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Activating FGFR3 mutations in human result in achondroplasia (ACH), the most frequent form of dwarfism, where cartilages are severely disturbed causing long bones, cranial base and vertebrae defects. Because mandibular development and growth rely on cartilages that guide or directly participate to the ossification process, we investigated the impact of FGFR3 mutations on mandibular shape, size and position. By using CT scan imaging of ACH children and by analyzing Fgfr3Y367C/+ mice, a model of ACH, we show that FGFR3 gain-of-function mutations lead to structural anomalies of primary (Meckel’s) and secondary (condylar) cartilages of the mandible, resulting in mandibular hypoplasia and dysmorphogenesis. These defects are likely related to a defective chondrocyte proliferation and differentiation and pan-FGFR tyrosine kinase inhibitor NVP-BGJ398 corrects Meckel’s and condylar cartilages defects ex vivo. Moreover, we show that low dose of NVP-BGJ398 improves in vivo condyle growth and corrects dysmorphologies in Fgfr3Y367C/+ mice, suggesting that postnatal treatment with NVP-BGJ398 mice might offer a new therapeutic strategy to improve mandible anomalies in ACH and others FGFR3-related disorders.
Achondroplasia (ACH) is the most common form of chondrodysplasia and is characterized by a rhizomelic dwarfism with short limbs, macrocephaly, frontal bossing and midface hypoplasia (1–3). ACH is caused by an activating mutation of Fibroblast Growth Factor Receptor 3 (FGFR3) that affects both endochondral and membranous ossification (3–5). FGFR3 over-activation disturbs the proliferation and differentiation of chondrocytes in the growth plate of long bones (6–8) and synchondroses of the cranial base (3,9). Other cartilaginous structures are affected in ACH, such as the inner ear (10), vertebral bodies and intervertebral disc (11) and joints (7).
Mandibular growth relies greatly on primary and secondary cartilages, even though their exact roles in the acquisition of the mandible final shape and size are not fully understood (12,13). Meckel’s cartilage (MC) is a rod-shaped primary cartilage that runs through the mandibular process of the first pharyngeal arch and acts as a morphogenic template for the membranous ossification of the mandible body. It is transiently present in the developing mandible and disappears at birth. Secondary cartilages (symphyseal, angular and condylar cartilages) form prenatally and eventually ossify by endochondral ossification during post-natal growth, serving as growth centers (12). The central role of cartilages in mandibular development and growth is exemplified by the mandibular phenotype observed in several chondrodysplasias where mutations in genes involved in chondrocyte function, such as Collagen II (responsible for Achondrogenesis type II), Sox9 (Campomelic dysplasia) or PTHrP (Jansen type of metaphyseal chondrodysplasia) result in abnormal mandibular shape, size or position (14–16).
Mutations of FGFR1, FGFR2 and FGFR3 genes are associated with various aspects of abnormal craniofacial development such as craniosynostoses and maxillary hypoplasia (2,17,18). FGFR2 and FGFR3 are expressed in MC (19,20) and throughout all phases of the chick mandibular development (21). FGFR3 signaling is required for the elongation of chick MC and FGFR2 and FGFR3 play a role during membranous ossification of mandible (22). Mandibular defects have also been observed in Fgfr1/2dckomice, with a shorter and smaller mandible at birth (20). The deletion in mice of the FGFR3 signaling molecule Snail (23) is associated with a growth retardation of MC and a reduction in mandibular length (24) and mice deficient for the FGFR3 ligand FGF18 display a reduced size of MC and a hypoplastic mandible (25). These data support the role of FGFR signaling in the elongation of MC and morphogenesis of the mandible.
However, it is unknown whether mandibular growth and development are affected in children with ACH and whether mandibular cartilages are disturbed by FGFR3 mutations. Analysis of lateral cephalogram from adult ACH patients has shown a prognathic mandible (i.e. anteriorly displaced) (26,27). It has also been reported that in humans, other skeletal dysplasia caused by activating mutations of FGFR3 can result in mandibular dysmorphogenesis as seen in thanatophoric dysplasia (TD) (28), and Muenke syndrome (MS) (29).
In this article, we studied the mandible growth and development in children with ACH and observed an abnormal shape, size and position of the mandible. We compared the human data with those obtained with a mouse model of ACH (Fgfr3Y367C/+) (10), for which we analyzed MC and secondary cartilages of the mandible. We observed proliferation and differentiation defects in chondrocytes of the mandibular cartilages and a delay in the replacement of MC by bone. By targeting the excessive activity of FGFR3 with a tyrosine kinase inhibitor NVP-BGJ398 (30), in organ cultures, we corrected the modifications of size and shape of the mandibles. Using NVP-BGJ398 in vivo, we observed a strong improvement of the mandibular phenotype. Taken together, these data bring out anomalies of the mandible in ACH and its mouse model and offer new perspective of treatment for ACH.
Achondroplasia results in mandibular hypoplasia and dysmorphogenesis
Although the craniofacial phenotype of patients with ACH has been described (2,3,26), no specific analysis of the mandible has been conducted. We took advantage of CT images acquired for assessment of risks for cervicomedullary-junction compression in infants with ACH (n = 8, mean age: 21.3 months) and compared these images with those of age-matched controls obtained after traumatic events (n = 9, mean age: 24.9 months). The length of the mandible, measured as the distance between condylion (Co) and gnathion (Gn), was significantly and consistently decreased in ACH patients compared to controls (−14%; P < 0.05; Fig. 1A and B). Mandibular body length [Gonion (Go) – Menton (Me)] and mandibular ramus length (Go – Co) were also significantly decreased in ACH children (−16%; P < 0.01 and −17%; P < 0.05, respectively).
The analysis of tridimensional coordinates of anatomical landmarks with geometric morphometrics (31) showed differences in mandible shape between ACH patients and controls. Principal components analysis (PCA) of the human mandible shape resulted in the separation of ACH patients and controls along PC1 accounting for 47% of total variance on the basis of shape features represented in Figure 1C. When compared with controls, mandibles of ACH children were characterized by a defective orientation and size of the ramus with prominent coronoid processes and relatively shorter condyles.
FGFR3 activation in mice results in mandibular hypoplasia and dysmorphogenesis
The role of Fgfr1 and Fgfr2 during mandibular development was demonstrated in mice with a mesenchyme-specific disruption of Fgfr1 and Fgfr2 (20), while the impact of an activating Fgfr3 mutation on the mandible has not been studied. Here, we compared the mandible size and shape of Fgfr3Y367C/+ mice that accurately mimic ACH (3,10,32) with wild-type (WT) mice at different ages. We sacrificed pregnant mice to collect Fgfr3Y367C/+ and control littermate embryos at gestational day E16.5 and E18.5. Mice were also sacrificed at postnatal day 0 and 21. The mandibles were dissected and stained with alcian blue for cartilage and alizarin red for mineralized tissue. We observed a significant reduction in the length of the mandible body in the mutant mice at all time-points: −11% P < 0.005, −9% P < 0.005, −10% P < 0.005, −16% P < 0.0001 compared with WT littermates at E16.5, E18.5, P0 and P21, respectively (Fig. 1D and E).
To identify potential dysmorphologies in Fgfr3Y367C/+ mandibles in addition to the hypoplasia, we measured anatomical landmarks on three-dimensional (3D) reconstructed micro-CT images of P21 mandibles and analyzed their coordinates with geometric morphometrics. The PCA of the mandible shape resulted in the separation of Fgfr3Y367C/+ mice and controls littermates along PC1 accounting for 78% of total variance on the basis of shape features (mainly located on the ramus) represented in Fig. 1F. Our results revealed that the shape changes observed in ACH patients, and Fgfr3Y367C/+ mice were relatively similar.
Chondrocytes homeostasis is disturbed in MC of Fgfr3Y367C/+ mice
We hypothesized that Fgfr3 activation disturbs chondrocytes homeostasis in MC, as it was shown in other cartilages, such as the growth plate (10), the inner ear (10), the cranial base synchondroses (3) or vertebral bodies (11), and that these defects could be responsible for the mandibular hypoplasia and dysmorphogenesis observed in Fgfr3Y367C/+ mice. We therefore collected embryos at gestational day E16.5 and E18.5 and analyzed markers of chondrocyte proliferation and differentiation on histological sections.
At E16.5, MC was readily identifiable on sections stained with safranin’O (Fig. 2A). We focused on the cartilage within the developing mandible. In its central part, chondrocytes are organized into layers (33,34), as in a growth plate or a synchondrosis. We used several markers to compare the spatial organization of the chondrocytes into distinct zones in WT and mutant embryos and observed that the chondrocyte differentiation was disrupted in mutant embryos. First, immunostaining for Collagen X showed that the size of the hypertrophic chondrocytes zone relative to the total size of the cartilage was reduced (−43% compared to WT, P < 0.05; Fig. 2A and B), as was the size of individual hypertrophic chondrocytes (−51% compared to WT, P < 0.0001; Fig. 2A and C) in Fgfr3Y367C/+ embryos. Immunostaining of the proliferation marker Ki67 indicated that more cells were proliferating in MC of mutant embryos (+81% compared to WT, P < 0.01; Fig. 2A and D). In WT and Fgfr3Y367C/+ embryos, the zones of proliferating and hypertrophic cells were clearly delimited and were almost completely mutually exclusive (Fig. 2A). We then observed that the number of Fgfr3-positive cells in this cartilage was increased in Fgfr3Y367C/+ embryos (+151%, P < 0.0001; Fig. 2A and D) as reported in the growth plate of the same mouse model (11,35) and ACH and TD fetuses (6). FGFR3 exhibits a specific pattern of expression during chondrocyte differentiation (5,36), and in the growth plate, its expression is mostly limited to the resting, proliferating and pre-hypertrophic chondrocytes (37). In WT embryos, Fgfr3 was expressed by proliferative and prehypertrophic chondrocytes (Fig. 2A), whereas in Fgfr3Y367C/+ embryos, the pattern of expression was less clearly delimited, with an overlapping of Collagen X and Fgfr3-positive cartilage areas.
Differentiation into hypertrophic chondrocytes precedes and contributes to the disappearance of MC and replacement by bone (38,39). At E18.5, large remnants of cartilage were detected in Fgfr3Y367C/+ embryos, in contrast with WT embryos where MC was mostly replaced by bone (Fig. 2E and F) and the size of MC was significantly increased in Fgfr3Y367C/+ embryos mandible (+129%, P < 0.05). Similar defects lead to an ossification delay in the growth plate (40).
Overall, these results suggest that Fgfr3 constitutive activation disturbed the chondrocytes proliferation and differentiation and delayed the ossification process in MC.
FGFR3 activation reduces condylar growth in humans and mice
Deviations in the growth of the mandibular condyle can have major functional and aesthetic consequences (41). Although the majority of the mandible is formed by membranous ossification, the upper part of the ramus is formed by endochondral ossification of the condylar cartilage. This process allows the mandible to elongate and grow upward and backward (42). In both humans and mice, endochondral ossification is severely disturbed by FGFR3 activating mutations and defects in condylar cartilage could contribute to mandibular hypoplasia and dysmorphogenesis. For these reasons, we characterized the condylar process, first in children with ACH, then in Fgfr3Y367C/+ mice. We observed on sagittal sections from CT images that the length of the condylar neck in children with ACH was reduced compared to age-matched controls (−21%, P < 0.005; ACH: n = 12, mean age= 32.4 months; controls: n = 14, mean age = 32.4 months) (Fig. 3A and B). This reduction was observed in children aged from 1 to 42 months. In Fgfr3Y367C/+ mice (P21), condylar hypoplasia was also present (Fig. 3C and D). In mutant mice, condyles were significantly reduced in length (−14.2%, P < 0.01) and width (−15.8%, P < 0.001).
To identify the mechanism that led to this shorter condylar neck, we examined the condylar cartilage on histological sections of E16.5 embryos and P21 mice. In Fgfr3Y367C/+ mice, the size of the hypertrophic zone in the cartilage was reduced compared to WT littermates as was the average size of individual hypertrophic chondrocytes (E16.5: −48%, P < 0.001; P21: −56,3%, P < 0.05) (Fig. 3E–H).
Tyrosine kinase inhibition corrects primary and secondary cartilages defects in Fgfr3Y367C/+ embryos
To test the hypothesis that the mandibular hypoplasia and dysmorphogenesis observed in children with ACH and Fgfr3Y367C/+ mice are direct consequences of an overactive FGFR3, we analyzed the effect on mandibular development and growth of pharmacological inhibition of FGFRs using NVP-BGJ398, a pan-specific FGFR inhibitor (30). We first developed an ex vivo system of mandible explant cultures, similar to the femur explant cultures we used to test pharmacological approaches of FGFR inhibition (11,32,35). We isolated the mandible from WT and Fgfr3Y367C/+ embryos at E16.5, dissected the mandibles into two hemi-mandibles and cultured those separately for 6 days. One hemi-mandible was treated with NVP-BGJ398, whereas the other served as control. As expected, the body and condyle of hemi-mandible from Fgfr3Y367C/+ embryos treated with DMSO were smaller than those of WT hemi-mandibles also treated with DMSO (−10,1%, P < 0.05; −18%, P < 0.05 respectively, Fig. 4A and B). Incubation with NVP-BGJ398 of Fgfr3Y367C/+ hemi-mandibles led to an increase in mandible body (+11%, P < 0.05) and condylar neck (+26%, P < 0.05) size, reaching the size of cultured WT hemi-mandibles treated with DMSO (Fig. 4A and B).
Increases in mandible length with NVP-BGJ398 treatment were associated with the correction of chondrocyte defects observed in primary and secondary mandibular cartilages of Fgfr3Y367C/+ embryos. Although the size of MC hypertrophic zone relative to the size of the cartilage was clearly reduced in Fgfr3Y367C/+ hemi-mandible treated with DMSO compared to WT (−40.2%, P < 0.05), this zone expanded following Fgfr3 inhibition in mutant mandible (+68.3%, P < 0.05) (Fig. 4C and D). This expansion was associated with an increased size of individual hypertrophic chondrocytes in hemi-mandible from Fgfr3Y367C/+ embryos treated with NVP-BGJ398 (+165% compared to Fgfr3Y367C/+ treated with DMSO, P < 0.0001) (Fig. 4C and E). We noted an increased replacement of MC by bone in the treated mandibles, compared to the untreated Fgfr3Y367C/+ mandible, rescuing the extinction delay of MC observed in Fgfr3Y367C/+ mice. The kinase inhibitor also corrected the overexpression of Fgfr3 and the increased proliferation of chondrocytes in MC of Fgfr3Y367C/+ hemi-mandible (Fig. 4F).
In the condylar cartilage, similar changes were observed. Treatment with NVP-BGJ398 increased the size of the cartilage hypertrophic zone and the size of individual hypertrophic chondrocytes in condyles of Fgfr3Y367C/+ embryos (+98.4%, P < 0.0005 compared to condyles from Fgfr3Y367C/+ hemi-mandibles treated with DMSO) (Fig 4G and H).
Altogether, we observed that the reduction of Fgfr3 activity with NVP-BGJ398 corrected the defective growth and cellular defects observed in the presence of an overactive Fgfr3. As those improvements occurred in the absence of any systemic regulation (ex vivo explants), the effect of the inhibitor is likely direct. This reinforces the view that the cartilage defects are responsible for the mandibular hypoplasia.
Tyrosine kinase inhibition improves the mandibular dysmorphogenesis and the size of the condyle in Fgfr3Y367C/+ mice
Finally, we tested the potential benefit of a pharmacological use of tyrosine kinase inhibitor to correct in vivo the mandibular hypoplasia and dysmorphogenesis of Fgfr3Y367C/+ mice. We performed daily subcutaneous injection of either NVP-BGJ398 (2 mg/kg) or vehicle on 1-day-old mice from P0 to P15 (11). The mandibles of P16 mice were then imaged by micro-CT. As in the ex vivo experiments, we observed phenotypic changes of the condylar neck in Fgfr3Y367C/+ mice treated with NVP-BGJ398 compared to Fgfr3Y367C/+ littermates treated with DMSO. The length (+20.9%, P < 0.005) and width (+22%, P < 0.005) of the condylar neck were increased by the reduction of the over-activation of FGFR3 (n = 7, n = 8 and n = 7 for WT vehicle, Fgfr3Y367C/+ vehicle and Fgfr3Y367C/+ NVP-BGJ398, respectively) (Fig. 5A and B). As expected, the length of mandible body was unchanged because MC had already been replaced by bone at the start of the injections. We confirmed the positive effect of NVP-BGJ398 on condylar cartilage with histological sections of the condyles (Fig. 6A and B). The PCA of the Procrustes shape coordinates of the landmarks measured on all mandibles was also run. As for P21 mice, PC1 separated the WT vehicle and the Fgfr3Y367C/+ vehicle mice (Fig. 5C and D). The Fgfr3Y367C/+ NVP-BGJ398 mice occupied a somewhat intermediate position though they overlap with the Fgfr3Y367C/+ vehicle mice (Fig. 5C and D). P-values from permutation tests (10 000 permutation rounds) for Mahalanobis distances among groups showed that the Fgfr3Y367C/+ NVP-BGJ398 mice were significantly different in the shape of the mandible from either the Fgfr3Y367C/+ vehicle mice or the WT vehicle. When comparing the centroid size (CS) used as a proxy for overall size of the mandible, WT mice displayed significantly larger mandible than Fgfr3Y367C/+ mice treated with the vehicle (t = −6.96; P < 0.005) or NVP-BGJ398 (t = −7.35; P < 0.005). Fgfr3Y367C/+ NVP-BGJ398 mice did not display significantly larger mandible than the Fgfr3Y367C/+ vehicle mice (t = −0.0887; P = 0.931). These data confirm that the length of the mandible was not corrected by the treatment, but that the overall shape of the mandible of Fgfr3Y367C/+ was improved with NVP-BGJ398.
Deviation from normal mandible growth can strongly affect the masticatory and respiratory functions, speech and aesthetic appearance of the face (41,43), and mandibular cartilages are recognized as critical growth centers for the mandible. Although many cartilages are affected in FGFR3-related chondrodysplasias, most strikingly the cartilaginous growth plate in the growing skeleton, mandibular cartilages and their impact on mandible growth had never been investigated in ACH. Our hypothesis was that mandibular cartilages would be affected by FGFR3-activating mutations, similar to other cartilages, and that these defects would cause modifications in the shape, size and/or position of the mandible.
We first observed differences in the size and shape of the mandible between ACH and control children. The mandible as a whole, or subunits such as the mandibular body and ramus were reduced in size at all ages in children with ACH and morphometric analysis revealed modifications in the shape of the ramus. Comparable changes were present in Fgfr3Y367C/+ mice, and in both children and mice, the mandibular hypoplasia worsened with age. Our findings illustrate again the similarity of this mouse model with ACH.
In addition to ACH, FGFR3-activating mutations cause four other types of chondrodysplasia (hypochondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans, and TD type I and II) (1,2) and two types of craniosynostoses (MS and Crouzon syndrome with acanthosis nigricans) (44). The presence of mandible abnormalities in these diseases has rarely been studied. A report showed mandibular clefting in a case of TD I (28), two cephalometric studies in adult ACH patients reported opposite results concerning the size of the mandible [normal for Cohen et al. (26) or decreased for Cardoso et al. (27)], whereas another study showed that MS patients had shorter mandibular body length than controls (29). Several mouse lines carrying Fgfr3 activating mutations have been generated but a very few comprehensive analysis of the mandible are available. The homozygous Fgfr3 G380R mouse, a model for ACH, has a reduced mandible length (79% of controls) (45), and the homozygous Fgfr3 P244R mouse, a model for MS, has a hypomorphic condyle (46).
The FGFR3 mutations responsible for chondrodysplasias and craniosynostoses were until recently known to primarily affect different ossification processes (endochondral versus membranous), but as observed in ACH (3), FGFR3 over-activation can consistently disturbs both ossification processes. The ossification of the mandible relies on these two types, the body being formed by membranous ossification, whereas the ramus is mainly formed by endochondral ossification. If the effect of FGFR3 mutations responsible for ACH on the ramus is expected, it is intriguing that the same mutations can affect the size of the mandibular body. In order to understand what led to these changes we studied a primary (Meckel’s) and a secondary (condylar) cartilage. The exact role of MC is not yet fully understood as it disappears before birth and only marginally participates in the ossification process of the mandible (47). It could play a role in the elongation of the mandible body as exemplified by the severe micrognathia present when MC is strongly perturbed as in the case of Sox9 haploinsufficiency (48) or deletion of Snai1 and Snai2 (24). Here, we found that Fgfr3 over-activation disturbed the differentiation of chondrocytes in MC. The size of the hypertrophic zone was markedly shortened, as was the size of individual hypertrophic chondrocytes. The same defects were observed in the growth plate of ACH patients (6) and Fgfr3Y367C/+ mice (10,40). As the chondrocytes differentiate in the growth plate, the volume enlargement to become hypertrophic determines the size of the growth plate elongation (49–51). The same mechanism could occur in MC and the disruption of MC chondrocytes differentiation caused by FGFR3 over-activation could lead to the shortening of the mandible body. Supporting this view, we observed that reduction of the tyrosine kinase activity of FGFR3 with NVP-BGJ398 not only corrected the hypertrophic chondrocyte phenotype in mandible ex vivo cultures but also improved the shortening of the mandible of Fgfr3Y367C/+ embryos. In contrast, the same molecule had no effect on the mandible body once MC was replaced by bone. We assume that this is due to the disappearance of MC before birth and, to us, reinforces the importance of MC in the elongation of the mandible. In mice of this age, ossification of the symphyseal is limited and therefore contributes marginally to the elongation of the mandible body (52).
The activity of FGFR3 must be precisely tuned for normal mandible elongation because the injection of dominant negative (DN) form of FGFR3 in the chick embryo leads to the truncation or shortening of MC (22). In the same study, the DN form reduced the proliferation of the cells in MC, whereas in our study, we observed that it was markedly increased by the activating mutation. Increased chondrocyte proliferation was also observed in the growth plate of embryos carrying either the Fgfr3 Y367C or K644E mutations (40,53). In MC, the expression of Fgfr3 was also increased by the Y367C Fgfr3 mutation. It could be the result of a prolonged half time of the protein because the mutation of the transmembrane domain could delay the turnover and degradation of the activated receptor (54,55).
FGFR3 mutations responsible for ACH could also affect the ossification of the mandibular process that was delayed in Fgfr3Y367C/+ embryos. It could be a consequence of the defective MC homeostasis and subsequent initiation of ossification. Alternatively, mesenchymal cells lateral to MC that directly differentiate into osteoblasts (20) could be independently affected by FGFR3 activation. Further work is needed to determine whether this delay affects mandible bone mass acquisition during growth, as reported in long bones (40,56).
Secondary cartilages are sites of late prenatal and early postnatal growth and eventually ossify. Here, we focused on the condylar cartilage. It develops adjacent to the intramembranous bone of the mandible, distinct from MC and in contrast with MC, directly contributes to the formation of the ramus by endochondral ossification and persists postnatally to function as a growth center (42). Genetic defects in chondrogenesis can cause abnormal condylar growth as in Pierre Robin sequence (41), an entity associated with genetic defects of SOX9 (57). We found that children with ACH had shorter condylar neck, a feature also found in Fgfr3Y367C/+ mice. Histological analysis of the condyle in Fgfr3Y367C/+ mice revealed a disturbed cartilage where the hypertrophic chondrocytes were smaller and the hypertrophic zone shortened, as in MC or in the growth plate of the same mice (10). Altered differentiation of chondrocyte is also present in condyles of TD infants (58) and in the hypolpastic condyle of homozygous Fgfr3 P244R mice (46). Here, cultures of hemi-mandibles and in vivo experiments with partial inhibition of Fgfr3 activity showed that it is likely that the defective chondrocyte differentiation and proliferation directly contributes to the reduced elongation of the condyle. The persistence of the condylar cartilage postnatally allowed us to observe that in vivo tyrosine kinase inhibition rescue condylar growth in Fgfr3Y367C/+ mice, increasing its length by 21% after 15 days of treatment. The same treatment improved the growth of appendicular and axial skeletons with a similar magnitude of change of long bones (11).
Current treatments of maxillofacial deformities and skeletal dysplasia are primarily surgical and can require multiple interventions during childhood (59,60). Innovative pharmacological treatments of these diseases are needed and reducing the excessive activity of the FGFR3 receptor in FGFR3-related chondrodysplasias or craniosynostoses with specific tyrosine kinase inhibitors, such as NVP-BGJ398, is an appealing approach. It potentially targets all downstream signaling pathways of the receptor, and in this aspect, NVP-BGJ398 has been shown to normalize in vitro and in vivo ERK1/2 and PLCγ pathways (11). Our pre-clinical experiments showed here that this inhibitor enters mandibular cartilages and improves craniofacial growth.
In summary, we showed in this article that a FGFR3 gain-of-function mutation disturbs the development and growth of the mandible, via structural anomalies of Meckel’s and condylar cartilages. These anomalies are likely related to the defective chondrocyte proliferation and differentiation, and the tyrosine kinase inhibitor NVP-BGJ398 corrects these defects and improves condyle growth. This suggests that postnatal treatment with the molecule could be a therapeutic strategy to improve mandible growth in ACH and others FGFR3-related disorders.
Materials and Methods
All patients with ACH (n = 12, mean age = 32.4 months) or age-matched controls (n = 14, mean age = 32.4 months) were examined and followed at the Craniofacial Surgery Unit of Necker-Enfants Malades Hospital. Ethics approvals were obtained from the institutional review Board of Necker-Enfants Malades Hospital.
All the experiments were conducted in Fgfr3Y367C/+ mice, a mouse model that display parts of the clinical hallmarks of ACH (32), or WT littermates, used as controls. WT and Fgfr3Y367C/+ mice were generated by crossing Fgfr3neoY367C mice (10) and Cmv-Cre mice (61). The mutant mice express the c.1100A > G (p.Tyr367Cys) mutation corresponding to the c.1118A > G (p.Tyr373Cys) in TD. All mice were on a C57BL/6 background. Mice were genotyped by PCR of tail DNA as described previously (10). Experimental animal procedures and protocols were approved by the French Animal Care and Use Committee.
CT images of human patients and mice
Human patients CT images were produced by a 64-slice CT system (LightSpeed VCT; General Electric Medical Systems, Milwaukee, WI, USA). The images were reconstructed in 3D using Carestream PACS v11.0 software (Carestream Health, Rochester, NY, USA). Cephalometric analysis of the mandible was carried on sagittal views of the 3D reconstructions.
Following 15 days of NVP-BGJ398 treatment, Fgfr3Y367C/+ mice and their control littermates were sacrificed, and the whole heads were imaged using a μCT40 Scanco vivaCT42 (Scanco Medical, Bassersdorf, Switzerland). The following settings were used: integration time: 300 ms, 45 E(kVp), 177 µA. The mandible images were reconstructed in 3D using OsiriX 64-bit version software (Pixmeo, Bernex, Switzerland).
The samples consisted of CT images of patients with ACH and unaffected age-matched individuals, and of high-resolution CT images of 7 P21 Fgfr3Y367C/+ mice and seven control littermates. Three-dimensional coordinates of 10 landmarks were recorded on the 3D reconstructed mandibles of humans and mice and analyzed with geometric morphometric methods. Standardization for position, scale and orientation was obtained by Procrustes superimposition (62,63), and shape information (Procrustes coordinates) and size (CS (63)) were extracted. Shape information was subsequently analyzed by PCA [for more information on geometric morphometrics applied to craniofacial birth defect, see (31)]. Wireframes are used to visualize the shape differences between positive and negative values of PC 1 corresponding to control and ACH mandibles respectively. Geometric morphometric analyses were run with Morpho J (64).
Whole-mount Alcian blue-alizarin red staining
Mandibles of Fgfr3Y367C/+ mice and their WT littermates at E16.5, E18.5, P0 and P21 were fixed in 95% ethanol and then stained with Alizarin Red and Alcian Blue, cleared by KOH treatment and stored in glycerol according to standard protocols. Size of the mandibles and the condyles was measured on images captured with an Olympus PD70-IX2-UCB using CellSens software (Olympus). Total length of the mandible as well as the length of the condylar and symphyseal cartilages were measured (identified with the alcian blue staining). The length of the body was calculated as the total length minus the condylar and symphyseal cartilages.
Mandibles of Fgfr3Y367C/+ and their WT littermates at E16.5, E18.5, P0 and P21 were fixed in 4% paraformaldehyde at 5 °C for 24 hours and decalcified in 0.5 M EDTA (pH 8.0) overnight or up to 1 week, depending on the age of the mice, and then dehydrated in graded series of ethanol, cleared in xylenes and embedded in paraffin. Five micometre sagittal sections were cut and stained with haematoxylin and eosin (H&E), safranin-O or subjected to immunohistochemical staining using standard protocols using an antibody against Collagen X (1:50 dilution; BIOCYC, Luckenwalde, Germany), FGFR3 (1:250; Sigma-Aldrich Co, St. Louis, MO, USA) or Ki67 (1:3000; Abcam, Cambridge, MA, USA) using the Dako Envision kit (Dako North America, Inc., CA, USA). Images were captured with an Olympus PD70-IX2-UCB microscope (Olympus, Tokyo, Japan), and morphometry was performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Ex vivo experiments
Mandibles from WT and Fgfr3Y367C/+ E16.5 embryos were dissected and cut at the symphysis to separate the two hemi-mandibles. For each embryo, the two hemi-mandibles were incubated for 5 days in DMEM medium with antibiotics and 0.2% BSA (Sigma), one supplemented with NVP-BGJ398 (100 nm), whereas the other served as control and was supplemented with DMSO (0.1%). The length of the hemi-mandibles was measured at the end of time course, following whole-mount Alcian blue-alizarin red staining.
In vivo experiments
Fgfr3Y367C/+ mice were 1-day old at treatment initiation and received daily subcutaneous administrations of NVP-BGJ398 at 2 mg/kg body weight or vehicle (HCl 3.15 mm, DMSO 2%) for 15 days. Cartilage and bone analyses were thus performed in 16-day-old male and female mice. Experimental animal procedures and protocols were approved by the French Animal Care and Use Committee.
Differences between experimental groups were assessed using analysis of variance or Mann–Whitney test. The significance threshold was set at P ≤ 0.05. Statistical analyses were performed using GraphPad PRISM (v5) (GraphPad Software Inc., La Jolla, CA, USA). All values are shown as mean ± SD.
M.B.D., D.K.E., Y.H. and L.L.M. designed research. M.B.D, D.K.E., Y.H., V.E., E.G., N.K. and C.B.L. performed research. I.K., M.K. and D.G.P. contributed new reagents. M.Z. and F.D.R. contributed human data. M.B.D., Y.H. and F.D.R. analyzed mouse and human data. M.B.D., D.K.E., Y.H. and L.L.M. prepared the figures and wrote the article.
Conflict of Interest statement. IK, DGP and MK work for Novartis. The other authors have declared that no conflict of interest exists.
Comité d’interface INSERM/Odontologie (MBD), YH was partly funded by the French National Agency of Research (ANR) through the program “Investissements d’avenir” (ANR-10-LABX-52). This project received a state subsidy managed by the National Research Agency under the “Investments for the Future” program bearing the reference ANR-10-IHU-01, the European Community’s Seventh Framework Programme Under grant agreement no. 602300 (Sybil) the Fondation des Gueules Cassées and the Association des Personnes de Petites Tailles. Funding to pay the Open Access publication charges for this article was provided by INSERM U1163.

domain: medical


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Investors of JJPTR are keeping silent out of fear they may lose their money.
They worry that the account of the company will be frozen by the authorities if police reports are made.
Some are also fearful that action will be taken against them for taking part in "illegal activities", since JJPTR operates forex trading, which was neither authorised nor approved under the relevant laws and regulations administered by Bank Negara.
A 65-year-old clerk, who declined to be named, stressed that he did not consider lodging a police report against JJPTR as he hoped to get back the US$2,000 (RM8,743) that he invested.
"I invested US$1,000 (S$1,394) and another US$1,000 under my wife's name. After I received three payments, I failed to get my fourth returns.
"I was told that the boss is figuring out a way to pay us back. I trust him since I got my earlier dividend payments on time," he said.
Another investor, Lau, said they do not want trouble, just their money back.
The 40-year-old who joined the scheme last year made a little profit but his two colleagues that he "recruited" did not.
"Both of them invested US$200 and US$1,000 early this month. They have not got back their money, and I feel that I have to help them," he said when met outside the JJPTR service centre in Perak Road yesterday which opened at 1pm.
He said an employee there told him that investors who have not received their returns might be transferred to a new plan.
The investors appeared calm and they accepted the explanation given by staff at the service centre, believed to be the headquarters in Penang.
One of the 20 staff members stationed at the centre said the investors were told about the risk before they joined.
"Now, the company is facing a crisis and lodging police reports will not help," a branch representative said yesterday.
Penang Commercial Crimes Investigation Department chief Asst Comm Abdul Ghani Ahmad said no reports against JJPTR had been received so far.

domain: economics


The lottery is a very old game with ancient roots. It originated in the Netherlands in the 17th century as a means of raising funds for the poor. Over time, the lottery became a popular alternative to taxes. The oldest continuously operating lottery is the Staatsloterij in the Netherlands, which was established in 1726. The word lottery derives from the Dutch word “lot,” which means “fate.”
In addition to distributing winnings to lottery players, the lottery can also help fund scholarship programs. The Kentucky Lottery, for example, provides money to the KEES Scholarship Program to help students pay for college. In Pennsylvania, the lottery also supports programs for the elderly. And recently, the lottery began operating in New Hampshire, which has an online lottery called the iLottery. The iLottery allows lottery players to purchase tickets online for major draws, and winnings are instantly deposited into their accounts.
The house edge in most lottery games is close to 50%. However, many lottery aficionados argue that this edge is not important compared to the fact that a lottery player has a chance to win a life-changing payout. In addition, the house edge in lottery games is much lower than in online slot machines, which can reduce a player’s bankroll to nothing in a matter of minutes. Because of this, lottery players should be aware of their limits and play responsibly.
Although the New York lottery doesn’t have an online version, there are apps available for iOS and Android devices that let players scan tickets and check results. The apps also allow lottery players to view prize drawings and current jackpot amounts, and display a map of retailers. Another popular lottery app is TheL11otter, which lets players pay for tickets using their smartphones.
A growing number of data hk lottery websites have made purchasing tickets online easy. In addition, some states offer geolocation technology to identify players’ locations. Those in the state of Illinois can purchase lottery tickets on their mobile devices online. The state lottery in Illinois now offers Mega Millions and Powerball lottery tickets. The lottery also offers virtual sports such as Keno.
The Connecticut Lottery is one of the oldest lottery organizations in the US. It offers several draw games and is a charter member of the Multi-State Lottery Association. Most of the lottery’s profits are donated to public schools and colleges. The Colorado Lottery, which was started in 1983, features several multi-state games. Ticket profits go to state education programs and park and open space conservation, as well as to local wildlife habitats.
The Powerball lottery is another popular lottery in the US. Although the jackpots are smaller compared to other lottery games, the jackpots have reached as much as $22 million. Tickets to the Powerball lottery cost just $2. A player needs to match five white balls and one green ‘cash ball’ number.


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Tucson Safe Place program for LGBTQ people
The Tucson Police Department (TPD) is starting a new program called Safe Place, where members of the lesbian, gay, bisexual, and transgender (LGBT) community can go if they’ve been the victim of a hate crime or fear they might become one. TPD is looking for businesses to sign up and agree to let people stay inside until police arrive. The department is working with the group Tucson Pride, launching the program this weekend during the group’s annual gay pride events. Police will give participating businesses a rainbow police badge decal to place on their windows.
The initial effort will focus on enrolling businesses in Downtown Tucson, which has a high concentration of people, shops, and restaurants.

domain: news


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SmutJ – Touch me MP3 Download
DOWNLOAD MP3 SmutJ – Touch me. We all need God’s Touch irrespective of our heights and accomplishments in life, They is no denial that they are certain situations we find ourselves that require only the touch of the most high as seen in Matt 8:3. “Touch me” is a song by SmutJ that depicts a lost soul who yearns for the touch of Jesus. It’s here for download
Stream And Enjoy “SmutJ – Touch me” “Mp3” “cdq” “320kbps” “Itunes” “torrent” download Song Below.

domain: artistic


On 17 March 2018 In Pictures
Explore 2020 Subaru Forester Sport, performance and technology features. We reviews the 2020 Subaru Forester Sport Redesign where consumers can find detailed information on specs, fuel economy, transmission and safety. Its EPA-estimated gas mileage is also very impressive. A comfortable ride and an energetic powertrain ...


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A seventy five year old lady from Harold Hill has written a poignant letter to Havering’s leader of the council, where she openly states she was sending the letter with ‘sadness, disillusionment, disgust and outrage.’
The two page letter written from the heart comes from Eileen who has lived in Harold Hill since 1952 and now feels ‘utterly let down’ by the politicians in the town hall and their treatment of the borough and its local residents.
Eileen exclusively told the Havering Daily, ‘I didn’t even start the letter with Dear Councillor White, I started it with Mr White as I am so outraged by the way he is treating residents in our borough.‘
It begins with ‘This letter is sent with sadness, disillusionment, disgust and outrage at the utter disrespect and contempt that your administration shows for the residents of this borough.’
Eileen then goes on to write ‘You seem to forget that you are public servants elected to serve the people of this borough who gave you the privilege and their trust to look after their welfare, environment and the future of their children.
‘I am seventy five years old and I have lived in this borough since I was seven years old and never have I seen such an incompetent lot within our town hall. Yes Mr White our town hall not yours. Yours is a position thanks to the courtesy of this borough’s resident’s.
‘It would appear all you lot do is feed your own ego while the borough’s services, amenities goes to the dogs. For example social services, mental health and social housing.
‘Parks in Havering received a million pounds six years ago to renovate and upgrade them for the use of the public. I use Raphael’s park most days and I am sadly am watching it deteriorate due to the lack of gardeners.
‘I wonder are you planning to steal our parks to build more houses or putting it up for privatisation?
‘It would seem that building houses is your thing. Ordinarily that would be great, but you don’t look after the existing residents. You are stealing every little piece of green space to build even more houses. For whom may I ask? Because the youngsters in our borough haven’t got a hope of getting onto the housing list. They are being forced out of the borough away from family and support.
‘Do you ever go out into this borough and speak to the ordinary everyday families? Families whose pockets you put your hand in at every opportunity i.e. parking charges. Not everyone has a privileged background.’
The seventy five year old is saddened at how Havering is changing and what she calls the ‘destruction’ of communities.’ She hopes her letter will have an impact now.
The Havering Daily has created some new facebook groups, where we will share our up-to-date stories happening in the borough.
Please feel free to share with us all the latest stories or views you have.
Don’t forget to share the groups and invite your friends to join too.
Thanks for the support!
GIDEA PARK NOW
COLLIER ROW NOW https://www.facebook.com/groups/1164902440564857/
HAROLD WOOD NOW
HAROLD HILL NOW
SOUTH HORNCHURCH NOW
ELM PARK NOW
UPMINSTER NOW https://www.facebook.com/groups/382525269084242

domain: sociology


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This is a post that should’ve been written after the 2014 regular season. And though it’s overdue, it’s no less deserved. As first reported by Darren Wolfson, Harrison Smith is expected to be named to his first Pro Bowl this week. ESPN’s Ben Goessling confirmed the report, making Smith the third Minnesota Vikings player represented in this year’s all-star contest. He’ll join Adrian Peterson and Everson Griffen in Hawaii next Sunday.
Goessling reports that two league sources confirmed Smith’s selection will be made official after tonight’s NFC Championship Game. Smith finished the season with 66 tackles, 1.5 sacks, one forced fumble, and two interceptions in 13 total games. Though he missed nearly four games with a knee injury, Smith proved invaluable to Mike Zimmer’s defense. And even if his stats weren’t as eye-popping in 2015 as they were in 2014, Smith’s greatness didn’t go unrecognized by the NFL.


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This page contains Warcraft 3: Reign Of Chaos Hints for PC called "Remove Blight" and has been posted or updated on May 26, 2007 by warious13.
- Developer: Blizzard Entertainment
- Publisher: Blizzard Entertainment
- Genre: Strategy Real-Time
- Release: Jul 3, 2002
- ESRB: Teen
When playing as the Humans, train some priests and make sure they have the Dispel Magic ability. If your enemy is the Undead, and has Blight, using the Dispel Magic spell will remove it and turn it to dirt.
Please Submit a Problem for any incomplete, non-working or fake cheat listed above.
If you know other secrets, hints, glitches or level guides, then please Submit your Cheats and share your insights with other players.
Stuck in this game? Set a specific, clearly defined question and let other gamers answer your question or view answers to previously asked questions. If you think you are an expert then please try to help others with their questions.

domain: game


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OIG: Medicare Advantage Prepayments Cost CMS Millions
That's according to a report from the Office of Inspector General, which says that Medicare Advantage plans are not legally prohibited from taking that prepaid money and earning interest on it, funds that are more appropriately retained by the Centers for Medicare and Medicaid Services.
For this report, the OIG reviewed $69 billion in prepayments made to 457 Medicare Advantage organizations in 2007. It found that the plans hold Medicare funds for approximately 46 days before paying for medical services.
The OIG recommended a change in payment policy for Medicare Advantage plans in the year 2000, but CMS rejected the idea saying that the plans would just raise rates to compensate for the loss of income.
"Medicare Advantage organizations and CMS officials have stated that if either of our previous legislative or regulatory recommendations were to be implemented, some Medicare Advantage organizations would increase their bid proposals to recoup investment income that they would lose," the OIG report says.
- Patient Harm Data to Remain on Medicare's Hospital Compare Site
- Quiet ORs Better for Patient Safety
- Tavenner Confirmed as CMS Administrator
- Leapfrog Hospital Safety Scores 'Depressing'
- Building a Better Healthcare Board
- CMS Seeks to 'Rapidly Reduce' Medicare Spending with $1B in Grants
- Hard-Nosed About Physician Teamwork
- Healthcare Leaders Sound Off on Organized Labor
- Esther Dyson's Population Health Dream
- Case Study: Advance Care Conversations

domain: health


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New Resident Evil: Revelations 2 Raid Mode Screenshots
Resident Evil: Revelations 2’s first chapter is set to release in less than a month, introducing the gaming world to the concept of genuine, episodic horror.
In a new batch of Revelations 2 screenshots, the beloved Barry Burton and a young girl, identified as series newcomer Natalia Korda, are featured, as well as numerous images of Claire tackling the game’s interesting Raid mode.
The Barry and Natalia screenshots showcase the impressive visuals of Revelations 2, with lifelike expressions of fear on each character’s face. The Raid mode screenshots are much more action-focused, which makes sense, considering Capcom’s announcement that the additional game mode will contain 15 different characters and over 200 levels.
Resident Evil: Revelations 2 will release on February 24, 2015 for the PlayStation 4, Xbox One, PlayStation 3, PC, and Vita.
“Making you a better geek, one post at a time!”


Utility demand charges—steep fees that commercial customers pay when their power consumption exceeds a certain level—are the bane of public charging operators, and most Charged readers are probably familiar with how they work. Average Joe and Jane, however, are blissfully ignorant of their existence, and might be shocked to learn that they represent a roadblock to more public charging deployment.
In a recent op-ed piece in the Tampa Bay Times, Florida Senator Jeff Brandes set out to educate the public about these nefarious charges. (Ironically, Republican Jeff Brandes, who represents Charged’s home town of St. Petersburg, is not known as a friend of EVs—in 2020, and again in 2021, he sponsored bills that would have imposed a special yearly tax on plug-in vehicle owners.)
Senator Brandes points out that, under the Bipartisan Infrastructure Law, Florida is expected to receive some $198 million in federal funding for charging infrastructure, and that the state needs to “allocate these funds in a way that prioritizes a competitive charging marketplace and supports private sector investments.” (One of Brandes’s colleagues has introduced a bill that would supposedly promote competition in the charging market.)
Demand charges are especially onerous for small businesses that want to install DC fast chargers. A small store or restaurant’s average energy use may be relatively low, but even moderate usage of a typical DC fast charging station can cause a spike in energy consumption that incurs a demand charge. “In many cases, these additional fees make it hard for charging station hosts to break even on charging costs, let alone begin recouping the costs associated with the initial installation,” writes Brandes.
Last June, the Florida Department of Transportation released an EV Infrastructure Master Plan, which listed utility demand charges as a primary barrier to public charging station deployment.
The document reads, in part:
EV charging station companies are concerned that through the rate structure, demand charges by utilities are an impediment to DC fast charging infrastructure. Fast charging stations are commercial customers billed under rate schedules that include an energy charge (based on the amount of energy consumed, or kWh) and a demand charge (dollar per kW). The demand charge is based on the highest usage, or demand, over a specified time interval (15 or 30 minutes). This peak usage determines the demand charge for the billing month.
Demand charges…challenge the economics of public fast charging stations that experience a high peak demand, but low levels of kWh energy sales, or utilization. Peak demand at an infrequently used site could be determined by the single customer of that site with the highest demand, rather than an aggregate from multiple users charging at the sites busiest time. At low levels of utilization, the bill incurred by the charging stations result in demand charges being spread over a low volume of energy sales. Stations with higher kWh sales spread the demand charge over more energy sales and are more likely to recover costs. In addition to evaluating whether demand charges are appropriate for EV charging, utilities may consider how rate structure can help manage the additional demand created by vehicle charging. Time-of-use rates, based upon the cost of producing energy during different segments of the day, can be a mechanism for encouraging EV charging during off-peak hours.
Brandes argues that charging stations should be exempt from demand charges, and any public charging operator would probably agree.
Last November, The Regulatory Commission of Alaska, which manages public utilities in the state, approved a set of rate structure changes that eliminate demand charges for EV chargers. (The revision also allows businesses hosting charging stations to charge for electricity by the kWh, a reform that Florida regulators made in 2012.)
“Electric vehicles are the future, and Florida needs to be ready to meet the needs of this growing industry with the infrastructure required to support it,” writes Brandes. “While Florida has taken promising steps toward an electrified future, lawmakers and utility regulators must work together to take the necessary steps to promote fair competition in the charging station marketplace.”
Source: Tampa Bay Times


Hey, remember that cover of “Mad World” by Tears for Fears from the end of Donnie Darko? Did it make you want to kill yourself, too? Well put down that razor; you’re doing it wrong, anyway! Jan Wayne says listen up!
Congratulations, Jan, for covering “Mad World” and taking the piss out of that lugubrious movie remake. As goofy as your song is, it’s more true to the Tears for Fears version than the one by…Gary Jules? Who the hell is that?
“Jan Wayne! Turn those turntables!”
Illegal downloading pays off again!
You can’t buy Schiller’s new album Tag und Nacht until tomorrow! And even then you have to be in Germany! I only got a hold of “I Feel You” from Schiller’s last album three months ago; it might take years for a legit “Day and Night” to hit our shores. As Americans it’s your patriotic duty to steal this music.
I haven’t yet heard the whole album; the standout track so far is “What’s Coming” with vocalist Jette von Roth. Her lusciously blissed out vocal style is very close to Tiff Lacey’s in songs like ATB’s “Ecstasy”.
Von Roth has a zonky advantage over Lacey, though: she’s a non-native English speaker! Like any good trance song, “What’s Coming” is filled with nonsense lyrics that don’t fit the melody; add von Roth’s vocals and the trippy gets even trippier. Good work!
I love you Schiller!
I’m Sally Struthers.
For every Paul Oakenfold there are thousands of struggling dance producers who lack the technical skill to produce a competent dance track. That’s why, for just pennies a day, you can help struggling Eurocheese artists like Bikini Girl and their song “Touch My Bikini”.
Bikini Girl and other artists are forced to use synthesizers from 1996, and use vocal artists that sound like a lobotomized Milky. In this song the group even steals a riff from Donna Summer’s “I Feel Love”—like so many bands before them.
There are thousands of acts just like Bikini Girl starving for a decent hit. And you can make a difference. Please, think of the Eurodance.
A three track single for $99.99?!?! What?!
Does it come with a blowjob? Maybe these Amazon people have a side project, and this is how they throw off the vice squad.
Or maybe not. iTunes is selling each track for $33.33. I can get two Frampton Comes Alive albums for that!
But not the blowjob.
Senor iTunes tells me that i have 57 songs, 5 hours, and 444 MB of music that is one artist “vs” another.
Over an hour of that is “Owner of a Lonely Heart”. Stupid Max Graham vs Yes!
But none of it is Pearljam.
Anybody else catch this full frontal male nudity in the video for Günther’s “Tutti Frutti Summer Love”?
Quite frankly, I’m offended. “The Ding Dong Song” video promised us roomfuls of oiled, naked girls, but the only real nudity we saw was Günther’s own backside. And in “Touch Me” he failed to get Samantha Fox naked like we’ve been trying to do since we were 13. Now there’s even less T&A and the most tra-la-la we’ve seen yet. Thumbs down!
PS: Mine’s bigger.
iTunes: That’s it, M. Stop playing “Greece 2000” over and over again.
M: Even the DJ Shog remix?
iTunes: Especially the DJ Shog remix. You only have two songs by Three Drives on a Vinyl and I’m sick of them.
M: I’m back from my car trip!
iTunes: M, I saw you through the window. You sat in the driveway for three hours listening to that song on CD.
Scooter, now celebrating their eleventh year of goofy techno cover tunes, has a pretty catchy single in “Hello! (Good To Be Back)”. In fact I though the chanting male chorus wouldn’t be out of place on an album by the twice-as-old Depeche Mode.
But who is really being sampled by Scooter? Read the fine print:
Yikes! It’s Gary Glitter!
So be careful. There’s only a thin line of good taste separating “Personal Jesus” from “Rock and Roll Part 2”.
Holy Undead Eighties Stars!
Is that really Laura Branigan in the video for Royal Gigolos cover of “Self Control”?!
Our dear Ms. Branigan died over a year ago. Does it really take that long for the Gigolos to rip her off? Somehow I doubt rival hacks the Vinylshakerz spend years contemplating their Wham! covers.
Or maybe they delayed release of “Self Control” with respect for the recently deceased. Because you wouldn’t want to squeeze that cheese over a fresh grave.
I stand in the shallow end of the deep ocean of trance music, but one of my favorite downloads is an unmixed compilation album called Trance Masters. It features name brand artists (Paul van Dyk, Armin van Buuren, and my sentimental favorites, Three Drives on a Vinyl) and quite a few crossover hits.
But what I did not know is that the download I have is just disc one of a three disc compilation. Wow!
It’s an import, but at $20 for three discs the price is right. This is just the latest example of copyright violation paying off for everybody.


There’s a new demographic that will greatly affect the housing market in both the near and distant future: Millennials.
Millennials, also known as Echo Boomers and Generation Y, are young adults who were born between 1980 and the early 2000s. This generation – you might be one! – is known for being skeptical, having grown up during an economic boom and then watching it slide into recession.
Contrary to stories seen in the media, 72 percent of Millennials say that homeownership is part of their personal American Dream. In a poll by the Joint Center for Housing at Harvard University, 96 percent of Millennials plan to own a home. Of those polled, 48 percent of those under 25 said that they even expect to buy in the next three to five years.
While this may not seem to have an immediate effect on the housing market, the Census Bureau recently reported that the annual household formation numbers are almost back to pre-2008 numbers, considered the Boom Years. (Between 2008 and 2011, only 650,000 households formed annually). In 2013, Freddie Mac is projecting the creation of 1,250,000 new households, which means an increase in potential homebuyers.
The current combination of great pricing and historically low interest rates have created a flood of first time homebuyers, and data consistently reports that these factors have already begun to encourage a positive change in the housing market. Furthermore, Chicagoland’s listing inventory for February averaged 4.7 months, which is down 51 percent from the 12.9 months in February 2011. This indicates a lack of supply, which encourages increased activity.
On the leasing front, rents are expected to reach record highs in 2013, and 31 percent of current renters plan to buy a home in the next two years. This will bring more buyers to the market during this time and add to an already positive trend.
Whether you’re looking to move up or downsize, now is the time to discuss listing your home and purchasing another at historically low interest rates. If you’d like more information about the best strategy for selling your current property in your specific market or purchasing your first home, I have the expertise to help you make it happen.
Spring has arrived! Let’s go look at some houses together! Let’s do some housing for good. Give me a call.
Check out my other site too and register for email alerts so you can know about each new listing as it comes on the market.


With robots replicating humans in every aspect of life, we are left to wonder whether they will eventually replace humans. Leaving the question to the scientists and psychiatrists, here are some of the famous robots that I fell in love with. If you love geeky gadgets and robots, you will certainly like these awesome robots that are going to alter the course of history. A giant leap for the mankind, they are going to fix many of the issues that are impeding our progress in future.
When I was a kid I was fascinated by the robots from the fictions and the movies who had protagonists and human-like autobots that excelled in faculties like computing skills and powers which could only possessed by a superman.
Some of these robots are good, some are monsters, all made by human beings displaying traits resembling humans. Enjoy the list.
Image Credit - Author prayitno via Wikimedia Commons


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domain: history


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By Marissa Yefsky
Needless to say, nobody ever expected COVID-19 to hit as hard as it has. At its beginning, many people hoped this virus would be contained in China, and everyone’s lives outside would not be affected. Every community has felt the impact of this virus as it sweeps the globe. There are places that have never seen a day without crowds of locals and tourists alike. Tourist spots and small communities are hurting. There are places we see in pictures or in person that always have crowds. Times Square, Las Ramblas, and the Trevi Fountain rarely see an empty day. Below, I’ve shared some haunting photos that show the impact of COVID-19 on every stretch of the globe.
Disney’s Magic Kingdom, Florida, USA
New York, USA

domain: geography


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- Put together for a “bumpy ride” in shares, longtime angel investor Mike Ghaffary advised CNBC on Thursday.
- Markets experienced a “fake feeling of hope” that the Federal Reserve wouldn’t embark on an aggressive rate-hike cycle, he claimed.
- The Federal Reserve has by now elevated fascination fees by 75 basis points this year, with more hikes in retail store.
Tech shares are main Thursday’s market place rout, and investors must brace for extra
Mike Ghaffary explained on CNBC on Thursday.‘s hawkish thrust in raising curiosity costs sales opportunities to repricing, Canvas Ventures’ basic companion
The Nasdaq Composite tumbled by far more than 5%, and the S&P 500 slid by additional than 3%. E-commerce stocks including eBay, Shopify, and Etsy and Wayfair were component of the selloff right after the corporations claimed mixed monetary benefits that often bundled weaker-than-predicted forecasts.
The rout erased Wednesday’s rally that took put after Fed Chairman Jerome Powell explained the central bank was not actively contemplating a rate hike of 75 basis points that would stick to its 50 percent-place improve to the benchmark lending rate to a vary of .75% to 1%.
“Yesterday, there was a untrue sense of hope that surrounded this thought that there wouldn’t be an intense amount hike in the long term. But I imagine the fact set in that we’re in the middle of a lengthy deleveraging cycle which is heading to continue to be unpleasant,” Ghaffary stated in an job interview on CNBC’s “TechCheck” present.
“In addition to that, you’ve acquired definitely inflation and labor difficulties. So I think you have bought to buckle up for a bumpy trip in advance,” said Ghaffary, who co-launched podcast distributor Stitcher and was a Yelp government before becoming a member of Canvas Ventures in 2019.
Development-oriented tech stocks in specific are susceptible as bond yields climb along with the Fed’s tightening cycle. On Thursday, the 10-calendar year Treasury produce soared as much as 17 basis factors to 3.09%, the optimum considering the fact that 2018.
And additional hawkishness is anticipated. Economists widely consider the Fed as staying driving the curve in taming inflation which in March had accelerated to 8.5%, the quickest enhance given that December 1981. Fed cash futures traders on Thursday priced in an 82.9% prospect the central lender will raise its key level by 75 basis points at the June 14-15 assembly.
However, Ghaffary was upbeat that some stocks will eventually arrive out of the current turbulence.
“For example, Amazon is a long-phrase keep,” he mentioned. “They face a good deal of headwinds now. They are priced incredibly aggressively. But it truly is difficult for me to imagine a potential 5 several years from now wherever Amazon is not a actually important business and continuing to boost sector share.”
There are inventory bargains to be had in the e-commerce room but that place is going through a complicated desire amount surroundings and increasing inflation that will put stress on customer expending.
“If you are seeking to working day trade and if you happen to be seeking to connect with the base market place, that is notoriously difficult. If you have a lengthy-expression check out, you can develop a situation close to a couple of distinct businesses if you have a purpose to believe they have bought a defensible prolonged-phrase moat that will give them an advantage,” Ghaffary said.


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Duke Ferguson continues to be educating companion dog entrepreneurs, police, armed forces K9 trainers and handlers and also other Pet training canine pros globe broad for a few years.
Start now to show your puppy not to leap up. Even tiny puppies can cause issues and harm to them selves and Other people whenever they leap and leap about.
Whining, crying and howling normally end result whenever a puppy is remaining alone. Puppies will whine and cry when separated from their homeowners.
The easiest way to do This is often to incorporate it as part of your vocabulary lessons as well as your respect classes. Should you instruct words and regard correctly, acceptance of being managed will occur The natural way – they go "hand-in-hand", so to talk!
You will find detailed housebreaking directions in my training reserve (see bottom of web page) – and Certainly, I protect Each and every a kind of housebreaking approaches so you're able to select which just one operates best to your Canine plus your Life-style.
Now, leadership does not imply hitting Puppy. No choke collars or shock collars. Just tiny points you need to say and do each and every time you connect with Puppy. These minimal things are considered as "Management" because of the canine head.
Create the correct pattern with the extremely commencing and Puppy are going to be housebroken the moment his inner organs can cooperate.
If you do not react correctly..... perfectly, unfortunately that's why lots of teenage canine are turned about to rescue groups and animal shelters.
These are generally all periods any time you – not Puppy – have to be the a person to come to a decision what is necessary. Puppy should stand quietly for nearly anything you'll want to do with him.
"The Pet Care Institute is a website managed by renowned Puppy Trainer, Jeff Coltenback. Now could be enough time for you to embark within the job in pet treatment that you've generally dreamed about. The Pet read more Treatment Institute delivers online certification and also customized fingers on training for individuals, shelters and rescues. The extensive curriculum contains research, essays, video clip tutorials and alternatives for hands on and virtual mentor ship. Other options like Holistic Pet Treatment Plans are available via the website. The systems have been made to supply students an available avenue to attain their pet care vocation goals.
Trainers which have received certification and keep the title CPDT are necessary to attain continuing education credits often in an effort to retain their title.
Introducing your puppy to his collar, leash along with the stairs can be quite a challenge. If the puppy is experienced effectively, It will likely be easy, satisfying and thriving. Generally use praise and lures in lieu of force.
Your puppy must take YOU since the chief in Your loved ones. Becoming the chief simply just means you will be the one particular who decides what is alright for Puppy to perform and what just isn't all right.
When you terminate throughout the initial 6 days of enrollment you can get a comprehensive refund. Soon after the primary 6 times your refund amount of money is going to be according to: Classes finished

domain: pet


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Subject: I have found a DVD that I think you would enjoy
Similarly Requested DVDs
Member Movie Reviews
Michelle C. from HERNDON, VA
Reviewed on 9/7/2011...
"Muppets From Space"
How can you resist the Muppets?! Love the celeb appearances and the sweet, innocent themes of the Muppet films. Cute.
In this movie, the muppets discover that Gonzo's family members are aliens from a distant planet AND they are coming for a visit!
Movie features Andie MacDowell, Ray Liotta, David Arquette and more.
Runs approximately 88 minutes.
* live video commentary with Kermit, Gonzo, Rizzo & director Tim Hill
* outtakes reel
* music video "Shining Star" by the Dust Brothers featuring Jaymes
Denise S. (stayhm) from NORTH BEND, OR
Reviewed on 4/29/2011...
Another great Muppets movie.
Melissa H. from PEORIA, IL
Reviewed on 2/5/2011...
Muppets from space

domain: entertainment


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domain: design


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Faced with the humanitarian disaster that befalls Afghanistan after the Taliban seizure of power, the UN called on Saturday to come to the aid of the country which is facing another crisis, drought, threatening 7 million people.
In a statement, the United Nations Food and Agriculture Agency (FAO) called on global donors to come to the aid of Afghanistan, especially farmers in the country whose lands are threatened by the drought.
In the event of an acute drought that would have an impact on harvests, 7 million people will be plunged into food insecurity, when the Taliban, who returned to power in mid-August, will not hesitate to steal the crops from farmers, as when they were in power in the country 20 years ago.
The UN warns by informing that already 1 Afghan in 3 is currently in a situation of acute food insecurity, a total of 14 million people. But animals are too, because of the drought. Three million animals are threatened.
The drought, the Covid-19 pandemic and the displacement of populations caused by the offensive of the Taliban, who took control of the country, “have hit the rural communities of Afghanistan hard, in particular its farmers and herders”, FAO said.
“If we do not provide assistance to the people most affected by the severe drought, many of them will have to abandon their farms and be displaced,” warned FAO Director General Qu Dongyu.
And to warn about the consequences of new displacements for the inhabitants. “This risks worsening food insecurity and further threatens the stability of Afghanistan,” he added.
To help the country and Afghan farmers, the UN is calling on donors to help purchase wheat seeds before seeds are scheduled for September. The FAO is asking for $ 18 million, which will be used to feed 250,000 farmers and their families, or 1.5 million people.
The FAO also indicates that forecasts in the country expect a harvest of 20% lower than 2020 because of the drought.


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domain: psychology


Media Buying Briefing: Another report calls for changes to the grueling pitch process
This Media Buying Briefing covers the latest in agency news and media buying for Digiday+ members and is distributed over email every Monday at 10 a.m. ET. More from the series →
If one research study didn’t get the message across, two should really do it — the message being that the agency pitch process needs to change irrevocably so as not to break the participants in the process.
Following Forrester’s Ditch the Pitch report, which Digiday wrote about in a recent Briefing, London-based MediaSense issued its assessment of the very flawed marketer/agency pitch process, citing all manner of factors that have made pitching arduous and almost not worth the effort.
Focused on global media agencies, MediaSense’s report, Pitch Smart, was helmed by managing director of strategy Ryan Kangisser, who was motivated to conduct it based on the fact that nearly every conversation he’s had with marketers and agencies revealed the biggest struggle is finding and keeping talent.
But other factors play a role in the need to conduct the process differently, he explained, such as technology’s impact on diluting the differences between agencies, as well as the increased length and complexity of pitching — the latter of which results in less of a payoff when a client is won.
“This perception of clients that they have a divine right where every agency is going to fall over to pitch for their business has clearly changed,” said Kangisser. “Perhaps it’s not as energizing for staff as much or it’s not so central to agency culture [anymore], when for most people who’ve worked in agencies [a long time], that adrenaline rush you got from a pitch was the thing you would look forward to.”
Some of the stats in the study confirm this. When asked if pitches are energizing for staff, only 44% of respondents agreed or strongly agreed, while 35% disagreed or strongly disagreed (21% neither agreed nor disagreed). Similarly, when asked if it’s becoming increasingly difficult to get people to work on pitches, 34% disagreed or strongly disagreed, while only 46% agreed or strongly agreed (20% remained neutral). Perhaps the most damning stat: 86% of respondents agreed the pitch process is excessively time- and cost-exhaustive.
So what does MediaSense recommend? For one, it argues that marketers should stop asking for theoretical planning exercises and quick turnaround work requests, because they place undue stress on the agencies while not accurately reflecting what the agency is capable of. It also stresses reducing virtual meetings in favor of true in-person chemistry-building. Finally, the study encourages marketers to offer clear feedback to the winner — as well as the losers — in a pitch, to encourage transparency.
“[Give] agencies feedback throughout the process, so that when it gets to the big reveal, the big final presentation, everyone has a very clear chance of being successful,” said Kangisser.
MediaSense plans to conduct a similarly aimed study but from the marketer (aka client) POV, but the lessons from this study apply evenly to client and agency. “Transparency, from a client perspective means [sharing] ‘This is what we look like, this is how ugly we are, this is our organization, this is where our dysfunction is,’” Kangisser added. “Fundamentally, it shows the agency where you’re looking for them to help accelerate transformation. So being really clear about what it is they’re actually looking for from an agency, which then will [empower] the agency to … respond in in a way that’s really aligned to the internal needs of the client.”
As broader-based targeting in programmatic continues, industry reckons with talent gap
Speaking of talent, media agencies spoke about the talent gap that’s been created as broader-based targeting in programmatic continues to grow and agencies work to meet client demand.
Agencies acknowledged at Digiday’s Programmatic Marketing Summit last week that recruiting and retaining specialized talent to handle clients’ diversified channel strategies has, in part, led them to create new training programs and rethink their benefit programs.
CMI Media Group adopted feedback from employees to create a flexible workweek in which employees could take summer Fridays year-long, with an eye toward retaining employees. “It’s listening to them and making sure they are heard and their opinion matters,” Greg Dreifus, svp of programmatic at CMI Media Group, said on May 22 in Palm Springs, California.
Otherwise, leadership has tried to keep employees engaged by encouraging getting together as a team whenever possible, creating on-demand, recorded video sessions for onboarding employees and allowing employees to travel and network.
Depending on client needs, Digitas also brings in external voices to speak to changing industry trends, said Leah Askew, svp and head of precision media at Digitas North America.
“Client funding is getting narrower and we’re spending more money in a workforce that’s expensive and specialized,” said Askew, who added that these specializations could range as client demands change from connected TV to optimization strategies.
Askew and Dreifus both reaffirmed the importance of encouraging their employees to ask questions — with clients and internally in this hybrid working environment. Curiosity among job applicants will often land them a second interview.
Speaking on another panel at the Programmatic Marketing Summit, JiYoung Kim, president of North America at GroupM Nexus, spoke on innovation and the importance of asking questions. “Be that toddler that continues to ask why,” she said.
While previous generations were forced to reckon with tech and data, this new cohort of workers will need to be won over by an industry that’s willing to make room for them.
“It used to be so exciting to be in programmatic and digital and social, search — this used to be cool. And if the thing we’re going to hang our hat on is we can reach millions of people, that’s not exciting,” Kim told Digiday’s senior media editor Tim Peterson. — Sara Jerde
Color by numbers
Nielsen and the Asian American Advertising Federation (3AF) released findings from their first joint Asian language media consumption study — showing that in-language media is the most trusted source of information among Asian Americans. The 2022 study focused on the three top Asian languages in the U.S.: Chinese, Vietnamese and Korean. Together these languages represent some 40% of the Asian American population and form the most spoken Asian languages at home. — Antoinette Siu
- More than 40% of survey respondents strongly agreed or agreed that Asian media offers programs and perspectives they trust, and more than 50% of respondents from those three language groups said they prefer to buy brands that advertise on programs reflecting their culture.
- When it comes to media consumption preferences, streaming TV is the most-watched Asian language platform for all groups, compared to broadcast and cable. But radio and social media remain important channels, with 34% of Chinese respondents reporting spending 7+ hours listening per week. Social media is used the most for Vietnamese (59%) compared to other groups.
- In-language media serves an important role for the Asian community, with most using it for current news — 67% of Korean respondents, 60% of Chinese respondents and 72% of Vietnamese respondents said they pay attention to ads in their preferred language.
Takeoff & landing
- Omnichannel ad platform Mediaocean will integrate Epsilon’s audience data through a partnership with the Publicis-owned data/martech firm, which has more than 1,000 different audience segments based off of 250 million U.S. consumers.
- Horizon Media has agreed to buy, plan and measure some of its clients’ upfront TV deals based off of VideoAmp’s data solutions, cementing the measurement service’s momentum in providing an alternative currency to Nielsen and other TV measurement services.
- Personnel moves: Former Media Kitchen CEO Barry Lowenthal joined AI data firm Inuvo as president … Randall Rothenberg and Martin Kristiseter joined the board of directors of performance media firm Digital Remedy.
“We spend a lot of time interrogating privacy-compliant data to get really granular in identifying our strategic audiences before we ever place a buy. By understanding where these audiences exist at a ZIP code level, we can be very choosy about applying data segments to our targeting. This both saves our clients money and protects us as the cookie pools dwindle and audiences become harder to find.”— Paul DeJarnatt, vp and head of digital at media agency Novus, discussing targeting and privacy changes this year with Antoinette Siu
- Both Digiday news editor Seb Joseph and senior ad-tech reporter Ronan Shields offered up distinct takes on the five-year anniversary of GDPR, given the looming importance of privacy regulations in the future.
- Think Web3 is history? Think again, explains media agency reporter Antoinette Siu, who covered Dentsu’s plan to codify Web3’s usage via its Readiness Index.
- And in his latest Future of TV Briefing, senior media editor Tim Peterson asks (and gets some answers to) all the right questions surrounding this year’s upfront marketplace, which will kick into deal-making gear now that all the presentations are over.
More in Media Buying
As women’s sports continue to capture audiences through March Madness and soccer, will agencies and brands be able to channel some of that momentum in the growing esports and gaming spaces?
Independent agency group Meet The People intends to acquire marketing and advertising group True Independent Holdings, Digiday has learned.
Casale outlined some of the main challenges to solving the problems of fraud in the programmatic world — the latest boogeyman being MFAs


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Australia in 8 days
Jan 27 - Feb 4, 2024
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domain: travel