HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.

The Effect of a Combined Exercise Program on Postural Control and Fine Motor Skills in Parkinson's Disease: Study Design.

Parkinson's disease (PD) is a progressive and neurodegenerative disorder defined by physical symptoms such as hand disability and postural instability. To counteract the detrimental effects of PD, physical activity programs showed improvements in overall aspects of physical functioning. Therefore, this protocol will aim to evaluate the effect a of postural and fine motor skills training program in older adults with PD. PD individuals, with mild to moderate stage PD, aged between 65 to 80 years, will be voluntary selected from the Nursing Home Residences and Rehabilitation Centers. Subsequently, they will be randomly assigned to intervention group (PD) to receive a combined training program (postural control and fine motor skills exercises) or to the Control group (CON) to receive a stretching program. Before (PRE) and after (POST) a 12-week program both groups will perform wobble board (WB) and grooved pegboard (GPT) tests. Different performances between groups will be expected: (1) no significant differences between PD and CON group for WB and GPT test values before the beginning of the training intervention (PRE); (2) significantly better WB and GPT test values in PD subjects after the training intervention (POST) when compared to the base values (PRE); and (3) no significant differences in WB and GPT test values in CON subjects after the training intervention (POST) when compared to the base values (PRE). The findings of the present study protocol could be used for future studies investigating clinical populations, such as PD, and the effects of different rehabilitative interventions aiming to improve postural control and fine motor skills performances assessed by WB and GPT tests.

Home-Based Physical Activity as a Healthy Aging Booster before and during COVID-19 Outbreak.

The role of physical activity in improving overall aspects of health regardless of age is well documented. Due to the coronavirus disease 2019 outbreak, preventive measures to limit airborne infection have been introduced, with people, especially older adults, advised to stay at home, thus increasing sedentary lifestyle and the risk of chronic diseases. As one of the few possible ways to stay active is home-based training, this review aims to provide evidence on alternative and feasible home-based activity programs as a tool to improve the fitness level in older adults, especially when preventive measures are needed to ensure isolation and limit interpersonal contacts. During quarantine, older adults, especially those with chronic diseases, are recommended to regularly exercise. Combined balance and muscle-strengthening training has proven to be particularly useful in limiting falls and mobility limitations. In addition, the use of virtual reality systems seems to be a potential strategy in remaining physically active, reducing physical inactivity time and significantly increasing the compliance of the older adults with physical activity programs. In conclusion, home-based programs induce improvements in physical functions in general and quality of life in older people with or without co-morbidities, and it can be considered in the future as one of the feasible and economic ways to increase physical well-being. This may be of unique importance in the setting of coronavirus disease 2019 enforced limitations in out-of-home activity.

Multi-national perceptions on challenges, opportunities, and support structures for Dual Career migrations in European student-athletes.

Despite Dual Careers (sports and education) and mobility of students being priorities in the funding policies of the European Commission, migrating student-athletes report severe challenges and decreased performance or dropouts at sport and academic levels. The objective of this study was to depict and assess the perceptions on challenges, support services, and their effectiveness in consideration of specific characteristics of participants and migrations. Based on a meta-synthesis and previous findings, a 50-items questionnaire was developed and completed by 245 student-athletes in 5 European countries. Participants with Dual Careers migration experience (n = 140) were considered for analyses of qualitative and quantitative (ordinal 5pt-Likert-scaled and metric) data on the Dual Career status, migration characteristics, received services, and outcomes. Chi-square-tests were conducted for differences between countries and genders at a significance level of p < .05. Country-related differences were found for experiences and intentions to migrate (X2(12) = 50.52, p<0.001), duration of the migration (X2(16) = 38.20, p = 0.001), financial support (X2(8) = 29.87, p<0.001), and decreased performances in academics (X2(16) = 56.12, p<0.001) and sports (X2(16) = 31.79, p = 0.01). Gender-related difference emerged in financial support (X2(4) = 10.68, p = 0.03), duration of the migration (X2(4) = 14.56, p = 0.01), and decreased academic performance (X2(4) = 10.57, p = 0.03). Tutoring and counselling support was ranked as the most effective support, especially when received from the academic field (4.0±1.0 pt) and others (4.1±0.8 pt), followed by online services from sport and academic sectors (both: 3.9±0.9 pt). Considering the pervasive globalization of sport and education, Dual Career migration can contribute to the development of a European sport culture. The high ratio of migrating student-athletes underlines the relevance of migrations in the field of Dual Careers. This study contributes to the literature by adding insights on practices, challenges, supports, and outcomes perceived by student-athletes migrating in Europe. Moreover, country- and gender-related differences support the consideration of specific characteristics and reveal critical factors in specific target groups. The findings contribute to identifying requirements and effective support measures in Dual Career migrations and can be used to improve support services.

Psychological determinants of physical activity across the life course: A "DEterminants of DIet and Physical ACtivity" (DEDIPAC) umbrella systematic literature review.

Low levels of physical activity (PA) are reported to contribute to the occurrence of non-communicable diseases over the life course. Although psychological factors have been identified as an important category concerning PA behavior, knowledge on psychological determinants of PA is still inconclusive. Therefore, the aim of this umbrella systematic literature review (SLR) was to summarize and synthesize the scientific evidence on psychological determinants of PA behavior across the life course. A systematic online search was conducted on MEDLINE, ISI Web of Science, Scopus, and SPORTDiscus databases. The search was limited to studies published in English from January 2004 to April 2016. SLRs and meta-analyses (MAs) of observational studies investigating the association of psychological variables and PA were considered eligible. Extracted data were evaluated based on importance of determinants, strength of evidence, and methodological quality. The full protocol is available from PROSPERO (Record ID: CRD42015010616). Twenty reviews (14 SLRs and 6 MAs), mostly of moderate methodological quality, were found eligible. Convincing evidence was found for self-efficacy (positive association with PA) in children and adolescents, and stress (negative association with PA) regardless of age. Most of the evidence revealing an association between psychological determinants and PA is probable and limited, mainly due to differences in the definition of PA and of psychological determinants across reviews. Thus, scholars are urged to reach a consensus on clear definitions of relevant psychological determinants of PA, subsuming cultural biases and allowing the possibility to obtain clear interpretations and generalizability of findings. Finally, most psychological determinants should be considered within a larger framework of other multi-level determinants that may interact or mediate some of the effects.

v-Src inhibits myogenic differentiation by interfering with the regulatory network of muscle-specific transcriptional activators at multiple levels.

The conversion of skeletal myoblasts to terminally differentiated myocytes is negatively controlled by several growth factors and oncoproteins. In this study, we have investigated the molecular mechanisms by which v-Src, a prototypic tyrosine kinase, perturbs myogenesis in primary avian myoblasts and in established murine C2C12 satellite cells. We determined the expression levels of the cell cycle regulators pRb, cyclin D1 and D3 and cyclin-dependent kinase inhibitors p21 and p27 in v-Src-transformed myoblasts and found that, in contrast to myogenin, they are normally modulated by differentiative cues, implying that v-Src affects myogenesis independent of cell proliferation. We then examined the levels of expression, DNA-binding ability and transcription-activation potentials of myogenic regulatory factors in transformed myoblasts and in myotubes after reactivation of a temperature-sensitive allele of v-Src. Our results reveal two distinct potential modes of repression targeted to myogenic factors. On the one hand, we show that v-Src reversibly inhibits the expression of MyoD and myogenin in C2C12 cells and of myogenin in quail myoblasts. Remarkably, these loci become resistant to activation of the kinase in the postmitotic compartment. On the other hand, we demonstrate that v-Src efficiently inhibits muscle gene expression by repressing the transcriptional activity of myogenic factors without affecting MyoD DNA-binding activity. Indeed, forced expression of MyoD and myogenin allows terminal differentiation of transformed myoblasts. Finally, we found that ectopic expression of the coactivator p300 restores transcription from extrachromosomal muscle-specific promoters.

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction.

FADD (Fas-associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1-2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.

Expression of AMPA-type glutamate receptors in HEK cells and cerebellar granule neurons impairs CXCL2-mediated chemotaxis.

We find that cerebellar granule neurons (CGN) obtained from newborn rats (p3) migrate in response to both CXC chemokine ligand-2 (CXCL2) and -12 (CXCL12), while CGN from p7 rats are unresponsive to CXCL2. The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor 1 (GluR1) greatly impairs the chemotaxis induced by CXCL2 in CXCR2-expressing HEK cells. By immunoprecipitation, we show that CXCR2 is associated with AMPA receptors (AMPARs) in p7 CGN, and with GluR1 co-expressed in HEK cells. Taken together, these results suggest that the association between CXCR2 and AMPARs results in the inhibition of CXCL2-dependent chemotaxis, and may represent a molecular mechanism underlying the modulation of nerve cell migration.

A two-tiered mechanism of EGFR inhibition by RALT/MIG6 via kinase suppression and receptor degradation.

Signaling by epidermal growth factor receptor (EGFR) must be controlled tightly because aberrant EGFR activity may cause cell transformation. Receptor-associated late transducer (RALT) is a feedback inhibitor of EGFR whose genetic ablation in the mouse causes phenotypes due to EGFR-driven excess cell proliferation. RALT inhibits EGFR catalytic activation by docking onto EGFR kinase domain. We report here an additional mechanism of EGFR suppression mediated by RALT, demonstrating that RALT-bound EGF receptors undergo endocytosis and eventual degradation into lysosomes. Moreover, RALT rescues the endocytic deficit of EGFR mutants unable to undergo either endocytosis (Dc214) or degradation (Y1045F) and mediates endocytosis via a domain distinct from that responsible for EGFR catalytic suppression. Consistent with providing a scaffolding function for endocytic proteins, RALT drives EGFR endocytosis by binding to AP-2 and Intersectins. These data suggest a model in which binding of RALT to EGFR integrates suppression of EGFR kinase with receptor endocytosis and degradation, leading to durable repression of EGFR signaling.

Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed. CONCLUSIONS/SIGNIFICANCE: miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits controlling skeletal muscle differentiation and maintenance.

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Cysteine residues are critical for chemokine receptor CXCR2 functional properties.

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.