Alloplastic total temporomandibular joint replacements: do they perform like natural joints? Prospective cohort study with a historical control

The aim of this study was to qualitatively and quantitatively describe the biomechanics of existing total alloplastic reconstructions of temporomandibular joints (TMJ). Fifteen patients with unilateral or bilateral TMJ total joint replacements and 15 healthy controls were evaluated via dynamic stereometry technology. This non-invasive method combines three-dimensional imaging of the subject's anatomy with jaw tracking. It provides an insight into the patient's jaw joint movements in real time and provides a quantitative evaluation. The patients were also evaluated clinically for jaw opening, protrusive and laterotrusive movements, pain, interference with eating, and satisfaction with the joint replacements. The qualitative assessment revealed that condyles of bilateral total joint replacements displayed similar basic motion patterns to those of unilateral prostheses. Quantitatively, mandibular movements of artificial joints during opening, protrusion, and laterotrusion were all significantly shorter than those of controls. A significantly restricted mandibular range of motion in replaced joints was also observed clinically. Fifty-three percent of patients suffered from chronic pain at rest and 67% reported reduced chewing function. Nonetheless, patients declared a high level of satisfaction with the replacement. This study shows that in order to gain a comprehensive understanding of complex therapeutic measures, a multidisciplinary approach is needed

Effects of Thyroxine Exposure on Osteogenesis in Mouse Calvarial Pre-Osteoblasts

The incidence of craniosynostosis is one in every 1,800-2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis. © 2013 Cray et al

Coordination of Cell Polarity during Xenopus Gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and naïve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity

GSK3β phosphorylation modulates CLASP–microtubule association and lamella microtubule attachment

Polarity of the microtubule (MT) cytoskeleton is essential for many cell functions. Cytoplasmic linker–associated proteins (CLASPs) are MT-associated proteins thought to organize intracellular MTs and display a unique spatiotemporal regulation. In migrating epithelial cells, CLASPs track MT plus ends in the cell body but bind along MTs in the lamella. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) directly phosphorylates CLASPs at multiple sites in the domain required for MT plus end tracking. Although complete phosphorylation disrupts both plus end tracking and association along lamella MTs, we show that partial phosphorylation of the identified GSK3β motifs determines whether CLASPs track plus ends or associate along MTs. In addition, we find that expression of constitutively active GSK3β destabilizes lamella MTs by disrupting lateral MT interactions with the cell cortex. GSK3β-induced lamella MT destabilization was partially rescued by expression of CLASP2 with mutated phosphorylation sites. This indicates that CLASP-mediated stabilization of peripheral MTs, which likely occurs in the vicinity of focal adhesions, may be regulated by local GSK3β inactivation

Effect of Combination L-Citrulline and Metformin Treatment on Motor Function in Patients With Duchenne Muscular Dystrophy: A Randomized Clinical Trial

Importance:Nitric oxide precursors, such as the amino acid l-arginine and the biguanide antidiabetic drug metformin, have been associated with metabolism and muscle function in patients with Duchenne muscular dystrophy (DMD). The treatment of DMD remains an unmet medical need.Objective:To evaluate the benefits and harms of a combination of l-citrulline and metformin treatment among patients with DMD.Design, setting, and participants:A single-center randomized double-blind placebo-controlled parallel-group clinical trial was conducted between December 12, 2013, and March 30, 2016, at the University Children's Hospital Basel in Switzerland. A total of 47 ambulant male patients aged 6.5 to 10 years with genetically confirmed DMD were recruited locally and from the patient registries of Switzerland, Germany, Austria, and France. Data were analyzed from April 6, 2016, to September 5, 2019.Interventions:Patients in the treatment group received 2500 mg of l-citrulline and 250 mg of metformin (combination therapy) 3 times a day for 26 weeks compared with patients in the control group, who received placebo.Main outcomes and measures:The primary end point was the change in transfer and standing posture, as assessed by the first dimension of the Motor Function Measure, version 32, from baseline to week 26. Secondary end points included assessments of timed function, quantitative muscle force, biomarkers for muscle necrosis, and adverse events. The 2 prespecified subgroups comprised patients who were able to walk 350 m or more in 6 minutes (stable subgroup) and patients who were not able to walk 350 m in 6 minutes (unstable subgroup) at baseline.Results:Among 49 ambulant male children with DMD who were screened for eligibility, 47 patients with a mean (SD) age of 8.2 (1.1) years were randomized to a treatment group receiving combination therapy (n = 23) or a control group receiving placebo (n = 24), and 45 patients completed the study. No significant differences between groups were found in the results of timed function and muscle force tests for overall, proximal and axial, and distal motor function. Among patients receiving combination therapy, the Motor Function Measure first dimension subscore decrease was 5.5% greater than that of patients receiving placebo (95% CI, -1.0% to 12.1%; P = .09). The administration of combination therapy had significantly favorable effects on the first dimension subscore decrease among the 29 patients in the stable subgroup (6.7%; 95% CI, 0.9%-12.6%; P = .03) but not among the 15 patients in the unstable subgroup (3.9%; 95% CI, -13.2% to 20.9%; P = .63). Overall, the treatment was well tolerated with only mild adverse effects.Conclusions and relevance:Treatment with combination therapy was not associated with an overall reduction in motor function decline among ambulant patients with DMD; however, a reduction in motor function decline was observed among the stable subgroup of patients treated with combination therapy. The statistically nonsignificant difference of distal motor function in favor of combination therapy and the reduced degeneration of muscle tissue appear to support the treatment concept, but the study may have lacked sufficient statistical power. Further research exploring this treatment option with a greater number of patients is warranted

A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis

Protein kinase B/Akt has been described as a central mediator of anti-apoptotic signals transduced by the PI3 kinase. Although the role of Akt in the suppression of apoptosis is well elucidated, a potential function of Akt in tumorigenesis and chemoresistance is less intensively documented. In this study, we describe the construction of a novel form of constitutively active Akt1, which relies on the deletion of its pleckstrin homology domain and the insertion of a C-terminal farnesylation sequence. Stable cell lines were generated with MCF10A mammary epithelial cells and A549 human NSCLC cells expressing constitutively active Akt1. Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis). We investigated the chemosensitivity of A549 cells expressing farnesylated Akt vs control cells. A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt. No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel. Our data suggest, that Akt is a central mediator in the suppression of anoikis and modulation of chemotherapy-induced apoptosis. Therefore it represents a promising target for small molecule inhibitors to shift the apoptotic threshold in cancer cells after treatment with standard chemotherapy

EB1 Is Required for Spindle Symmetry in Mammalian Mitosis

Most information about the roles of the adenomatous polyposis coli protein (APC) and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells

Supporting first-grade writers who fail to learn: multiple single-case evaluation of a response to intervention approach

We report a multiple-baseline single-case study, based in the response to intervention framework, evaluating transcription-only and transcription-and-planning interventions for young, struggling writers. In a baseline phase, 8 classes of Spanish children at the start of their first year of primary (elementary) education completed short, probe writing tasks twice-weekly over the first 120 days of their school career. During this period, all students received researcher-developed classroom instruction in spelling, handwriting, and text-planning. Students then completed a battery of tests including measures of spelling, handwriting and composition quality. On the basis of writing probe tasks and test scores we identified 12 struggling writers for whose written composition performance was below the 15th percentile, relative to the full sample, whose spelling performance was below 25th percentile, and whose handwriting was poor. For the next 72 days, these students received twice-weekly, parent-delivered training in transcription skills (handwriting and spelling) or transcription skills plus text planning. Researcher-developed classroom instruction and regular probe tasks continued during this phase. All students, in both intervention conditions, showed improvement in handwriting quality relative to Phase 1. 10 students also showed improvement in composition quality, with 8 performing, post intervention, within normal range relative to peers. Our findings demonstrate the value of a response-to-intervention approach to identification and remediation for struggling writes in their first school year

Down-Regulation of HtrA1 Activates the Epithelial-Mesenchymal Transition and ATM DNA Damage Response Pathways

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways