The range of interobserver kappa values was −0 008 to 1 00 When

The range of interobserver kappa values was −0.008 to 1.00. When the frequency of a certain trait is low, such as for agenesis of the maxillary

central incisors, a single disagreement can have a major effect on the kappa. The negative kappa value for the interobserver agreement of maxillary central incisors in the non-cleft side was the result of only 2 disagreements between the 2 observers. Furthermore, this had no effect on the Dapagliflozin cell line reliability of our data, as an uncertain observation concerning the presence or absence of a tooth at one point in time, could be verified on other OPTs at later time points. We choose to analyze our data separately for the cleft side and non cleft side as differences between sides may be expected. A recent meta-analysis confirmed that the majority of publications on tooth agenesis in OFCs did not do so. In their meta-analysis the authors attributed higher quality scores to studies that took the side, jaw and tooth type into consideration.18 In this cohort, we identified, in total, 13 different tooth agenesis patterns. The lateral incisor in the cleft quadrant was involved in 7 of these 13 different patterns. The maxillary lateral incisor at the non-cleft quadrant was absent in 8.7% of the patients, and was part of only two patterns. The most common www.selleckchem.com/products/gsk1120212-jtp-74057.html symmetric patterns in the maxilla were the lateral

incisors (5.2%), and the second premolars (0.9%) in the mandible. Mannose-binding protein-associated serine protease Our study confirmed the earlier observation that left-sided clefts are more common than right-sided clefts.9 We also found a statistically significant difference for the number of missing teeth in the cleft and the non-cleft quadrants (p = 0.020). Our findings regarding the sidedness of the cleft and tooth agenesis are confirmed by the existing literature. 9, 19 and 20 In our study however, children with a cleft on the right side were far less likely to have missing teeth. Although the prevalence of a cleft and tooth agenesis is significantly and consistently higher on the left side, as were clefts and tooth

agenesis separately, as for the combined phenotype, the underlying genetic aetiology for this general finding has not yet been explained. One way to speculate on this preferable sidedness of clefts and tooth agenesis, could be the observation of cleft sidedness and tooth agenesis of cleft syndromes, where clefts are associated with congenital defects of sided organs, like heart defects. An example is the OFCD (Occulo-facio-cardio-dental) syndrome, in which it has been shown that the causative gene (BCOR-gene) contributes to the left/right sidedness of organ development. 21 and 22 If the interaction of BCOR with clefting genes can be demonstrated, this could provide at least one of the clues for the higher prevalence of left sided clefts.

It is possible that the low elevation, higher temperature, and hi

It is possible that the low elevation, higher temperature, and high SEC Sunny Spring taps a similar confined aquifer, with flow through natural fracture pathways, possibly associated with the Belham Valley fracture network (Fig. 1). SEC of 1703 μS/cm suggests some component of mixing with more conductive waters, possibly sea water; spring water SEC is 3% of local seawater conductivity. Interestingly, the temperature of the northern and western CH springs is lower than the local ambient annual average temperature of 25.9 °C (see Fig. 2) indicating that recharge occurs at a lower temperature. Spring temperatures lower than ambient

air temperatures are not uncommon in volcanic terrain and are normally attributed to recharge occurring at higher elevation (e.g. Nathenson et al., 2003). Ponatinib Using the estimate of 0.6 °C temperature decrease per 100 m elevation (Blume et al., 1974), the average temperature at a recharge elevation between 400 and 700 m amsl would be between 21.7 and 23.5 °C. Spring temperatures of 22–24 °C are consistent with this. CH spring temperatures reported here are consistent with data from previous studies (Jones et al., see more 2010, Chiodini et al., 1996 and Davies and Peart, 2003), however previous authors have not commented on the anomalous temperatures

in the southern CH springs. The warmer springs are those closest to the active SHV; however, at elevations above 190 m (over 250 m, excluding Bessy Mack) and more than 4 km from the active vent the mechanism for this local but systematic elevation of temperature is unclear. One possible mechanism is a contribution not from a deeper, hotter fluid component delivered through a fracture network from a deeper aquifer. The potential of this mechanism is supported by our SEC measurements; SEC in the warmer springs is slightly elevated, compared to the western springs, towards the level observed in the deep Belham well aquifer (Fig. 17). A number of the lower yielding springs in the north also display higher SEC, but these springs are fed by slow flowing seeps emanating through soils. A series of 200 m deep boreholes,

drilled for geophysical installation as part of the CALIPSO project (Mattioli et al., 2004), provide rare access to the geology beneath Montserrat’s forested and highly weathered surface. Permeability measurements were made on 16 one-inch-diameter (2.54 cm) core samples of various lithology collected from depths ranging from 27 to 151 m in the Trants CALIPSO borehole (TRNT in Fig. 1). Five samples were tested in a liquid permeameter at constant flow rate and confining pressure of 2 MPa to simulate approximate lithostatic conditions. Pressure restrictions of the permeameter and the fragility of the samples meant that upstream pressure was limited to 700 kPa. Flow through some lower permeability samples was not possible at these pressures.

The event

provided a unique opportunity to assess the dis

The event

provided a unique opportunity to assess the dispersal and potential effects of contaminated sediment released during a major spill BIBW2992 mouse (Parsons Brinckerhoff Australia, 2009 and Queensland Government, 2012a) on a previously non-impacted ephemeral river system (Fig. 1). The contaminated spill was large, with at least 447 Ml of water released downstream during the event, an equivalent volume to approximately 178 Olympic-sized swimming pools (Queensland Government, 2012a). This study is significant in that the spill provided a unique opportunity to evaluate the dispersal and potential environmental impacts of contaminated materials on an ephemeral system in the absence of historical mining influences. In addition, the principal creeks affected (Saga and Inca creeks; Fig. 1) drain into one of Australia’s last vestiges of wilderness: the Lake Eyre catchment basin. The Eyre catchment is significant for a multitude of reasons: it drains ∼1.2 million km2 of land, approximately 1/6th of the Australian continent; it is considered to be one of the world’s last and largest

unregulated wild river systems (Lake Eyre Basin Ministerial Forum, 2010); and it is Australia’s (and one of the world’s) major endorheic (interior) drainage basins. Within the State of Queensland, the system is protected by unique Australian legislation, the Wild Rivers Act 2005 (Queensland), which is designed to preserve the natural values of rivers in the Lake Eyre Basin. Remote northwest Queensland has been classified as BCKDHB having one of the lowest identifiable impacts from human selleck screening library activities on the Earth’s surface (Sanderson et al., 2002). It is likely, however, that the more spatially linear

impacts arising from diffuse mining-related metal contamination of Australia’s remote river systems have not been captured for two main reasons: (i) The lack of basic research due to the remoteness and difficulty of access to Australia’s interior. (ii) Environmental assessments and reporting of the impacts from mining activities are captured predominantly in industry reports, which are not readily available to the public because they are commercial-in-confidence documents. Furthermore, the challenges of mining in remote areas is increasing in response to resource sector demands, leading to a greater need for data and the proper planning and regulation of mining exploration, extraction and logistics (Brannock and Tweedale, 2012 and NSW Government, 2014). Besides mining, cattle grazing is the dominant industry within northwest Queensland. Despite the high worth of Queensland beef cattle products (∼$3.3 Australian) billion each year (Queensland Government, 2012b), the impacts or risks associated with mine-related contamination remain largely unknown.

The cytotoxic

effect of 20(S)-Rg3 in MCF-7 cells unexpect

The cytotoxic

effect of 20(S)-Rg3 in MCF-7 cells unexpectedly showed no significant difference. These results were consistent when Rg3 was treated in MDA-MB-453 cells (Figs. 4A, 4B). The results from flow cytometric analysis [i.e., fluorescence-activated cell sorting (FACS)] indicated that Rg5 significantly induced cell cycle arrest (Figs. 5A, 5B). This was further confirmed by the cell cycle assay with the data representing suppressed cell proliferation in MCF-7 cells after Rg5 treatment. Rg5 increased the number of cells in the G0/G1 phase and decreased the number of cells in the S phase. Based on these results, Rg5 may induce cell cycle arrest at the G0/G1 phase. Protein expression of cyclin D1, cyclin E2 and CDK4 was decreased, whereas the expression of p15INK4B, buy PF-02341066 p53 and p21WAF1/CIP1 was increased (Figs. 6A, 6B). As Fig. 7A shows, treatment with Vorinostat Rg5 induced caspase-8 and caspase-9, caspase-7, caspase-6. The full-length Bid consequently disappeared in a dose-dependent manner. Poly (ADP-ribose) polymerase

(PARP) cleavage was detected in Rg5-treated MCF-7 cells, which indicated that Rg5 reduced cell viability by inducing apoptosis. Promotion of mitochondria-mediated intrinsic apoptotic pathway by Rg5 was evidenced by Bax/Bcl-2 dysregulation, activation of caspase-9, and release of cytochrome C (Fig. 7A). Apoptosis was evaluated by annexin V/FITC/PI dual staining. After 48 h, Rg5 significantly increased apoptosis at 25μM and 50μM and reduced apoptotic cells at 100μM, whereas necrotic cells were increased (Fig. 7B). The increased expression

of DR4 and DR5 on the cell surface was obvious when cells were treated at the 100μM concentration of Rg5 (Fig. 8A). Activation of p38 mitogen-activated protein kinases (MAPKs) is necessary for apoptosis induced by exposure to ultraviolet radiation, cytokines, chemotherapy, ceramide, and serum deprivation [24]. When ifenprodil cells were treated with Rg5 (50μM and 100μM), p38 MAPKs were activated with the generation of reactive oxygen species (data not shown) (Fig. 8C). Survivin, an inhibitor of apoptotic proteins, is highly expressed in most types of cancer and is a regulator of mitosis; survivin-targeting cancer treatment is validated with great efficacy and no serious toxicity [25]. The expression of survivin was suppressed at high concentrations of Rg5 (Fig. 8D). Apoptotic cells were visualized with DAPI as fluorescent probes. When cells were incubated for 48 h with Rg5 at indicated concentrations (i.e., 0μM, 50μM, and 100μM), the cells displayed the typical apoptosis morphology such as fragmented and condensed nuclei with cellular shrinkage (Fig. 9B). Cells treated with Rg5 at the 100μM concentration showed a necrosis-like morphology (Fig. 9C). Red ginseng is fresh ginseng that is dry-steamed once using water vapor. Black ginseng refers to ginseng that is steamed nine times. Fine Black ginseng refers to the fine roots (i.e., hairy roots) of BG steamed nine times. As Fig.

Sediment with excess 210Pb depletion was found in the river chann

Sediment with excess 210Pb depletion was found in the river channel bank areas and uplands and surficial sediment contained excess 210Pb accumulation. Sunitinib price In the urban river, excess 210Pb accumulated in the river sediment area but was depleted in the river sediment from the more rural stream (Feng et al., 2012). Additionally, no detectable 137Cs was found in either river channel bank or river channel bottom sediment. Previous studies determined the activity of these radionuclides in fluvial sediment, and use either

their depletion or concentration to interpret the watershed processes. As these radionuclides are atmospherically-deposited and fix readily to fine-grained particles, they can indicate deposition processes that concentrate them or erosional processes that deplete them. Using radionuclides as tracers, this study addressed Rigosertib price the following questions. First, what is the origin of fine-grained fluvial sediment draining into a reservoir that supplies drinking water? Second, how do the sources vary longitudinally along the river channel? Third, what do the sediment records reveal regarding the continuity of sedimentation? In other words, does

the accumulated sediment originate from different sources over time? While it is more common to sample depositional environments such as deltas or lakes, or suspended sediment, this study focused on the sediment present in the river channel. Our approach provides snapshots of the sources of sediment along the river channel and how those sources may change along the river. As this sediment can still impact water quality and aquatic habitat (e.g., burial of gravel

beds needed for fish spawning) and is still being transported downstream during floods, this approach offers a different perspective from the usual method of sampling suspended sediment and retrieving samples from depositional environments. The Rockaway River (5th order), in northern New Jersey, supplies the Boonton Reservoir. This reservoir is a major source of drinking water and part of a larger regional water supply system that provides water for over five million New Jersey residents. Samples were collected at three sites along the main stem in order to ascertain the spatial variability of the sediment sources. Site 1 (39 km2 upstream drainage Amino acid area; 40.954233° N, 74.571099° W), the farthest upstream site, is mostly surrounded by forested land with little impervious coverage (Fig. 1). The channel bed sediment was mostly gravel and sand. Site 2 (288 km2 upstream drainage area; 40.907533° N, 74.419322° W) is downstream of an urban area with more impervious surfaces (Fig. 1), but upstream of the steep gorge where site 3 is located. Site 2 had mostly sand and silt (Fig. 1). Site 3 (289 km2 upstream drainage area; 40.904172° N, 74.414586° W) is just upstream of the Boonton Reservoir, and is located less than one kilometer from Site 2.

The oxidative pattern in REVS observed in this study (level of ca

The oxidative pattern in REVS observed in this study (level of carbonylation higher at the end of the storage) differs from the one published by Kriebardis

et al. [73]. In their study, they normalized the oxidation level to the corresponding RBC membranes, which may explain the differences in case of accumulation of oxidized proteins at the Bortezomib order membranes. Delobel et al. also identified mechanisms of oxidation of proteins and postulated that REVS may have a role, as cargos leading to their elimination [75]. Finally, the group of Bosman provided very recently interesting data dealing with the physiology of RBC removal and alloimmunization [76]. The authors investigated if RBC storage leads to the increased expression of non-physiological antigens, and for that purpose, used immunoprecipitation, testing RBCS and REVS from blood units of increasing storage periods with patient plasma containing RBC autoantibodies. The immunoprecipitate was analyzed using proteomic tools. With such

an approach, the authors observed that patient plasma autoantibody binding increased with RBC storage time, which was not the case with healthy volunteer plasma, and showed that pathology-associated antigenicity changes with storage. They also identified several membrane proteins as candidate antigens (namely band 3, adducing, ankyrin, band 4.1, band 4.2 and other HSP cancer protein of the band 3 complex) for immunization. The protein

complexes that were precipitated by the patient autoantibodies were different whether RBCS or vesicles formed during RBC storage were Gefitinib research buy used, indicating that the storage-associated REVS have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the controls. The fascinating observations made by the group of Bosman support the theory that disturbed RBC aging during storage of blood units contributes to transfusion-induced alloantibody and autoantibody formation. Using flow cytometry analysis, Almizraq et al. observed a significant increase in the mean number of EVS per mL during storage, with significant decreases in the amount of phospholipids and total lipids within the RBC membrane. These results clearly indicate that lipidomics of the storage lesion of RBCS is certainly a new avenue of research [77]. The proteome of REVS from patients with beta-thalassemia/hemoglobin E has been explored by Chaichompoo et al. [78]. In these patients, aggregability and oxidative damage of RBCS, combined with platelet activation and increased amount of REVS may provoke thrombotic events.

O podofilino, o ácido tricloroacético e o imiquimod de aplicação

O podofilino, o ácido tricloroacético e o imiquimod de aplicação tópica, apesar de eficazes em condilomas perianais simples e pequenos, não têm eficácia comprovada nas lesões de TBL, pelo que não devem ser utilizados5 and 10. É necessário um seguimento periódico para identificar e tratar precocemente as recorrências. O risco de recorrência após a excisão é de 60-66%

(tempo médio de 10 meses) e as complicações não são desprezáveis, nomeadamente infeção da ferida operatória e dificuldade de cicatrização10. Erismodegib ic50 A taxa de mortalidade é de 20-30%. O prognóstico é mau nos doentes não tratados, uma vez que o tumor, apesar de ter crescimento lento, é fatal por envolvimento dos órgãos adjacentes. Quando adequadamente tratado, o prognóstico é favorável4 and 6. No

presente caso, as características clínicas, com volumoso tumor ocupando toda a região perianal e canal anal, associadas aos achados no exame histológico de lesões de hiperqueratose, papilomatose e coilocitose, confirmam o diagnóstico de TBL. A presença de coilócitos, alterações celulares típicas da infeção pelo HPV, apoia a hipótese do papel deste vírus como agente etiológico. A imunossupressão marcada do doente (67 células CD4/μl) resultante da infeção pelo VIH terá também contribuído como fator PLX4032 purchase predisponente para o aparecimento da lesão. Neste caso, a idade de apresentação foi cerca de 10 anos inferior à idade média referida na literatura, acompanhando a tendência atual para o TBL ocorrer em idades mais precoces5. O facto de ser uma doença de curta duração (10 meses de evolução) terá contribuído para

a inexistência de focos de malignidade e para a eficácia duradoura da terapêutica cirúrgica. Este caso reforça o papel da coinfeção VIH e HPV no desenvolvimento do TBL, bem como da importância do tratamento cirúrgico no tratamento destes tumores. Os autores declaram não haver conflito de interesses. “
“A colangite esclerosante primária (CEP) é uma manifestação extra-intestinal da doença inflamatória intestinal idiopática, em geral do cólon, relativamente rara. Não tem tratamento médico e o seu prognóstico é reservado, a menos que o doente seja submetido Ponatinib mouse a um transplante hepático1, 2 and 3. Há uma variante de CEP, de pequenos ductos (CEP-PD), que pressupõe colangiograma normal, sendo por isso diagnosticada por biopsia hepática. Trata-se, segundo parece atualmente, de uma entidade própria, com melhor prognóstico que a CEP de grandes ductos4 and 5. Apresentamos o caso de uma mulher de 18 anos com doença de Crohn do cólon agudizada e colestase sem icterícia com colangiograma normal. A biopsia hepática foi sugestiva de CEP, fazendo-se por isso o diagnóstico da variante de pequenos ductos. Revemos o estado atual do conhecimento sobre esta doença. Relata-se o caso clínico de uma mulher de 18 anos, caucasiana, natural do Brasil, com o diagnóstico de colite de Crohn aos 8 anos.

The MicroSprayer delivers more aerosolized nanoparticles to the c

The MicroSprayer delivers more aerosolized nanoparticles to the cells than the VITROCELL/PARI BOY system, which is important for cytotoxicity testing. On the other hand application with the MicroSprayer might damage cells by generation of shear stress because high flow rates are needed for effective particle deposition. Decreases in cell viability due to impaction of aerosols have been shown by Mühlhopt et al. see more (Mülhopt et al., 2007). Although adverse effects on cells cannot be excluded this

study do not provide any indication for cell damage by using the MicroSprayer. Both aerosol generating systems were assessed with respect to cytotoxicity testing. This assessment is an important first step in the toxicological assessment of compounds. Routine cytotoxicity testing, the exposure by addition of the test compounds to the medium above cells seeded in plastic wells (submersed culture), is not physiological for respiratory cells. It may lead to a sub-estimation of their cytotoxicity because a direct contact of the nanoparticles with the plasma membrane is not likely. Therefore, cells cultured in

ALI and exposed to aerosols are recommended for physiologically relevant in vitro testing. This recommendation is supported by data showing the higher induction of the anti-oxidative enzyme HO-1 Selleckchem ATR inhibitor in A549 upon exposure to ZnO nanoparticles in ALI than in submersed culture (Lenz et al., 2009). The higher cytotoxicity of aerosolized polystyrene nanoparticles reported in this study also suggests a stronger effect upon aerosol application. It may be suspected

that for nanoparticles with a greater tendency for aggregation, Morin Hydrate like CNTs, the exposure condition (aerosol or suspension) has a much smaller influence on the cytotoxicity. For cytotoxicity testing, where high concentrations have to be tested to determine safety margins, the use of the MicroSprayer appears indicated because much higher doses than with the VITROCELL/PARI BOY system can be applied and the application itself did not cause adverse effects on cells. These data together with data from other groups (Fiegel et al., 2003, Knebel et al., 2001 and Savi et al., 2008) show that higher aerosol delivery rates can only be obtained by a less physiological application mode. To assess the efficacy of aerosolized nanoparticles at therapeutic doses the VITROCELL/PARI BOY system appears better because it mimics better the low flow velocities in the alveoli. Providing every compartment with one nebulizer could decrease the differences in the deposition rates between the compartments. This work was financed by the Austrian Research Science Grant P22576-B18. The Federal Ministry Transport, Innovation and Technology provided student grants for this work. The authors thank Dr. S. Mautner for help with the manuscript. “
“The level and quality of UV protection provided by sunscreen products have improved considerably over the past three decades.

In NMR studies of isotope labeling protein dynamics the 15N isoto

In NMR studies of isotope labeling protein dynamics the 15N isotope is the preferred nucleus because its relaxation times

are relatively simple to relate to molecular motions. The types of motions, which can be detected, are motions that occur on a time-scale ranging from about 10 ps to about 10 ns. The T1 and T2 relaxation times can be measured using various types of HSQC based experiments. In addition slower motions, which occur on a time-scale INK 128 ranging from about 10 μs to 100 ms, can also be studied. However, since nitrogen atoms are mainly found in the backbone of a protein, the results mainly reflect the motions of the backbone, which is the most rigid part of a protein molecule. Thus, the results obtained from 15N relaxation measurements may not be representative for the whole protein. Therefore

techniques utilizing relaxation measurements of 13C and 2H have recently been developed, which allow systematic studies of motions selleck chemicals llc of the amino acid side chains in proteins. Relaxation dispersion (RD) spectroscopy is emerging as a very interesting NMR method to measure the relationship between molecular motions and the limiting-steps in catalysis (Henzler-Wildman and Kern, 2007). With this methodology movements in time scale between 50 μs and 10 ms can be measured. This complements the events measured through the relaxation times T1 and T2 as explained before. For example, RD has been used to measure the movement of interdomains and its relation Teicoplanin with catalysis in adenylate cyclase ( Henzler-Wildman et al., 2007). IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN) and Nomenclature Committee of lUBMB (NC-IUBMB) published in 1999 a newsletter in the journal Folia Microbiol (44, 243–246) with recommendations for presentation of NMR structures of proteins and nucleic acids where they mentioned three articles with the recommendations published

in 1998 (these articles were published in Pure Appl. Chem. 70, 117–142 (1998); Eur. J. Biochem. 256, 1–15 (1998) and J. Biomol. NMR 12, 1–23 (1998)). The recommendations published in Pure Appl. Chem contain general recommendations for publication and communication of NMR data and NMR structures of proteins and nucleic acids through a common nomenclature and reporting standards. This is suitable for publishing of NMR studies of enzymes structures but the binding of substrates and the catalytic process are not covered. In order to describe the molecular events involved in the enzymes function necessarily the knowledge of the relationship between the binding of the substrates and the catalytic steps with the dynamic of the protein structure is required. As shown before, all of these processes can be determined through NMR spectrosocopy where the use of different methods requires a special nomenclature for each of them. Many of these methods were mentioned before with their respective nomenclature.

Sinograms were framed into 25 frames (6 × 10 seconds, 4 × 15 seco

Sinograms were framed into 25 frames (6 × 10 seconds, 4 × 15 seconds, 2 × 30 seconds, 2 × 120 seconds, 1 × 180 seconds, 6 × 300 seconds, and 4 × 600 seconds) and reconstructed with an ordered subset expectation maximation (OSEM) two-dimensional iterative algorithm. Images were summed from 60 to 80 minutes, and volumes of interest were drawn over whole tumors with Inveon Research Workplace image analysis software 4.1 (Siemens Medical Solutions), using the CT template as an anatomic reference. Radioactivity uptake was calculated as the percentage of injected dose per gram tissue (%ID/g) in whole tumor. After PET/CT scans, mice were killed, tumors were collected,

and paraffin-embedded tumor sections were stained with antibodies raised against CA IX (ab15086; Abcam, Cambridge, UK, 1:8000), Glut-1

(GT12-A; Immune Diagnostics and Research, Hämeenlinna, Finland, 1:1000), and Hif-1α (610959; BD Transduction Laboratories, Franklin check details Lakes, NJ, USA, 1:100). Immunostaining was performed as described previously [11]. The expressions of CA IX, Glut-1, and Hif-1α were visually analyzed from 10 different areas in each tumor section using a × 20 microscope objective. The percentage of positively stained tumor cells was counted, and staining intensity was described as weak (1), moderate (2), or strong (3). Each tumor was scored (range = 0-300) by multiplying the average intensity value by the average percentage of positively stained cells. Analyses were performed independently by two investigators (J.S. and K.K.). Digital autoradiography

was used to measure the http://www.selleckchem.com/products/Gefitinib.html uptake of [18F]EF5 and [18F]FDG in cell lines grown on eight-well chamber slides (Nunc™, Thermo Scientific, Waltham, MA, USA) under normoxia and different time periods of hypoxia (1% O2). Four days before tracer incubation, cells were plated onto chamber slides in duplicate (two wells per cell line) and cultured under normal culturing conditions. Medium SPTLC1 was changed on the third day. Cells were seeded at various densities according to their growth rates to ensure that there would be equal amount of cells at the time of tracer incubation. On day 4, one chamber slide per time point was transferred to a hypoxia workstation (Invivo2; Ruskinn Technology Ltd, Pencoed, United Kingdom) at 24, 12, 6, 3, and 1 hour before the end of tracer incubation time. Chambers were removed, and slides were washed with phosphate-buffered saline before being incubated with [18F]EF5 or [18F]FDG for 60 or 30 minutes, respectively, in 50 kBq/ml Dulbecco’s modified Eagle’s medium ([18F]EF5) or physiological saline ([18F]FDG) under hypoxia. The workstation and all solutions used were stabilized in 1% O2 before the experiment. Control cells were incubated with tracers under normal culture conditions (normoxia, 0 hour). Cells were then washed with phosphate-buffered saline and fixed in 4% paraformaldehyde for 10 minutes at room temperature.