In a 2005 ecological model, Didier Gascuel demonstrated the effect of differing trophic location of target catch on fishery health. Gascuel concluded that high trophic

levels are the most sensitive to fishing pressure, noting that a 40% fishing effort would be considered full exploitation of species with a trophic level greater than 4. In contrast, full exploitation would be achieved at a fishing effort of 100% for trophic level 3 [27]. This demonstrated sensitivity of high trophic level species makes them especially prone to stock collapse. In a top-down driven ecosystem, where predator-prey interactions are the primary influence in ecosystem biomass and relative species abundance, an ecological extinction of top predators could create a trophic cascade [27]. Trophic cascades are defined by indirect effects of the

removal of a predator on the relative PLX4032 cost species abundance of lower trophic levels. While trophic cascades have been demonstrated in several marine ecosystems [28], [29] and [30], an especially relevant and well-documented example of a trophic cascade is that of the Atlantic Cod (Gadus morhua) in the Northwest Atlantic. Scientists, including both Pauly and Essington agree that the primary mechanism leading to the cod collapse is Nutlin-3a order that of intense overfishing. Upon the collapse of the cod fishery, fishing effort was redirected toward smaller pelagic species and macroinvertebrates, clearly illustrating the scenario of fishing down the food web [31]. In a 2005 study, Frank et al., explored the relationship between

biomass shifts of trophic levels on the Scotian Shelf of the North Atlantic. The researchers compared biomass estimates before the collapse of the cod fishery to estimates following the collapse. Abundance of top predators, including cod and other commercially important species experiencing significant declines in landings, was found to significantly correlate in the negative direction with abundance of small pelagic fishes and benthic macroinvertebrates. These small fishes and macroinvertebrates are the primary diet of top predators within the ecosystem. A positive correlation between top predator abundance and zooplankton abundance was identified, indicating a decrease in zooplankton abundance. Additionally, a negative correlation between top predator abundance and phytoplankton abundance was diglyceride evident. These interactions suggest that a decrease in the biomass of top predators caused an increase in abundance of their prey species (benthic macroinvertebrates), one trophic level lower. An increase in the abundance of benthic macroinvertebrates likely led to increased predation on zooplankton, thus decreasing zooplankton abundance. A decrease in the predation pressure by zooplankton would lead to the witnessed increase in phytoplankton biomass. To further support the hypothesis of a trophic cascade, Frank et al., examined the change in abundance of seals, a direct competitor of cod.

Because no guidelines exist for volumetric tumor response criteria, we deliberately selected the same cutoff values that are currently used in RECIST and mRECIST for vRECIST and qEASL to unify and simplify response assessment in a clinical setting. Thus, by using the formula: Volume = 4/3πr3, where r is the radius and π is the mathematical

constant representing the ratio of a circle’s circumference to its diameter, a decrease of 30% defining partial response (PR) using the unidimensional RECIST and mRECIST corresponds to a decrease of 65% of tumor volume. Table 2 summarizes tumor response criteria. Objective response was defined as complete response (CR) and PR. The AC220 patients with objective response were classified as responders, and the other patients [with stable disease Decitabine mw (SD) and progressive disease (PD)] were classified as non-responders. As no data exist for the response assessment in uveal melanoma metastatic to the liver with regard to the inclusion of target and non-target lesions, the final response assessment was based on the target lesions alone and also determined by incorporating the target and non-target lesion responses (overall response) [10], [11], [12] and [14]. Data were summarized

using descriptive statistics (count and frequency for categorical variables and mean and range for continuous variables). Significance levels and confidence intervals (CIs) were calculated, when possible, with exact methods that do not rely on normal approximations, to increase validity of the findings. A paired Student’s t test with Sinomenine its exact permutation distribution was used to compare size, tumor volume, and tumor enhancement before and after TACE to evaluate tumor response to treatment. To evaluate the change in signal intensities on T2- and T1-weighted images before versus after TACE, the

ratio of the sample proportion of “T2 = 2” and “T1 = 2” in all target and non-target lesions before versus after treatment was calculated. The significance level of this ratio was obtained as twice its tail probability from its exact permutation distribution [23], where permutations were performed, for each patient independently, between the pretreatment and the posttreatment vector of the T2 and T1 signal values of the target and non-target lesions. The overall survival was calculated from the date of the first TACE until death. The median overall survival of the entire cohort was estimated from the 50% point of the Kaplan-Meier curve, and its standard error and 95% CI were obtained using the jackknife technique. The predictive value of each response criterion was evaluated on its own (univariate) and then in a multivariate analysis.

This study has several limitations. check details There is a need to further substantiate the validity and limitations of the mentalising paradigm as applied to music, and the relation between elementary emotion processing and the attribution of more complex or ambiguous affective states to music. There is no universally agreed ‘lexicon’ of musical emotions and more information is needed about musical mentalising in the healthy brain. Mentalising in music should

ideally be studied in the context of a comprehensive assessment of mentalising abilities in different modalities; this would enable evaluation of the specificity and sensitivity of the music-associated deficit. In a related vein, it would be relevant to manipulate musical stimulus parameters such as familiarity, valence and complexity as well as perceptual characteristics to assess the extent to which these may modulate mentalising on musical stimuli. From a clinical perspective, there is a need for detailed neuropathological correlation in bvFTD populations, both to establish disease associations and to correlate behavioural deficits

with histopathological features. It would, for example, be intriguing to evaluate the role of Von Economo neurons in this very specifically human ability (Seeley et al., 2012). In addition, the promise of early disease detection requires further substantiation of the timing of development of mentalising deficits in the course of bvFTD evolution. selleck inhibitor It would be of great interest, for example, to establish whether such deficits (and particularly, deficits of more abstract ToM processes, such as those embodied in music) might lead other features in presymptomatic carriers of mutations causing bvFTD. This will require longitudinal study of individuals affected and at-risk Sitaxentan of developing bvFTD. Taking these caveats into account, the present findings provide evidence that music can represent surrogate mental states and that the ability to construct such mental representations is impaired

in bvFTD. The findings have potential implications for our understanding of the biology of this disease and human social cognition more broadly. LED, CJM, SJC and JDW were each involved in designing and conducting the study, and in drafting and critically revising the manuscript. LED collected and analysed the data and CJM also provided technical assistance with the neuroimaging analysis. AB and RO created the stimulus sets, collected pilot data and were involved in drafting the manuscript. HLG assisted with collection of neuropsychological data and was involved in drafting the manuscript. JN designed and supervised the statistical analysis and was involved in drafting the manuscript. LED, CJM, HLG, SJC and JDW receive salary and research support from the Medical Research Council, Alzheimer Research UK and the Wellcome Trust.

4 g l− 1) in the receiving seawater pond (P1). The pH decreases very gradually with increasing

salinity gradient (Pearson’s r = 0.89, p < 0.05), fluctuating between 6.37 in SB431542 P5 and 7.72 in P1. Nitrate concentrations were the highest (6.16 μmol l− 1) in the crystallizer pond, while levels in the other ponds varied between 3.12 μmol l− 1 and 4.80 μmol l− 1 (Pearson’s r = 0.95, p < 0.05). Concentrations of phosphates fluctuated between 0.93 μmol l− 1 in P3 and 2.54 μmol l− 1 in P1. 42 species of phytoplankton were identified in the whole saltern system; they consisted primarily of cyanobacteria (16 species), diatoms (12 species) and dinoflagellates (11 species), in addition to two species of Euglenophyceae and one species of Chlorophyceae (Table 2). Each pond was characterized by a specific phytoplankton community structure that varied in the number of species, total phytoplankton density and type of dominant species. As shown in Figure 3, GSI-IX mw the community structure in terms of the number of species decreased rapidly and significantly with increasing salinity in the ponds (Pearson’s r = − 0.95, p < 0.05), starting with a maximum of 33 species in the first pond (P1) and ending with only one species (Dunaliella salina) in the crystallizer pond (P5). Conversely,

the total phytoplankton density, except that recorded in P1, increased significantly with rising salinity (Pearson’s r = 0.96, p < 0.05), fluctuating between a minimum value of 8.7 × 105 individuals l− 1 in P2 and a maximum of 56 × 105 individuals l− 1 in P5 ( Figure 3). Marked differences were observed between the

ponds in terms of the species richness of each group of phytoplankton. There was a conspicuous decrease in the number of diatoms and dinoflagellates with Edoxaban increasing salinity. They were well represented in the first and second ponds, but poorly represented in P3 and absent altogether in P4 and P5. Cyanobacteria were more diversified in P3 and were likewise so in P4, albeit with a lower number of species, but were absent in P5 (Figure 4). In terms of cell density, dinoflagellates and diatoms followed by Euglenophyceae appeared to be the predominant components in the first pond. They respectively contributed 45.6%, 33.1% and 15.6% of the total phytoplankton population (Figure 5). Among the most dominant dinoflagellate species were Karenia brevis contributing about 9.3 × 105 individuals l− 1 (32.7% by number to the total density of phytoplankton) and Scrippsiella trochoidea (4.9%). Diatoms were represented mainly by Cylindrotheca closterium (8 × 105 individuals l− 1, 28%), while Lepocinclisacus (4.2 × 105 individuals l− 1, 14.7%) was the dominant species in Euglenophyceae. In the second pond, diatoms ranked first (42.7%) and were dominated mainly by C. closterium with about 25.4% of the total percentage abundance. Cyanobacteria and dinoflagellates came second with similar percentages (23.2% and 20.9% respectively).

Esteban-Fernández et al. [54] führten In-vivo-Experimente an Ratten aus, denen Pt-Medikamente injiziert wurden. Die Autoren untersuchten die Bindung von Platin an Proteine in der Niere und im Innenohr, um die nephrotoxischen und ototoxischen Effekte von Pt-Medikamenten zu charakterisieren. Nach Behandlung von Ratten mit Cisplatin, Carboplatin und Oxaliplatin wurde die Pt-Akkumulation in den beiden Organen analysiert. Die Ergebnisse zeigten deutlich, dass nicht nur der (Gesamt-) Pt-Gehalt, sondern vielmehr die Struktur des Medikaments (die tatsächliche ABT-199 Pt-Spezies) für die Änderung

der Organfunktion verantwortlich ist. Speziationsstudien an Proben der Niere und des Innenohrs mittels 2D-Flüssigchromatographie (Größenausschlusschromatographie + FPLC) in Kombination mit ICP-MS demonstrierten eine vollständige Bindung des Platin an Proteine. Ein Metallothionein-Standard eluierte bei derselben Retentionszeit wie einige der cytosolischen Pt-Biomoleküle.

Peaks des freien Pt-Medikaments wurden nicht beobachtet. Urin wird als Matrix für das Pt-Biomonitoring verwendet, ZVADFMK um den Zeitverlauf der Pt-Exkretion nach der Verabreichung zu verfolgen und die biologische Halbwertszeit zu bestimmen. Außerdem lassen sich die Pt-Metaboliten (Spezies), die letztlich vom Organismus ausgeschieden werden, charakterisieren. Auf diese Weise könnte sich eine Beurteilung des in-vivo-Metabolismus Pt-haltiger Medikamente durchführen lassen. Speziation des Urins von Krebspatienten zeigt, dass etwa 40 % der Ausgangssubstanz (Cisplatin) in hydrolysierter Form als Monoaqua-Cisplatin exkretiert werden [21]. Der restliche Teil wird als (natives)

Cisplatin exkretiert, das dann entsprechend der für hohe Chloridkonzentrationen ermittelten Kinetik hydrolysiert wird. In einer weiteren Arbeit, durchgeführt von Tang et al. [55], wurde die Speziation von Platinverbindungen in Urin von Patienten, die mit Cisplatin behandelt worden waren, mittels HPLC– ICP-MS untersucht. Bei der Analyse trat als Hauptkomponente Cisplatin auf, jedoch wurden auch ein Monoaqua-Cisplatinkomplex und ein Pt-Creatininkomplex im Verhältnis 1:1 identifiziert. Letzterer, so wurde festgestellt, war die zweithäufigste TCL Pt-Spezies im Urin. Weitere Peaks entsprachen Cisplatin-Harnstoff und Cisplatin-Harnsäure, die beide durch Vergleich ihrer Retentionszeiten mit der von Standardsubstanzen identifiziert wurden. Bei einem parallel durchgeführten Experiment wurde Urin von Carboplatin-behandelten Patienten untersucht. In diesem Fall war die hauptsächliche Pt-Spezies im Urin die Ausgangssubstanz Carboplatin [55]. Keine der seltener auftretenden Spezies stimmte mit einer derjenigen überein, die sich in Proben nachweisen ließen, welche nach einer Cisplatin Behandlung genommen worden waren.

Over time there is, for a number of patients at least, diminished recruitment of right hemisphere structures for language tasks. Eventually, for some patients with chronic aphasia, significant language recovery is associated with Lumacaftor mw redistribution of language processing back to left hemisphere perisylvian areas. Intervention with noninvasive brain stimulation may work in several different ways. To date, most therapeutic stimulation studies have employed inhibitory stimulation of right hemisphere structures. This approach may modulate

both right and left hemisphere components of chronically reorganized language networks in ways that allow them to function more efficiently. The effect of stimulation in the right hemisphere may be to down-regulate local inhibition of right hemisphere regions engaged in language-related tasks. Concurrently, inhibitory stimulation of intact contralesional cortical areas may facilitate increased recruitment of perilesional regions of the left hemisphere into reorganized language networks by diminishing the impact of transcallosal inhibitory inputs to those areas. Finally, although it has been proposed that the effects of noninvasive brain stimulation are specific with respect to their

effect on reorganized language networks, it may be the case that the changes in language performance observed after brain stimulation may relate to alterations in cerebral function that are less focal and that may affect a variety AZD2281 supplier of neural functions

in ways that have not yet been described. Further investigations will be critical to further clarifying the impact of noninvasive brain stimulation on different mechanisms of aphasia recovery. Noninvasive brain stimulation provides a potentially promising set of tools for understanding and enhancing aphasia recovery. Future investigations BCKDHB involving noninvasive brain stimulation may be able to further characterize the roles of the left and right hemispheres in aphasia recovery by employing a variety of experimental manipulations. For example, noninvasive brain stimulation techniques could be paired with behavioral techniques that are believed to facilitate right hemisphere involvement in language tasks (Crosson et al., 2007 and Schlaug et al., 2009). Other investigations may explore the degree to which reorganized language networks in the right hemisphere share functional homology with perisylvian language circuits in the left hemisphere. Administration of therapeutic rTMS to different regions in the right hemisphere could result in manipulation of specific linguistic processes, further elucidating structure–function relationships in reorganized language networks. Additional noninvasive stimulation studies could further characterize temporal aspects of language recovery by stimulating the right and left hemispheres at different timepoints relative to stroke onset.

END was defined as 1-point and 2-point increase in NIHSS (during the first three and five days of ictus respectively) in the Australian and German study, respectively. Recent

studies have shown that END is an independent predictor of poor outcomes in the setting of AIS. More specifically, the investigators of SORCan (Stroke Outcomes Research Canada) registry have reported BEZ235 datasheet that END (defined as 1-point decrease in CNS) was an independent predictor of 7-day, 30-day and 1-year case fatality rate in a cohort of 3631 patients [7]. Similarly, END was associated with higher rates of death during hospitalization, longer duration of hospitalization and lower rates of functional independence in an Australian study [5]. The causes of END can be classified

into two major groups: hemodynamic and non hemodynamic [1]. Several non-hemodynamic mechanisms can lead to ischemic lesion extension and subsequent neurological worsening, including infections, cerebral edema/increased intracranial pressure, hemorrhagic conversion of infarction and metabolic disorders (hypoxia, hyperglycemia and fever) [1]. The most common hemodynamic causes related to infarct expansion, leading to END in the setting of ACI are the following: (i) cardiac complications, (ii) arterial reocclusion, CB-839 in vivo (iii) intracranial arterial steal phenomenon and (iv) cerebral microembolization. Patients with severe disabling strokes are particularly vulnerable to cardiac complications because stroke can provoke disturbances in autonomic and neurohormonal control and predispose patients to severe cardiac adverse events (SCAEs). It is well-known that acute stroke may lead to a variety of cardiac abnormalities such as myocardial infarction, electrocardiographic changes, cardiac arrhythmias, cardiac arrest, stress cardiomyopathy (tako-tsubo syndrome) and intracardiac thrombus [8]. SCAEs can medroxyprogesterone hinder functional recovery and contribute to cardiac morbidity and mortality [8]. They are common in the

acute period after stroke onset (19.0% of all patients experience at least one SCAE) and are responsible for 2–6% of the total mortality three months after acute ischemic stroke [9]. The main predictors of SCAE are outlined below: history of heart failure, diabetes mellitus, baseline creatinine >115 μmol/L, severe stroke, and a long QTc (>450 ms in men and >470 ms in women) or ventricular extrasystoles on ECG, low admission systolic blood pressure (<110 mmHg) and right insular stroke [8] and [9]. Right insular region has been shown to moderate the autonomic control of the heart and this may partly explain the potential relationship of right insular stroke with SCAEs. Moreover insular infarction is associated with abnormal cardiac repolarization and increased risk of vascular mortality [9].

Our data are therefore not inconsistent with Karsenty’s conclusion but neither do they support it. In conclusion, the data presented here indicate that the expression of the human Lrp5 G171V HBM mutation is associated in both cortical and cancellous bone with an increased osteogenic responsiveness to supra-physiological loading, which is more marked in females than males, and with some protection against the bone loss associated with neurectomy-induced disuse. Absence of normal Lrp5 activity is associated in both males and females with greater neurectomy-induced bone loss in cancellous bone than in WT controls but there is no difference between these genotypes in the selleck products level of bone loss in the cortex.

Absence of Lrp5 activity abolished the percent increase in cortical bone gain in response to loading in males but similar experiments in females showing no difference in loading-related response between those with and without functional Lrp5 were inconclusive since for most parameters neither the female Lrp5−/− mice nor their WT+/+ littermate controls, showed a statistically significant dose:response to loading. This work was supported by a programme grant to LEL and JP from the Wellcome Trust. The mice were the kindly donated by Wyeth Research, Monmouth, New Jersey. USA. The authors are grateful to Kristien

Verheyen for her advice on statistical analysis and Behzad Javaheri for selleck chemicals llc his insightful comments. “
“In the author line, the names of Songlin Peng, Ge Zhang, Yixin He, Xinluan Wang, Pingchung Leung, Kwoksui Leung and Ling Qin were listed incorrectly. The correct author line appears above. “
“The authors regret that in the original manuscript title, the expression ‘osteoclast plasma proton pump’

was incorrect. The correct article title is ‘Murine ameloblasts are immunonegative for Tcirg1, the v-H-ATPase subunit essential for the osteoclast plasma membrane proton pump. “
“The iliac crest bone marrow aspirate (ICBMA) was the first source from which multipotential stromal cells (MSCs), also termed mesenchymal stem cells, were isolated [1]. This anatomical site has become the most frequently accessed in harvesting MSCs for bone tissue engineering pheromone and is generally accepted as the ‘gold-standard’. Whilst this source is readily accessible and has good handling properties it has a low frequency of MSCs (0.001–0.01%) [1]. This is of significance as many regenerative medicine uses of MSCs including putative bone repair applications require large cell numbers [2], [3] and [4]. High MSC yields can be achieved by in vitro culture with relative ease, with a 1000-fold increase in numbers within 2–3 weeks [5]. However, this results in daughter cells that have reduced differentiation capacity [5] and impaired cell function including gradual accumulation of senescence-related markers [6] and [7] and increased potential for transformation [8].

The pairing of heavy and light chain V-genes from each family occurs in proportion to their abundance in the library (data not shown), indicating random pairing as expected with the library construction AG-014699 cost method that was employed.

Previous data suggests random pairing also occurs in the human repertoire (de Wildt et al., 1999). Each library was assessed by selection against seven targets: gastrin (a 14 amino acid peptide), β-galactosidase (a bacterial protein, β-gal), human proteins insulin receptor in complex with insulin (InsR + Ins), TIE1, TIE2, TIE2 in complex with angiopoeitin 1 (ANG1), and TIE2 in complex with angiopoeitin 2 (ANG2). Three rounds of panning were performed for each target using previously described panning methods (Hoet et al., 2005 and Bhaskar et al., 2012). For each target, five to ten 96-well HSP inhibitor plates of clones were screened either by ELISA (gastrin, β-gal, TIE1, TIE2, and TIE2 complexes) or flow cytometry (InsR + Ins (Bhaskar et al., 2012), TIE2, and TIE2 complexes) for binding to the target. The clones that bound to their target were sequenced to identify unique clones. The unique clones were then analyzed for VH and VL family representation (Fig. 4), for CDR3 length of the VH and VL, and to assess the germline representation in FR1–FR3 of the selected clones (Table 2). Once unique clones were identified for each

target, further Florfenicol characterization of those clones was performed. For both libraries, the unique clones that bound to β-gal and TIE1 were prepared

as soluble antibody fragments in periplasmic extracts (PPE) and the KD (equilibrium dissociation constant) was determined using Biacore. For both targets, multiple antibody fragments with high affinities (single-digit nM to triple-digit pM) were identified. Table 2 lists the best KD identified for each target per library (Fig. S3). When screening the panning campaigns of TIE2 in complex with either of its ligands (ANG1 or ANG2), antibody fragments in PPE were screened by flow cytometry for binding to TIE2 or TIE2–ligand complex, and screened by ELISA for binding to ANG1 or ANG2. Binders in three categories emerged: single-protein binders (TIE2, ANG1 or ANG2), TIE2/ANG1-complex binders, or TIE2/ANG2-complex binders (Table 3). A subset of 10 Fab clones and 8 scFv clones that bound TIE2 was reformatted as IgG and the KD for each clone was determined with Biacore (Table 2 and Fig. S3). For clones from XscFv2, 6 out of 8 clones have KDs > 8 nM. For clones from XFab1, 9 out of 10 have KDs > 11 nM with two of these clones having KDs in the pM range (Fab09 = 800 pM and Fab10 = 500 pM). The sequences of the 591 unique selected clones for both libraries were compared to each other and aligned to the closest germline sequence.

Blood was collected from the abdominal aorta in order to quantify the number of circulating mononuclear cells and the membrane expression of adhesion molecules. The BALF was collected according to De Lima et al. (1992). Total and differential cell numbers in the blood and BALF were determined in Neubauer chambers and in smears stained by the Romanowsky stain (Panótico®). In order to characterize the mononuclear cell population

in the BALF, the cells were incubated with the monoclonal antibodies anti-F4/80-PE and CD11b-FITC (macrophages) and CD3e-FITC (T lymphocytes) or CD19-PE (B lymphocytes; 30 min; 37 °C) and analysed in a FACSCalibur Flow Cytometer (Becton & Dickinson, San Jose, CA, Trichostatin A supplier USA). Alveolar macrophages (1 × 105/well) were isolated from the BALF and placed in a 24-well plastic microplate containing RPMI-1640 medium supplemented with 10% FBS for 3 h to allow them to adhere. Then, non-adherent cells were removed learn more and adhered cells were stimulated or not with LPS (1 μg/ml) and IFN-γ (10 ng/ml) and incubated at 37 °C, 5% CO2, for 24 h. Tracheal tissue was collected and placed in a 24-well plastic microplate containing DMEM medium (2 ml). The tissue was incubated in the absence or presence of LPS (1 μg/ml) and maintained at 37 °C, 5% CO2 for 24 h, according to Lino-dos-Santos-Franco et al. (2010a). The AM and tracheal tissue culture supernatants

were collected in order to evaluate inflammatory mediators. The animals were exposed to aerosolized HQ at 25 ppm (1.5 mg/60 ml) for 1 h, once a day for 5 days, according to the method of Ribeiro et al. (2011). Control animals were exposed to a vehicle (5% ethanol in saline). An ultrasonic nebulizer that generated particle PtdIns(3,4)P2 sizes within the range of 0.5–10 μm (NS®, Sao Paulo, Brazil) was used to nebulize the solutions. The efficacy

of the exposure system has been detected in several models of in vivo intoxications and for the induction of systemic and local inflammation ( Lino-dos-Santos-Franco et al., 2006, Lino-dos-Santos-Franco et al., 2009, Lino-dos-Santos-Franco et al., 2010b, Riffo-Vasquez et al., 2007 and Ribeiro et al., 2011). The concentration of HQ in the chamber was measured according to NIOSH, Protocol No. 5004. Extracts of cellulose ester membrane filters exposed for 1 h to 25 ppm HQ were analysed by HPLC, which resulted in a concentration of 0.20 mg/m3 ± 0.09 in the box, equivalent to 0.04 ppm. This concentration is 10 times lower than those allowed by international regulatory agencies (0.4 ppm; Ribeiro et al., 2011). Blood, BALF, AM and trachea were collected and used as described earlier. Tracheal tissue or AMs obtained from the BALF of naive animals were incubated with 1 μM, 10 μM or 100 μM HQ or RPMI-1640 medium supplemented with 10% FBS (control) for 1 h.