The biomechanics of maintaining effective force application across cycling positions.

Cyclists are known to change their cycling position to reduce aerodynamic drag. Research has shown that this compromises their physical capacity to perform, but there is considerable inter-individual variability present. Proposed training specificity effects by cycling position do not explain all of the observations in the literature, so a search for other influencing parameters is warranted and might help practitioners to further optimise cycling position. This study captured full-body kinematics and 2D crank forces in 19 Time-Trial (TT) and 36 Road trained cyclists. Data in preferred and standardised cycling positions were systematically evaluated and showed that, amongst other kinematic differences, TT cyclists prefer a more forwardly positioned hip joint over Road cyclists. Despite their different setup, no effects in mechanical effectiveness were seen between the groups when tested in their preferred position. Across the standardised positions, the full cohort showed lower mechanical effectiveness when lowering trunk angle. However, significant group by position interactions showed this effect to be less extreme for the TT group. Kinematic data revealed that an increased pelvic tilt resulted in increased hip flexion and induced a more dorsiflexed ankle angle. In addition, linear hip position acutely responded to positional changes by moving forwards when the trunk angle was lowered. A more forwards hip position is thus associated with maintaining a better mechanical effectiveness in aerodynamic cycling positions. This suggests that there is potential to mitigate the effect of negative crank forces in aerodynamic positions by acutely adjusting the saddle placement to facilitate linear hip movement.

A compartmentalized phosphoinositide signaling axis at cilia is regulated by INPP5E to maintain cilia and promote Sonic Hedgehog medulloblastoma.

Sonic Hedgehog (SHH) signaling at primary cilia drives the proliferation and progression of a subset of medulloblastomas, the most common malignant paediatric brain tumor. Severe side effects associated with conventional treatments and resistance to targeted therapies has led to the need for new strategies. SHH signaling is dependent on primary cilia for signal transduction suggesting the potential for cilia destabilizing mechanisms as a therapeutic target. INPP5E is an inositol polyphosphate 5-phosphatase that hydrolyses PtdIns(4,5)P2 and more potently, the phosphoinositide (PI) 3-kinase product PtdIns(3,4,5)P3. INPP5E promotes SHH signaling during embryonic development via PtdIns(4,5)P2 hydrolysis at cilia, that in turn regulates the cilia recruitment of the SHH suppressor GPR161. However, the role INPP5E plays in cancer is unknown and the contribution of PI3-kinase signaling to cilia function is little characterized. Here, we reveal INPP5E promotes SHH signaling in SHH medulloblastoma by negatively regulating a cilia-compartmentalized PI3-kinase signaling axis that maintains primary cilia on tumor cells. Conditional deletion of Inpp5e in a murine model of constitutively active Smoothened-driven medulloblastoma slowed tumor progression, suppressed cell proliferation, reduced SHH signaling and promoted tumor cell cilia loss. PtdIns(3,4,5)P3, its effector pAKT and the target pGSK3ß, which when non-phosphorylated promotes cilia assembly/stability, localized to tumor cell cilia. The number of PtdIns(3,4,5)P3/pAKT/pGSK3ß-positive cilia was increased in cultured Inpp5e-null tumor cells relative to controls. PI3-kinase inhibition or expression of wild-type, but not catalytically inactive HA-INPP5E partially rescued cilia loss in Inpp5e-null tumor cells in vitro. INPP5E mRNA and copy number were reduced in human SHH medulloblastoma compared to other molecular subtypes and consistent with the murine model, reduced INPP5E was associated with improved overall survival. Therefore our study identifies a compartmentalized PtdIns(3,4,5)P3/AKT/GSK3ß signaling axis at cilia in SHH-dependent medulloblastoma that is regulated by INPP5E to maintain tumor cell cilia, promote SHH signaling and thereby medulloblastoma progression.

Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation.

Embryonal cancer can arise from postnatally persistent embryonal remnant or rest cells, which are uniquely characterized by the absence of p53 mutations. Perinatal overexpression of the MycN oncoprotein in embryonal cancer precursor cells causes postnatal rests, and later tumor formation through unknown mechanisms. However, overexpression of Myc in adult tissues normally activates apoptosis and/or senescence signals as an organismal defense mechanism against cancer. Here, we show that perinatal neuroblastoma precursor cells exhibited a transiently diminished p53 response to MycN oncoprotein stress and resistance to trophic factor withdrawal, compared with their adult counterpart cells from the TH-MYCN(+/+) transgenic mouse model of neuroblastoma. The adult stem cell maintenance factor and Polycomb group protein, Bmi1 (B-cell-specific Moloney murine leukemia virus integration site), had a critical role at neuroblastoma initiation in the model, by repressing p53 responses in precursor cells. We further show in neuroblastoma tumor cells that Bmi1 could directly bind p53 in a complex with other Polycomb complex proteins, Ring1A or Ring1B, leading to increased p53 ubiquitination and degradation. Repressed p53 signal responses were also seen in precursor cells for other embryonal cancer types, medulloblastoma and acute lymphoblastic leukemia. Collectively, these date indicate a general mechanism for p53 inactivation in some embryonal cell types and consequent susceptibility to MycN oncogenesis at the point of embryonal tumor initiation.

Canonical Notch signaling is not required for the growth of Hedgehog pathway-induced medulloblastoma.

Current treatment for medulloblastoma is successful in more than half of all cases but results in substantial disability in survivors. Accordingly, there is considerable interest in drugs that may target specific signaling pathways activated in the tumors, with inhibitors of both the Hedgehog and Notch pathways currently proposed as possible therapeutics. Here, we tested the hypothesis that Notch pathway inhibition in vivo may block the formation of Hedgehog-dependent medulloblastoma. We took the general approach of using a cre recombinase under the control of the GFAP promoter to generate medulloblastoma in mice carrying a conditional Ptc1 allele and introduced a conditional RBP-J allele to ablate canonical Notch signaling. Loss of RBP-J from the developing cerebellum led to a modest loss of stem cells and an overall developmental delay. These phenotypes could be partially compensated by activation of the Hedgehog pathway. Hedgehog-dependent medulloblastoma were not blocked by loss of RBP-J, indicating that canonical Notch signaling is not required for tumor initiation and growth in this model.

Patched1 deletion increases N-Myc protein stability as a mechanism of medulloblastoma initiation and progression.

Medulloblastoma tumorigenesis caused by inactivating mutations in the PATCHED1 (PTCH1) gene is initiated by persistently activated Sonic Hedgehog (Shh) signaling in granule neuron precursors (GNPs) during the late stages of cerebellar development. Both normal cerebellar development and Shh-driven medulloblastoma tumorigenesis require N-Myc expression. However, the mechanisms by which N-Myc affects the stages of medulloblastoma initiation and progression are unknown. Here we used a mouse model of Ptch1 heterozygosity and medulloblastoma to show that increased N-Myc expression characterized the earliest selection of focal GNP hyperplasia destined for later tumor progression. Step-wise loss of Ptch1 expression, from tumor initiation to progression, led to incremental increases in N-Myc protein, rather than mRNA, expression. Increased N-Myc resulted in enhanced proliferation and death resistance of perinatal GNPs at tumor initiation. Sequential N-Myc protein phosphorylation at serine-62 and serine-62/threonine-58 characterized the early and late stages of medulloblastoma tumorigenesis, respectively. Shh pathway activation led to increased Myc protein stability and reduced expression of key regulatory factors. Taken together our data identify N-Myc protein stability as the result of loss of Ptch1, which distinguishes normal cerebellar development from medulloblastoma tumorigenesis.

Sonic Hedgehog regulates Hes1 through a novel mechanism that is independent of canonical Notch pathway signalling.

Aberrant regulation of signalling mechanisms that normally orchestrate embryonic development, such as the Hedgehog, Wnt and Notch pathways, is a common feature of tumorigenesis. In order to better understand the neoplastic events mediated by Hedgehog signalling, we identified over 200 genes regulated by Sonic Hedgehog in multipotent mesodermal cells. Widespread crosstalk with other developmental signalling pathways is evident, suggesting a complex network of interactions that challenges the often over-simplistic representation of these pathways as simple linear entities. Hes1, a principal effector of the Notch pathway, was found to be a target of Sonic Hedgehog in both C3H/10T1/2 mesodermal and MNS70 neural cells. Desert Hedgehog also elicited a strong Hes1 response. While Smoothened function was found necessary for upregulation of Hes1 in response to Sonic Hedgehog, the mechanism does not require gamma-secretase-mediated cleavage of Notch receptors, and appears to involve transcription factors other than RBP-Jkappa. Thus, we have defined a novel mechanism for Hes1 regulation in stem-like cells that is independent of canonical Notch signalling.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

DHPLC analysis of patients with Nevoid Basal Cell Carcinoma Syndrome reveals novel PTCH missense mutations in the sterol-sensing domain.

Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 h of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AP1 transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon.

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, deltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551) K18-GFP-CFTR(+/-), designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

G551D CF mice display an abnormal host response and have impaired clearance of Pseudomonas lung disease.

Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.

Murine CFTR channel and its role in regulatory volume decrease of small intestine crypts.

Cystic fibrosis (CF) is caused by mutations in the secretory Cl(-) channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl(-) channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other 'environmental' factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl(-) channel and as a regulator of volume-dependent homeostatic cell mechanisms.

The spectrum of patched mutations in a collection of Australian basal cell carcinomas.

Inactivating mutations in the human patched (PTCH) gene have been identified in both familial and sporadic basal cell carcinomas (BCCs). In some tumors mutations have been detected in both alleles thereby supporting the role of PTCH as a tumor suppressor gene. We have analyzed 22/23 coding exons of PTCH for mutations in 44 sporadic BCCs, and detected 10 novel mutations in nine tumors. In two of the mutant tumors the remaining allele was inactivated by loss of heterozygosity. Five novel PTCH polymorphisms were also identified. Most of the variations found were C>T substitutions at dipyrimidine sites, supporting previous studies which indicate a role for ultraviolet-B in the genesis of sporadic BCCs.

G551D cystic fibrosis mice exhibit abnormal regulation of inflammation in lungs and macrophages.

The major cause of death in cystic fibrosis (CF) is chronic lung disease associated with persistent infection by the bacterium, Pseudomonas aeruginosa. S100A8, an S-100 calcium-binding protein with chemotactic activity, is constitutively expressed in the lungs and serum of CF patients. Levels of S100A8 mRNA were found to be three to four times higher in the lungs of mice carrying the G551D mutation in CF transmembrane conductance regulator compared with littermate controls. Intravenous injection of bacterial LPS induced S100A8 mRNA in the lung to a greater extent in G551D mice than in wild-type littermates. Localization of S100A8 mRNA and protein in the lung indicate that it is a marker for neutrophil accumulation. Bone marrow-derived macrophages from G551D mice were shown to also exhibit hypersensitivity to LPS, measured by induction of TNF-alpha. These results provide evidence that the pathology of CF relates to abnormal regulation of the immune system.

Sequence variants of DLC1 in colorectal and ovarian tumours.

Loss of heterozygosity occurs frequently on the short arm of chromosome 8 in many neoplasms, including colorectal and ovarian cancer. Monochromosome transfer experiments into colorectal tumour cell lines have provided functional evidence for a tumour suppressor gene located at 8p22-23. One of the genes from this region that is expressed by our suppressed hybrids is a candidate tumour suppressor gene, DLC1 (deleted in liver cancer), which has homology to rat RhoGAP. We have delineated the structure of the DLC1 gene and used single-stranded conformation polymorphism analysis (SSCP) to look for sequence variants in 126 colorectal and 33 ovarian primary tumours and cell lines. One exonic missense mutation and three intronic insertions/deletions were identified in primary colorectal tumours, as well as many polymorphisms present in germline DNAs. The rarity of exonic missense mutations, and the absence of protein-truncating mutations, indicates that DLC1 is not the target of 8p LOH in colorectal or ovarian tumours. The delineation of the gene structure allows mutation analysis of DLC1 in other tumour types for which it remains a candidate tumour suppressor gene based on its location and homology to rhoGAP.

The effect of longer-term creatine supplementation on elite swimming performance after an acute creatine loading.

We investigated the effect of an acute creatine loading (25 g per day for 4 days) and longer-term creatine supplementation (5 g of creatine or 5 g of placebo per day for 2 months) on the performance of 22 elite swimmers during maximal interval sessions. After the acute creatine loading, the mean of the average interval swim times for all swimmers (n = 22) improved (44.3+/-16.5 s before vs. 43.7+/-16.3 s after supplementation; P<0.01). Three of the 22 swimmers did not respond positively to supplementation. After 2 months of longer-term creatine supplementation or placebo, neither group showed a significant change in swimming performance (38.7+/-13.5 s before vs. 38.7+/-14.1 s after for the creatine group; 48.7+/-18.0 s before vs. 48.7+/-18.1 s after for the placebo group). We conclude that, in elite swimmers, 4 days of acute creatine loading improves swimming performance significantly when assessed by maximal interval sessions. However, longer-term supplementation for 2 months (5 g of creatine per day) did not benefit significantly the creatine group compared with the placebo group.

Isolation and characterization of human patched 2 (PTCH2), a putative tumour suppressor gene inbasal cell carcinoma and medulloblastoma on chromosome 1p32.

Mutations of the human Patched gene ( PTCH ) have been identified in individuals with the nevoid basal cell carcinoma syndrome (NBCCS) as well as in sporadic basal cell carcinomas and medulloblastomas. We have isolated a homologue of this tumour suppressor gene and localized it to the short arm of chromosome 1 (1p32.1-32.3). Patched 2 ( PTCH2 ) comprises 22 coding exons and spans approximately 15 kb of genomic DNA. The gene encodes a 1203 amino acid putative transmembrane protein which is highly homologous to the PTCH product. We have characterized the genomic structure of PTCH2 and have used single-stranded conformational polymorphism analysis to search for mutations in PTCH2 in NBCCS patients, basal cell carcinomas and in medulloblastomas. To date, we have identified one truncating mutation in a medulloblastoma and a change in a splice donor site in a basal cell carcinoma, suggesting that the gene plays a role in the development of some tumours.

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects.

Previous studies have indicated that milrinone, a specific type III phosphodiesterase inhibitor, may be able to induce chloride secretion in cystic fibrosis (CF) tissues. We have now assessed the effect of this agent in vivo on the nasal epithelium of CF mutant mice and also in the nose and lungs of human subjects with CF. Wild-type mice showed a small hyperpolarization of the nasal potential difference (PD) in response to milrinone (100 microM, 1.6 +/- 0.6 mV, n = 8, P < 0.05). In contrast, CF mice carrying either the most common human mutation of the gene for the CF transmembrane regulator (CFTR), DeltaF508 (protein mislocalized), or the G551D mutation (protein normally localized) failed to demonstrate this response. Milrinone perfused alone had no significant effect on the baseline nasal PD of human subjects without CF (14.7 +/- 4.0 mV preperfusion; 15.3 +/- 4.6 mV postperfusion), but significantly (P < 0.05) augmented the hyperpolarization induced by a subsequently perfused low-chloride solution (with milrinone, 36.8 +/- 3.0 mV, n = 6; without milrinone, 18.1 +/- 2.2 mV, n = 19). In contrast, in human subjects with CF (n = 6), milrinone alone significantly (P < 0. 05) altered the nasal baseline PD (52.2 +/- 3.3 mV preperfusion; 57. 4 +/- 4.2 mV, postperfusion) but not the subsequent responses to the low-chloride solution (with milrinone, 1.1 +/- 2.2 mV, n = 4; without milrinone, 0.6 +/- 0.5 mV, n = 28) or to isoproterenol (100 microM). In a separate study in subjects (n = 6) with the DeltaF508 mutation, nasal coadministration of milrinone with isoproterenol produced no effect in the presence of amiloride and a low-chloride solution (-0.8 +/- 0.5 mV). This was also the case in the nasal epithelium of CF subjects (n = 4) carrying at least one G551D allele (-0.3 +/- 0.8 mV). Similarly, milrinone did not hyperpolarize the PD of either the tracheal (n = 6) or segmental (n = 6) airways of CF subjects (DeltaF508) when applied topically in vivo in the presence of amiloride, isoproterenol, or adenosine triphosphate (all 100 microM) in a low-chloride solution. These data do not support the use of milrinone to induce chloride secretion in CF airways in vivo.