3B″) and at this point the implant was clearly osseointegrated. The maximum amount of osseointegration was achieved by day 21 (Fig. 3E). Of 23 implants placed, 21 had primary stability and by histologic assessment,

17 achieved osseointegration (a 74% success rate). We evaluated the peri-implant tissue reaction to the surgery and implant placement, and focused on samples harvested on day 14, when implants had osseointegrated. The peri-implant mucosa appeared healthy and devoid of inflammatory cells (Fig. 4A). A junctional epithelium, composed of non-keratinized, invaginating epithelium had CYC202 formed around the neck of a non-enclosed implant (Fig. 4A). The connective tissue attachment was well organized and was in direct contact with the implant surface (Fig. 4A). In regions closer to the native bone, new osteoid matrix was forming adjacent to the maxillary periosteum (arrows, Fig. 4A). In mice, most implants projected through the maxillary bone into the olfactory epithelium (e.g., Fig. 3). Murine olfactory tissue, which is considerably larger in rodents, occupies the position of the nasal fossae in humans. We evaluated how these tissues responded to the implant. Fibroblasts had infiltrated the glandular olfactory epithelium and adhered to the implant without evidence of inflammation (Fig. 4B).

In other cases, ATM/ATR phosphorylation new bone formation was detectable in the fibrous tissue attached to the implant surface (Fig. 4B′). We also analyzed cell viability in the maxillary bone. Using DAPI to detect cell nuclei and DIC to illustrate the osteocyte lacunae, we noted areas of extensive cell death in the cortical bone adjacent to the implant (dotted

yellow line, Fig. 4C). The empty Farnesyltransferase lacunae were exclusively found near the cut edge of the maxillary bone (dotted yellow line, Fig. 4C) and along the alveolar ridge where the flap was raised during the surgery (Fig. 4C′). This same DAPI staining indicated abundant new cells on the (unperturbed) nasal surface of the bone, along the new bone in contact with the implant surface, and along the periosteum (Fig. 4C,C′). Thus, the observed changes in peri-implant tissues are remarkably similar to the mucosal responses observed in large animals [28]. Furthermore, the results demonstrate how the standard surgical procedure of implant placement affects cell viability in the native bone. We were particularly interested in the impact of the osteotomy on the viability of osteocytes in the maxillary bone, because this has implications for long-term bone regeneration and bone remodeling at the site of implant placement. Using samples from day 14, we first distinguished between mature osteocytes of the maxillary bone (dotted line, Figs. 5A,B) and new osteoid matrix: Mature maxillary bone had a lamellar organization whereas the new bone was characterized by a woven appearance (arrows, Figs. 5A,B).

, 2011 and Qian et al., 2009) that can induce increased cell size and hypertrophy. These considerations prompted us to verify if the progression of the cell cycle in curcumin-treated HT-29 cells was deranged. Indeed, long-term exposure to 5.0–20 μM curcumin induced G1-phase arrest and S-phase depression (Fig. 10) in

HT-29 PLX-4720 solubility dmso cells. While the cell cycle arrest may explain the increased volume observed in curcumin-treated HT-29 cells, the underlying mechanisms leading to the deranged progression of cell cycle in these cells need to be elucidated. It is worth to note however, that an arrest of cell growth in the G0/G1 phase is often associated with a significant decrease in IClswell (He et al., 2011, Klausen et al., 2007 and Shen

et al., 2000). Curcumin induces apoptosis through a wide variety of mechanisms (Reuter et al., 2008). These mechanisms include the activation of the mitochondrial pathway via activation of Bax/Bak (Shankar and Srivastava, 2007b) or BID (Anto et al., 2002). Moreover, evidence exists that curcumin activated caspase 3 and 8 with no activation of caspase 9, raising the hypothesis of an activation of a death receptor-dependent (non-mitochondrial) pathway via FasL-independent aggregation of Fas receptors (Bush et al., 2001). In addition, activation by curcumin of novel apoptosis-like pathways, independent of mitochondria and caspases, was described (Piwocka et al., 1999). Therefore, it is likely that curcumin could click here induce apoptosis also when the mitochondrial pathway is blocked. From the presented G protein-coupled receptor kinase data we conclude that curcumin is able to affect cell survival and cell volume in a dose-dependent manner. At lower concentrations (<5.0 μM), curcumin indirectly activates IClswell, which is most likely the result of apoptosis induction. Higher curcumin concentrations (≥5.0 μM) indirectly lead to an inhibition of IClswell, an arrest of cell cycle in G1-phase and hence to cell swelling. Charity Nofziger is supported by the Lise Meitner stipend of the Fonds zur Förderung der Wissenschaftlichen Forschung (FWF) (M11108-B11).

The experimental work was further supported by the FWF and the FP-7 to M.P. (P18608; PIRSES-GA-2008-230661). None. We greatly appreciated the helpful discussion with M. Ritter. The authors acknowledge the expert secretarial assistance of Elisabeth Mooslechner. “
“Although the organophosphorus compounds (OPs), employed as insecticides exhibit preferential toxicity to insects, they are also toxic to humans and other animals due to the inhibition of AChE and the subsequent accumulation of acetylcholine at the neuron synapses (Johnson et al., 2000). In addition, some OPs can inhibit and age another esterase, known as the neuropathy target esterase (NTE) (Johnson, 1988), and cause a delayed effect that is known as organophosphorus-induced delayed neuropathy (OPIDN).

An Italian RCT of older (≥70 years) patients with CKD who were close to starting dialysis117 showed that a very low protein diet with 0.3 g/kg BW/d, supplemented with keto-analogues, amino acids, and vitamins, delayed the start of dialysis by approximately 11 months compared with a control group who followed a nonrestricted protein diet and immediately started dialysis. Compared with the control group, patients who were prescribed

a very low protein diet had similar mortality rates and their nutritional status was maintained. It is important to mention that patients enrolled in the study were not malnourished at baseline, and that they received nutritional counseling and follow-up nutritional care to maintain intake at 35 kcal/kg BW/d. In a retrospective

Dutch study of older patients Cabozantinib price (average age 65) with uncomplicated advanced CKD, a diet of 0.6 g protein/kg BW/d with nutritional counseling helped delay the start of dialysis by 6 months, with no difference in mortality compared with a control group not receiving a low-protein diet.112 Nonetheless, some experts remain selleckchem concerned about prospects for survival in older patients with CKD with sarcopenia, or depleted muscle mass. These experts call for 0.8 g protein/kg BW/d as a measure to help maintain fat-free mass and improve survival prospects (Table 6).113 and 118 The International Society of Renal Nutrition and Metabolism (ISRNM) has recently developed new dietary recommendations for people with CKD, including patients not on dialysis as well as those on peritoneal or hemodialysis.119 Because patients with kidney disease are at risk of protein-energy wasting, 30 to 35 kcal/kg BW/d is recommended.

In patients not on dialysis, protein intake of 0.6 to 0.8 g/kg BW/d is recommended for people who are well and 1.0 g/kg BW/d for those with disease or injury. Once maintenance dialysis begins, a diet with higher protein is necessary to overcome nutritional depletion of the dialysis procedure. Experts currently recommend more than 1.2 g/kg BW/d to compensate for the spontaneous decline in protein intake and the dialysis-induced catabolism.119 It is recommended that more than 50% of the protein consumed be of high Sulfite dehydrogenase biological value (ie, complete protein sources containing the full spectrum of amino acids). PROT-AGE recommendations for older people reflect the ISRNM guidelines, providing as much protein as possible for patients not no dialysis based on actual kidney function (measured as GFR).119 In a recent year-long study of older people with CKD (65 ± 14 years) on hemodialysis, patients were offered high-protein, multinutrient ONS during their thrice-weekly dialysis sessions.102 The “as-treated” patients receiving ONS had a 34% reduced risk of 1-year mortality (hazard ratio 0.66; 95% confidence interval [CI] 0.61–0.71), a significant and important improvement.

subcapitata check details in suspension was poured into an individual mold (disposable UV–vis cuvette), CaCO3 nanoparticles were gently placed on the surface of the liquid, 10 μL of 2.0% sodium alginate solution was poured on top and 0.2 M CaCl2 solution was immediately added in the form of a mist by means of a nebulizer machine. The second step of the immobilization procedure consisted of a silicate (sodium silicate, Riedel-de Haën; NaOH 10%, SiO2 27%) sol–gel process in the presence of commercial silica nanoparticles (LUDOX HS-40, 40% in water, obtained from Aldrich), leading to a nanoporous monolithic structure.

Monoliths were prepared at room temperature by mixing volumes of the different precursor solutions to obtain a SiO2:H2O molar relation of 0.038 with a fixed proportion of polymeric to particulate silica precursors (1:4) at constant pH 7.0, adjusted with HCl. As described in Section 2, daphnids and microalgae are co-immobilized in calcium alginate capsules of (8.5 ± 0.5) mm diameter and are further immersed in tubes where a mixture of sodium silicate

and colloidal silica is vigorously mixed. This colloidal solution undergoes a rapid sol–gel transition, and alginate capsules are quickly covered with a nanoporous silica gel (time of gelation: 2–3 min). As a result, silica biomaterials with liquid macrocavities containing daphnids and microalgae in M4 culture medium are formed. After 20 min (necessary time for the consolidation of the find more silica matrix), the biomaterials are immediately rinsed with distilled water, and fresh M4 medium is added to the tube (see Fig. 1). The high biocompatibility of this silica encapsulation procedure with P.subcapitata microalgal cells

is well established [16]. In this work, only the assessment of initial viability (1 h after encapsulation) is conducted by averaging the content of 10 macrocavities. To this end, the silica hydrogel is removed and samples are exposed to 0.05% potassium citrate to solubilize OSBPL9 the calcium alginate shell. The total number of cells inside individual cavities (2.35 × 105) was determined by counting cells in a Mallassez counting chamber; (99.2 ± 1.1)% of P.subcapitata cells remains intact, in good agreement with previous published results [16]. To evaluate the effect of the encapsulation procedure on D. magna, the content of each macrocavity was observed under an optical microscope (100× magnification) and the mobility of daphnids was recorded. The analysis reveals that 98% of the D. magna population (52 out of 53 total daphnids tested) remains active 1 h post-encapsulation, but this percentage drops to ∼32% only 6 h post-encapsulation (17 out of 53 total daphnids tested), and at 24 h post-encapsulation daphnids present no mobility. The complete set of results is presented in Fig. 2. Apart from the possible deleterious effect of the confinement itself, we hypothesized that the low biocompatibility towards D.

It was found that the winter NAO index varied in the same way as the mean annual water level variation (Figure 6) in the lagoons under study in 1961–2008. The correlation

analysis showed a positive correlation between the winter NAO index and the annual water level variations in the lagoons. Correlation coefficients between the NAO index and water level variations at Klaipėda/Memel, Baltiysk/Pillau and Zingst were 0.58, 0.62 and 0.43 respectively, with a statistical significance of 99.9%. This suggests that the changes in air mass dynamics in the North Atlantic are partly reflected in the interannual fluctuations of the water level on the coasts and in the lagoons of the south-eastern Baltic Sea. The Veliparib present-day water level variations on Baltic Sea coasts are determined by three main factors: the post-glacial uplifting of the Fennoscandian land mass, the global rise in eustatic water level, and the atmospheric circulation. Highly influential in this respect is the mesoscale atmospheric variation of circulation, which determines the air masses flowing into the North Atlantic region, as well as the formation and development of cyclones and anticyclones. The predominance of westerly inflows air masses leads to higher water levels in the eastern Baltic. When comparing the long-term tendencies in water

level selleck chemicals rise in the Baltic lagoons, we see that the rate of this rise increases as we move from the southern to the south-eastern shores: it is approximately 4 mm year−1 in the CL and VL, but only 1 mm year−1 in the DZBC. However, the structure of seasonal water level variations remains the same, independently of the average climate scale period, and the mean monthly level increased by 3–10 cm in nearly all

months. On the basis of an analysis of seasonal variations of monthly averaged water level, we see that the trend in annual mean water levels is influenced by high water level in the January–March months. Some of the most important factors affecting the long-term mean water level Aprepitant change in the coastal lagoons on the southern and south-eastern Baltic are land uplift, the rise in the global eustatic mean sea level, the prevailing wind with respect to the shore, and changes in freshwater gain. The eustatic change of sea level has a global influence, whereas tectonic movements can change the response on a regional scale. According to recent investigations, a land subsidence of –1 mm year−1 (Vestøl 2006) for southern and southeastern Baltic shores should be taken into consideration when calculating the absolute water level rise in these lagoons. If we take these trends into account when calculating water level rises for longer periods (1937–2008, Table 2), land subsidence practically cancels out any climatically induced water level changes in the DZBC, but not in the CL or VL, where the trend is strongly pronounced.

Consensus sequences were analyzed using the DnaSP 5.19 software (Librado and Rozas, 2009) to calculate nucleotide and haplotype diversity. Molecular analysis of variance (AMOVA) and neutrality tests were calculated using the Arlequin software (Schneider et al., 1999). An intraspecific phylogeny of COI haplotypes was inferred using the network algorithm median-joining in the Network program ( Bandelt et al., 1999). In the alignment of

60 partial COI sequences were observed 19 polymorphic Raf inhibitor sites along 751 bases, all corresponding to silent mutations, resulting in the formation of 15 mitochondrial haplotypes (for GenBank accession numbers see Supplementary material). Table 1 shows the number of D. willistoni specimens from each location analyzed, the COI haplotypes, genetic diversity estimates and Wolbachia DAPT clinical trial infection status. Of the 60 individuals tested, 33 (55%) were positive and 27 (45%) were negative for Wolbachia infection. Infection frequencies varied between populations but there was no discernible geographical pattern ( Fig. 1A). The partial sequence of the wsp gene was identical in 33 amplicons, corresponding to the sequence observed in strains wWil and wAu. This finding differs from the observations by Miller and Riegler (2006), who suggested that Wolbachia would be fixed in continental D. willistoni populations.

Nevertheless, it should be stressed that samples analyzed by those authors were composed mostly by laboratory strains. As previously described for D. melanogaster, there is polymorphism for infection rates in natural populations ( Hoffmann et al., 1994). The relationship between mitochondrial haplotypes and the association with Wolbachia is shown

in Fig. 1B. Haplotype C1 is ancestor of the other haplotypes, is the most frequent total, and is shared across all samples (except for the sample collected in São João do Polêsine). Wolbachia was observed to be associated click here to 10 of the 15 mitochondrial haplotypes generated. Yet, haplotypes C1, C4 and C9 were detected in both infected and uninfected individuals. The chi-square analysis showed no statistical difference between infected and uninfected in C1 and C4 haplotypes. However, statistically significant difference was found for haplotype C9 (P < 0.02). This haplotype was the most frequent in places where it was sampled (Guaratuba and Laguna) and this may be related to this deviation to a greater number of infected. The highest haplotype diversity was found in the Torres sample, while the lowest was seen in the Laguna sample. AMOVA revealed that 70.63% of variation occurs within populations and 39.98% between populations. The star network arrangement, with several rare haplotypes (C3, C5, C6, C7, C8, C10, C11, C12, C13 and C14) and the low nucleotide diversity indicate populational expansion (Mirol et al., 2008). Analyses of neutrality tests of Tajima D (−1.82193, P < 0.05) and Fu and Li F (−3.52798, P < 0.02), also support this scenario.

95 At least part of this effect was attributed to the effect of LIN28B on expression of BCL11A. Similarly, microRNA-486-3p was shown

to bind to the BCL11A messenger RNA 3′-untranslated region and downregulate its expression concomitant with OSI-744 manufacturer upregulation of ɣ-globin gene expression in cultured human erythroid cells. 96 The role of epigenetic changes in the actions of either LIN28B or microRNA-486-3p remains unknown. Any discussion of epigenetic regulation of globin gene expression must account for the interplay between transcription factors and coregulatory complexes with which they interact and which in turn often contain both “writers” (eg, histone acetylases and deacetylases), and “readers” (eg, methylcytosine-binding proteins) of epigenetic chromatin marks. Several transcription factors that are involved in embryonic fetal β-type globin gene silencing are known to associate with one or more corepressor complexes. Among these, Cell Cycle inhibitor BCL11A has emerged as a dominant regulator of developmental globin gene silencing in mice and is also implicated as a strong mediator of ɣ-globin gene silencing in

cultured human primary erythroid cells.19 BCL11A has been shown to associate with the MBD3-NuRD complex, as well as the LSD1/CoREST complex, Sin3A, NCoR/SMRT, and DNMT1.86 Another transcription factor complex associated with embryonic globin gene silencing, the TR2/TR4/DRED orphan nuclear check details receptor complex, has been shown to associate with a number of epigenetic coregulatory proteins, including the MBD3-NuRD, LSD1/CoREST, Sin3A complexes, and DNMT1.87 Thus, the effectors of these transcription factors may be in large part epigenetic. Another connection

between epigenetic regulators and transcription factors that are involved in ɣ-globin gene silencing is through epigenetic regulation of expression of the transcription factors themselves. It was recently shown that Mi2β/CHD4 (chromodomain helicase DNA–binding protein 4), independently of the NuRD complex, is required for high level expression of both KLF1 and BCL11A in primary human adult erythroid cells and that Mi2β/CHD4 binds directly to BCL11A 67 (see Fig 1). It is important to note that virtually all the epigenetic and transcriptional regulatory factors that are discussed here and depicted in Fig 1 have been shown to play a role in normal developmental globin gene switching. However, the relative effect of a given factor in the totality of ɣ-globin gene silencing appears to vary considerably in developmental globin silencing or “switching” vs maintenance of silencing in the adult erythroid compartment.

The disease has been known in the Indian sub-continent for over a century (Crawford, 1912 and Husain and Nath, 1927). In the United States, HLB is now established in Florida and has resulted in substantial economic losses, estimated to be about US$3.6 billion in economic activity, in a 5 year period (Hodges and Spreen, 2012). Because of the significant financial Erastin in vivo implications associated

with HLB, the citrus industries and the regulatory agencies in USA, Brazil, and other countries, are interested in early, rapid detection of the pathogen and subsequent management strategies required to mitigate the disease. Three fastidious gram negative bacteria have been associated with citrus HLB: ‘Candidatus Liberibacter asiaticus’ (Las), ‘Candidatus Liberibacter americanus’ (Lam) and ‘Candidatus Liberibacter africanus’ (Laf). Las is the most prevalent HLB-associated bacterium in Asia as well as in the Western hemisphere. Asian citrus psyllid (ACP; Diaphorina citri Kuwayama), the vector of Las has been reported from most citrus growing regions. The first report of ACP in the United

States was from Florida in 1998 ( Halbert et al., 2000). In Brazil, the psyllid vector prevailed for about 60 years without CB-839 nmr the pathogen and did not cause significant damage to the citrus industry ( Bové, 2006 and Lima, 1942). Suggested actions for mitigation of citrus HLB include: a) planting of disease-free nursery stock, b) constant scouting for visual detection of symptomatic trees and subsequent removal and, c) ADP ribosylation factor control of psyllid vector by pesticide sprays (Belasque et al., 2010, Bové, 2006, Grafton-Cardwell et al., 2013 and Hall et al., 2013). Starting a citrus grove with HLB-tested disease-free nursery stock is an excellent method of disease control and is currently being implemented by regulatory agencies in the United States and Brazil. Reduction of inoculum by removing infected plants based on visual detection of HLB symptoms was followed in many citrus industries including

Brazil (Belasque et al., 2010 and Bové, 2006). It has been shown that infected plants can remain non-symptomatic for an extended period of time, and hence tree removal will not be very effective since the pathogen is known to have a lengthy incubation and latent period (Chiyaka et al., 2012 and Gottwald, 2010). In several locations in Florida, Las was first recorded in psyllids and the subsequent detection in field plants was verified 6 months to 3 years after the initial find in psyllids (Manjunath et al., 2008). Under controlled conditions, Pelz-Stelinski et al. (2010) have demonstrated that it may take one year or longer to detect Las in plants that are successfully inoculated by Las-positive D. citri. HLB disease management based on constant monitoring of the psyllids for Las may be a suitable approach.

Before experimental procedures, animals were submitted to handling for five consecutive days to adapt to the experimenter and minimize stress. Thermocoagulation of the blood in the submeningeal blood vessels of the motor and sensorimotor cortices was used to induce ischemic lesion as previously described (Giraldi-Guimarães et al., 2009; Szele et al., 1995). Briefly,

animals were anesthetized with ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (10 mg/kg) and placed in a stereotaxic apparatus (Insight Ltda., Ribeirão Preto, SP, Brazil). Skull was exposed, and a craniotomy was performed, exposing the frontoparietal FK228 research buy cortex contralateral to the preferred forelimb in the adhesive test (see Section 2.4.) (+2 to −6 mm A.P. from bregma; according to the atlas of Paxinos and Watson (2005). Blood was thermocoagulated transdurally by approximation of a hot probe to the dura mater. Sham operated animals suffered only the craniotomy. Trichostatin A clinical trial After procedure, skin was sutured, and animals were kept warm under a hot lamp and returned to colony room after recovery from anesthesia. The flavonoid rutin was purchased commercially (Sigma-Aldrich, St. Louis, MO, USA). Rutin was diluted in propylene glycol. To facilitate the dissolution of rutin, the solution

was made to stand for 15 min in a water bath at 50 °C for 10 min. Rutin solution or vehicle (propylene glycol) was administered by intraperitoneal (i.p.) injection. D-malate dehydrogenase Ischemic animals were divided into three experimental groups: one that received vehicle (control group), one that received the dose of 50 mg of rutin/kg of body weight (R50 group) and one that received the dose of 100 mg/kg (R100 group). These

doses were chosen from previous studies showing protective effect of rutin in models of global brain ischemia (Abd-El-Fattah et al., 2010 and Pu et al., 2007). For behavioral analyses, all groups were used and the protocol of treatment was a daily injection during five consecutive days, starting just after the end of surgical procedure. In other analyses, as explained below, control and R50 groups were used with changes in protocol of treatment. Functional recovery of the forelimb contralateral to the ischemic cortical hemisphere was evaluated using two sensorimotor tests: cylinder test and adhesive test (Schallert, 2006). Their effectiveness to assess sensorimotor function has been shown after thermocoagulatory cortical lesion (de Vasconcelos dos Santos et al., 2010, Giraldi-Guimarães et al., 2009). All animals were tested one day before ischemia and at post-ischemic day (PID) 2, and then weekly. Pre-ischemic day was plotted in graphs as PID 0. Tests were performed as previously described (de Vasconcelos dos Santos et al., 2010, Giraldi-Guimarães et al., 2009). Briefly, in the forelimb use asymmetry (cylinder) test, a trial consisted in placing the animal inside a glass cylinder (20 cm diameter X 30 cm height).

pASARM and npASARM peptides were added to ATDC5 cells and metatarsal organ cultures at concentrations Ibrutinib mw of 10, 20 and 50 μM, with controls treated with a DMSO (Sigma) carrier only. In further studies, peptides were added at a final concentration of 20 μM with experiments being performed at least 3 times. Embryonic metatarsal organ cultures provide a well‐established model of endochondral bone growth [22], [23] and [24]. Metatarsal bones were cultured in a humidified atmosphere

(37 °C, 5% CO2) in 24-well plates for up to 10 days. Each culture well contained 300 μl α-minimum essential medium (MEM) supplemented with 0.2% BSA Fraction V; 1 mmol/l β-glycerophosphate (βGP); 0.05 mg/ml L-ascorbic acid phosphate; 0.05 mg/ml gentamicin and 1.25 μg/ml fungizone (Invitrogen, Paisley, UK) as previously described [22]. For the E17 bones, the medium was changed every second or third day and for the E15 bones, the medium was not changed throughout the culture period [25]. Concentrations of peptide and DMSO carrier were however added every second day. The total length of the bone through the centre of the mineralizing zone was determined using image analysis software (DS Camera Control Unit DS-L1; Nikon) every second or third

day. selleck The length of the central mineralization zone was also measured. All results are expressed as a percentage change from harvesting length which was regarded as baseline. Metatarsals were fixed in 70% ethanol, stained with eosin dye (for visualisation) and then embedded in paraffin blocks. Samples were then were scanned

with a high-resolution μCT (μCT40; Scanco Medical, Southeastern, PA) as previously described [13] and [16]. Data were acquired at 55 KeV with 6 μm cubic voxels. Three-dimensional reconstructions for bone samples were generated with the following parameters: Gauss Sigma = 4.0; Support = 2, Lower Threshold = 90 and Upper Threshold = 1000. Tissue mineral density was derived from the linear attenuation coefficient of threshold bone through precalibration of the apparatus for the acquisition voltage chosen. The mafosfamide bone volume (BV/TV) was measured using sections encompassing the entire metatarsal on a set of 85 sections that was geometrically aligned for each sample. On day 7 of culture, 3 μCi/ml [3H]-thymidine (Amersham Biosciences, Little Chalfont, UK) was added to each metatarsal for the last 6 h of culture [22]. After washing in PBS, the unbound thymidine was extracted using 5% trichloroacetic acid (Sigma). Metatarsals were then washed in PBS before being solubilised (NCS-II tissue solubiliser, 0.5 N, Amersham) at 60 °C for 1 h. [3H]-thymidine incorporated into DNA was determined using a scintillation counter.