Tks5 Regulates Synaptic Podosome Formation and Stabilization of the Postsynaptic Machinery at the Neuromuscular Junction.

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.

Involvement of unconventional myosin VI in myoblast function and myotube formation.

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier.

Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed 'chemically inducible diffusion trap at cilia' to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 nm to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

The Proteasome and Cul3-Dependent Protein Ubiquitination Is Required for Gli Protein-Mediated Activation of Gene Expression in the Hedgehog Pathway.

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

PP2A phosphatase regulatory subunit PPP2R3C is a new positive regulator of the hedgehog signaling pathway.

Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored. This study unveils a novel role for the B″gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.

Effectiveness of the Use of Polymers in High-Performance Concrete Containing Silica Fume.

The incorporation of polycarboxylate ether superplasticizer (PCE)-type polymers and silica fume (SF) in high-performance concretes (HPC) leads to remarkable rheological and mechanical improvements. In the fresh state, PCEs are adsorbed on cement particles and dispersants, promoting the workability of the concrete. Silica fume enables very well-compacted concrete to be obtained, which is characterized by high mechanical parameters in its hardened state. Some PCEs are incompatible with silica fume, which can result in slump loss and poor rheological behavior. The main objective of this research is to study the influence of three types of PCEs, which all have different molecular architectures, on the rheological and mechanical behavior of high-performance concretes containing 10% SF as a partial replacement of cement. The results show that the carboxylic density of PCE has an influence on its compatibility with SF.

Synergistic effect of metformin and doxorubicin on the metastatic potential of T24 cells.

BACKGROUND: The motor ability of cancer cells to cross the basement membrane contributes to their implantation in a new location. Metastasis is a significant factor that worsens the prognosis of cancer patients. Thus, reducing cell invasiveness is an important aspect of anticancer therapy, also in bladder cancer treatment. MATERIAL: The study material was the T24 cell line of human urinary bladder cancer. The migratory potential of the cells and the effect of the treatment with individually doses and synergistic combination of doxorubicin and metformin in the 500:1 ratio for 24 h were analyzed. RESULTS: The results obtained show a compound-initiated decrease in the motor abilities of bladder cancer cells compared to controls. A decrease in the rate of colony formation was observed, as well as inhibition of migration through inserts. The visualized reorganization of the vimentin and actin networks confirms the drug-initiated limitation of the metastatic potential of T24 cells. CONCLUSION: According to our knowledge, we are the first to show, that combination of doxorubicin and metformin also worth considering in the treatment of bladder cancer. We showed that simultaneous administration of these cytostatic enhances the antiproliferative effect of drugs, but also limits cells' migratory potential.

Pituitary adenylyl cyclase-activating peptide counteracts hedgehog-dependent motor neuron production in mouse embryonic stem cell cultures.

Pituitary adenylyl cyclase-activating peptide (PACAP; ADCYAP1) is a neuropeptide that regulates a wide array of functions within the brain and periphery. We and others have previously demonstrated that PACAP and its high-affinity receptor PAC1 are expressed in the embryonic mouse neural tube, suggesting that PACAP plays a role in early brain development. Moreover, we previously showed that PACAP antagonizes the mitotic action of Sonic hedgehog (Shh) in postnatal cerebellar granule precursors. In the present study, we demonstrate that PACAP completely blocked Shh-dependent motor neuron generation from embryonic stem cell cultures and reduced mRNA levels of the Shh target gene Gli-1 and several ventral spinal cord patterning genes. In vivo examination of motor neuron and other patterning markers in embryonic day 12.5 spinal cords of wild-type and PACAP-deficient mice by immunofluorescence, on the other hand, revealed no obvious alterations in expressions of Islet1/2, MNR2, Lim1/2, Nkx2.2, or Shh, although the Pax6-positive area was slightly expanded in PACAP-deficient spinal cord. Caspase-3 staining revealed low, and similar, numbers of cells undergoing apoptosis in embryonic wild-type vs. PACAP-deficient spinal cords, whereas a slight but significant increase in number of mitotic cells was observed in PACAP-deficient mice. Thus, although PACAP has a strong capacity to counteract Shh signaling and motor neuron production in vitro, corresponding patterning defects associated with PACAP loss may be obscured by compensatory mechanisms.

Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus.

Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is modulated by SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.

Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling.

The Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.

Cement Paste Mixture Proportioning with Particle Packing Theory: An Ambiguous Effect of Microsilica.

Recently, the research of innovative building materials is focused on applying supplementary materials in the form of micro- and nanopowders in cementitious composites due to the growing insistence on sustainable development. Considering above, in paper, a research on the effect of microsilica and SiO2 nanoparticles addition to cement paste, designed with Andreasen and Andersen (AA) packing density model (PDM), in terms of its physical and mechanical properties was conducted. Density, porosity, compressive strength, hardness, and modulus of indentation were investigated and compared regarding different amount of additives used in cement paste mixes. Microstructure of the obtained pastes was analyzed. The possibility of negative influence of alkali-silica reaction (ASR) on the mechanical properties of the obtained composites was analyzed. The results of the conducted investigations were discussed, and conclusions, also practical, were presented. The obtained results confirmed that the applied PDM may be an effective tool in cement paste design, when low porosity of prepared composite is required. On the other hand, the application of AA model did not bring satisfactory results of mechanical performance as expected, what was related, as shown by SEM imaging, with inhomogeneous dispersion of microsilica, and creation of agglomerates acting as reactive aggregates, what as a consequence caused ASR reaction, crack occurrence and lowered mechanical properties. Finally, the study found that the use of about 7.5% wt. of microsilica is the optimum in regards to obtain low porosity, while, to achieve improved mechanical properties, the use of 4 wt. % of microsilica seems to be optimal, in the case of tested cement pastes.

Prediction of Compressive Strength of Fly Ash Based Concrete Using Individual and Ensemble Algorithm.

Machine learning techniques are widely used algorithms for predicting the mechanical properties of concrete. This study is based on the comparison of algorithms between individuals and ensemble approaches, such as bagging. Optimization for bagging is done by making 20 sub-models to depict the accurate one. Variables like cement content, fine and coarse aggregate, water, binder-to-water ratio, fly-ash, and superplasticizer are used for modeling. Model performance is evaluated by various statistical indicators like mean absolute error (MAE), mean square error (MSE), and root mean square error (RMSE). Individual algorithms show a moderate bias result. However, the ensemble model gives a better result with R2 = 0.911 compared to the decision tree (DT) and gene expression programming (GEP). K-fold cross-validation confirms the model's accuracy and is done by R2, MAE, MSE, and RMSE. Statistical checks reveal that the decision tree with ensemble provides 25%, 121%, and 49% enhancement for errors like MAE, MSE, and RMSE between the target and outcome response.

A Comparative Study for the Prediction of the Compressive Strength of Self-Compacting Concrete Modified with Fly Ash.

Artificial intelligence and machine learning are employed in creating functions for the prediction of self-compacting concrete (SCC) strength based on input variables proportion as cement replacement. SCC incorporating waste material has been used in learning approaches. Artificial neural network (ANN) support vector machine (SVM) and gene expression programming (GEP) consisting of 300 datasets have been utilized in the model to foresee the mechanical property of SCC. Data used in modeling consist of several input parameters such as cement, water-binder ratio, coarse aggregate, fine aggregate, and fly ash (FA) in combination with the superplasticizer. The best predictive models were selected based on the coefficient of determination (R2) results and model validation. Empirical relation with mathematical expression has been proposed using ANN, SVM, and GEP. The efficiency of the models is assessed by permutation features importance, statistical analysis, and comparison between regression models. The results reveal that the proposed machine learning models achieved adamant accuracy and has elucidated performance in the prediction aspect.

Enhancement of direct electron transfer in graphene bioelectrodes containing novel cytochrome c553 variants with optimized heme orientation.

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec553(cytc553)peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc553holoprotein and thesurface anchoring C-terminal His6-tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers.

Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures.

BACKGROUND: Hedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. METHODS: Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. RESULTS: Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. CONCLUSIONS: Primary tumorspheres derived from ptch1+/-/p53+/- mice exhibit constitutive HH pathway activity. PACAP antagonizes HH signalling in these cells in a manner blocked by the PKA antagonist H89. PACAP and pharmacological activation of PKA also inhibited proliferation. Our data suggests that regulation of HH signaling by PACAP/PKA signaling may provide an alternative to SMO inhibition for the treatment of MB.

Arhgef5 Binds α-Dystrobrevin 1 and Regulates Neuromuscular Junction Integrity.

The neuromuscular junctions (NMJs) connect muscle fibers with motor neurons and enable the coordinated contraction of skeletal muscles. The dystrophin-associated glycoprotein complex (DGC) is an essential component of the postsynaptic machinery of the NMJ and is important for the maintenance of NMJ structural integrity. To identify novel proteins that are important for NMJ organization, we performed a mass spectrometry-based screen for interactors of α-dystrobrevin 1 (aDB1), one of the components of the DGC. The guanidine nucleotide exchange factor (GEF) Arhgef5 was found to be one of the aDB1 binding partners that is recruited to Tyr-713 in a phospho-dependent manner. We show here that Arhgef5 localizes to the NMJ and that its genetic depletion in the muscle causes the fragmentation of the synapses in conditional knockout mice. Arhgef5 loss in vivo is associated with a reduction in the levels of active GTP-bound RhoA and Cdc42 GTPases, highlighting the importance of actin dynamics regulation for the maintenance of NMJ integrity.

Disruption of the PACAP gene promotes medulloblastoma in ptc1 mutant mice.

Hedgehog (Hh) proteins and cAMP-dependent protein kinase A (PKA) generally play opposing roles in developmental patterning events. Humans and mice heterozygous for mutations in the sonic hedgehog (Shh) receptor gene patched-1 (ptc1) have an increased incidence of certain types of cancer, including medulloblastoma (MB), a highly aggressive tumor of the cerebellum. Despite the importance of PKA in Hh signaling, little is known about how PKA activity is regulated in the context of Hh signaling, or the consequences of improper regulation. One molecule that can influence PKA activity is pituitary adenylyl cyclase-activating peptide (PACAP), which has been shown to regulate cerebellar granule precursor proliferation in vitro, a cell population thought to give rise to MB. To test for a PACAP/Hh interaction in the initiation or propagation of these tumors, we introduced a PACAP mutation into ptc1 mutant mice. Deletion of a single copy of PACAP increased MB incidence approximate 2.5-fold, to 66%, thereby demonstrating that PACAP exerts a powerful inhibitory action on the induction, growth or survival of these tumors. Tumors from PACAP/ptc1 mutant mice retained PACAP receptor gene expression, and exhibited superinduction of Hh target genes compared to those from ptc1+/- mice. Moreover, PACAP inhibited proliferation of cell lines derived from tumors in a PKA-dependent manner, and inhibited expression of the Hh target gene gli1. The results provide genetic evidence that PACAP acts as a physiological factor that regulates the pathogenesis of Hh pathway-associated MB tumors.

Differential Expression of Mitochondrial Biogenesis Markers in Mouse and Human SHH-Subtype Medulloblastoma.

Medulloblastoma is a brain tumor that arises predominantly in infants and children. It is the most common pediatric brain malignancy. Around 25% of medulloblastomas are driven by constitutive activation of the Hedgehog signaling pathway. Hedgehog-driven medulloblastoma is often studied in the laboratory using genetic mouse models with overactive Hedgehog signaling, which recapitulate many of the pathological features of human Hedgehog-dependent tumors. However, we show here that on a molecular level the human and mouse HH-dependent MB are quite distinct, with human, but not mouse, tumors characterized by the presence of markers of increased oxidative phosphorylation and mitochondrial biogenesis. The latter suggests that, unlike for many other types of tumors, a switch to glycolytic metabolism might not be co-opted by human SHH-MB to perpetuate their survival and growth. This needs to be taken into consideration and could potentially be exploited in the design of therapies.

Gli Proteins: Regulation in Development and Cancer.

Gli proteins are transcriptional effectors of the Hedgehog signaling pathway. They play key roles in the development of many organs and tissues, and are deregulated in birth defects and cancer. We review the molecular mechanisms of Gli protein regulation in mammals, with special emphasis on posttranslational modifications and intracellular transport. We also discuss how Gli proteins interact with co-activators and co-repressors to fine-tune the expression of Hedgehog target genes. Finally, we provide an overview of the regulation of developmental processes and tissue regeneration by Gli proteins and discuss how these proteins are involved in cancer progression, both through canonical regulation via the Hedgehog pathway and through cross-talk with other signaling pathways.

Mice deficient in both pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide survive, but display growth retardation and sex-dependent early death.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two closely related neuropeptides exhibiting overlapping activities which have actions on almost every organ system of the body. To determine if these peptides exert essential but redundant functions, we interbred VIP- and PACAP-deficient mice to obtain VIP/PACAP double knockout (DKO) mice. DKO mice had normal birth weights and survived to weaning, but exhibited a dramatic postnatal growth rate reduction. Analyses at postnatal day 16 indicated that all organs examined except the brain were reduced in mass by 40-70% compared to mixed background controls, with the thymus and spleen most profoundly affected. Brain size was also significantly reduced, but by only 10%. The reduced growth rate of DKO mice was associated with reduced serum concentrations of insulin-like growth hormone-1 (IGF-1), but unchanged levels of growth hormone. Despite the normal survival of DKO mice up to the weaning stage, many subsequently experienced early sudden death, with only 48% of females and 82% of males surviving past 6 months. The results indicate that a significant percentage of mice deficient in both VIP and PACAP survive to adulthood, but their growth rate is profoundly affected, and that females in particular exhibit high rate of mortality after about 3 months of age.