Bioengineering Unit, University of Strathclyde. All participants provided written informed consent prior to participation in the study. Eighty-four healthy older adults aged 60–88 years (mean age 73.2 years (SD 7.3); height 1.66 m (SD 0.1); body mass 73.7 kg (SD 13.1)); 41 males and 43 females were recruited through posters placed in older adult organizations in the Greater Glasgow area, Stirlingshire and Ayrshire in Scotland, Regorafenib order UK. Participants were categorized into three sub-groups (60–69 years, 70–79 years and 80 years and over) based on their age and were from a wide range of social, economic and educational backgrounds as reported through an initial screening questionnaire. The inclusion and exclusion

criteria published previously (Greig et al., 1994) were adopted for inclusion of older adults. Those with neurological conditions, musculoskeletal disease or systemic disorders affecting multiple joints such as Rheumatoid Arthritis were excluded from the study. Participants attended the Biomechanics Laboratory at the University of Strathclyde for two, 2-h sessions, one Selleckchem Temsirolimus for muscle strength tests and one for whole body biomechanical assessment. A torque dynamometer attached to a purpose-built plinth was utilized to measure isometric muscle moments. The device consisted of a strain-gauged metal bar referred to as the transducer attached to a circular indexing wheel. The transducer and indexing wheel were attached to an aluminum base which was secured to the frame of a custom-built plinth. The output from the transducer was amplified using a strain-gauge amplifier and was input into a 16-channel analog to digital data collection system, housed inside a PC computer.

Evidence that serum calcium increases with the dose of vitamin D administered but calcium absorption reaches a plateau once normalized by a small dose of vitamin D [21] suggests the existence of a safer, side effect-free therapeutic window for vitamin D and its analogs. Many attempts have been made to synthesize EPZ5676 price a compound that would retain only the differentiation and anti-proliferative effects of 1α,25-(OH)2D3, thus allowing for safer usage and less side effects [22] and [23]. Eldecalcitol, formerly known as ED-71, is an analog of 1α,25-(OH)2D3 bearing a hydroxypropyloxy residue at the 2β position that was developed

in the early 80s and is currently awaiting approval as a drug for treatment of osteoporosis in Japan [24] and [25]. It has been reported that eldecacitol lowered biochemical

and histological parameters of bone resorption in a rat ovariectomized (OVX) model of osteoporosis [26] and that its effects on bone were observed without sustained hypercalcemia or hypercalciuria [27]. Examinations focusing on the effects of vitamin D analogs at the tissue level have been relatively neglected despite the therapeutic potential of these compounds for the treatment of bone diseases. A recent report involving bone marrow ablation showed that eldecalcitol may promote bone formation and angiogenesis in addition to inhibiting bone resorption [23]. Further data on the histological subtleties and on the interplay among bone cells selleck inhibitor after eldecalcitol treatment are not yet available. In the present study, we used histological, histochemical, histomorphometrical and ultrastructural analyses as tools for investigating the tissue events following the administration of eldecalcitol in OVX rats. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd., Niigata University and Hokkaido University, and were conducted in accordance with Isotretinoin accepted standards of humane animal care. Eight-months old Crl:CD(SD)(IGS)

rats were obtained from Charles River Laboratories Japan, Inc., and acclimated until 11 months of age under standard laboratory conditions (23 ± 3 °C, humidity 35%–75%, light–dark cycle 12 h), with ad libitum access to food (1.25% calcium, 1.06% phosphate, CE-2, Clea Japan, Inc., Tokyo, Japan) and water. Rats were then divided in three groups: 1) the Sham group, whose animals were sham-ovariectomized and received only vehicle (medium chain triglyceride, MCT) after the procedure (n = 8), 2) the OVX group, where animals underwent standard ovariectomy but received only MCT after the surgical procedure (n = 8), and 3) the eldecalcitol group, where animals underwent standard ovariectomy and were given eldecalcitol by gavage (n = 8, 30 ng/kg, 5 times per week, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan).

Effects on algae and fish were only observed at extremely high SAS concentrations that exceed current cut-off values for classification as hazardous. No effects on growth and reproduction parameters were found in daphniae or aquatic midge. Even after direct injection into the yolk of zebrafish embryos, no adverse effects were seen with spherical silica particles, while nanowires caused malformations. Toxicity to bacteria and damage to the cell membrane in yeast were observed only at very

high silica concentrations CDK inhibitor of ≥1000 ppm. In humans, SAS did not induce silicosis, lung cancer or any other form of cancer. There is no evidence that SAS induces mutations either in vitro or in vivo. Though genotoxicity was observed in a few in vitro test systems, this was generally at dose levels and concentrations that also induced cytotoxicity. No genotoxicity was found after in vivo exposure of experimental animals. In rats, SAS produced transient lung inflammation, and reversible increases of pro-inflammatory cytokines and chemokines at exposure levels of 5 mg/m3 (respirable dust) or higher with

1 mg/m3 (respirable dust) being the No-observed-effect-level (NOEL). As elimination mechanisms include the clearance of particles by macrophages and since human macrophages have about four times DNA Damage inhibitor the volume of rat macrophages ( Krombach et al., 1997), the rat is assumed to respond with more chronic inflammation and epithelial responses as compared to humans. Important insight into the mechanisms and modes of action of SAS, including CYTH4 colloidal silica, has been gained from mechanistic studies (e.g., via intratracheal instillation in experimental animals) and from in vitro models. In this context, it has to be considered that results of studies using a suspension medium to apply silica particles either to animals via intratracheal instillation or in in vitro studies, are strongly influenced not only by the particle characteristics but also by the protein and lipid content of the suspension medium which may influence the degree of

particle aggregation. Furthermore, using intratracheal instillation or pharyngeal aspiration as the delivery route to the respiratory tract of experimental animals involves administration of high doses as a bolus, i.e., within a very short time period whereas it would take much longer (hours, days or even weeks) to deliver the same dose via inhalation exposure. This bolus administration implies that many physiological defence mechanisms may be disrupted and artificial health responses be generated that would not occur under physiological in vivo conditions. Interestingly, milder effects have been shown after intratracheal instillation of “nano” silica as compared to micrometre-sized silica particles, possibly because of a faster translocation and elimination ( Chen et al., 2004). Findings from studies employing the intratracheal route can nevertheless be useful as proof-of-principle studies.