Separate transcriptionally regulated pathways specify distinct classes of sister dendrites in a nociceptive neuron.

The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches.

Cell shape-dependent early responses of fibroblasts to cyclic strain.

Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000µm(2)) were micro-contact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size.

Electrospinning With Lyophilized Platelet-Rich Fibrin Has the Potential to Enhance the Proliferation and Osteogenesis of MC3T3-E1 Cells.

Platelet-rich fibrin (PRF) as a reservoir of various growth factors plays an essential role in wound healing and tissue engineering at present. Electrospinning technology is an efficient approach to acquire artificial scaffold which has large specific surface area and high porosity. The goal of this study was to investigate the potential of electrospinning on the proliferation and osteogenesis of osteogenic precursor cells in vitro, with lyophilized PRF added as a component for electrospinning preparation. The surface structure of lyophilized PRF and nanofibers were investigated, and the proliferation, osteogenesis of MEC3T3-E1 cells with lyophilized PRF or nanofibers extract were studied. The results showed that the diameters of the lyophilized PRF pores were 1.51 ± 0.75 µm, and lyophilized PRF medium promoted the proliferation and osteocalcin (OCN) and osteopontin (OPN) genes expression of MEC3T3-E1 cells. Furthermore, the diameters of the polyvinyl alcohol/sodium alginate/lyophilized PRF (PVA/SA/PRF) fibers were 201.14 ± 40.14 nm. Compared to PVA/SA nanofibers extract and control medium, PVA/SA/PRF nanofibers extract also enhanced the proliferation and mineralization activity of MEC3T3-E1 cells. These results might be instructive to future therapeutics with PVA/SA/PRF electrospinning for bone tissue engineering or other applications.

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3.

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.

Protocadherins branch out: Multiple roles in dendrite development.

The proper formation of dendritic arbors is a critical step in neural circuit formation, and as such defects in arborization are associated with a variety of neurodevelopmental disorders. Among the best gene candidates are those encoding cell adhesion molecules, including members of the diverse cadherin superfamily characterized by distinctive, repeated adhesive domains in their extracellular regions. Protocadherins (Pcdhs) make up the largest group within this superfamily, encompassing over 80 genes, including the ∼60 genes of the α-, ß-, and γ-Pcdh gene clusters and the non-clustered δ-Pcdh genes. An additional group includes the atypical cadherin genes encoding the giant Fat and Dachsous proteins and the 7-transmembrane cadherins. In this review we highlight the many roles that Pcdhs and atypical cadherins have been demonstrated to play in dendritogenesis, dendrite arborization, and dendritic spine regulation. Together, the published studies we discuss implicate these members of the cadherin superfamily as key regulators of dendrite development and function, and as potential therapeutic targets for future interventions in neurodevelopmental disorders.

Genetic regulation of mec-3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans.

The mec-3 gene, a member of the LIM-homeodomain transcription factors, is required for touch receptor, FLP and PVD neurons to differentiate in the nematode Caenorhabditis elegans. Stably integrated transgenic strains with mec-3-lacZ fusion were generated by irradiating UV light to an unstable transgenic strain with the extrachromosomal DNA. Expression patterns of the mec-3-lacZ fusion were examined in mutant backgrounds (lin-4, lin-14, egl-44, egl-46 and sem-4 genes) which alter touch receptor-specific gene expression. In the lin-4 mutant background, ectopic mec-3-lacZ positive AVM/PVM-like cells were observed in 9% of the animals. By contrast, in the lin-14 mutant background, mec-3-lacZ staining in AVM/PVM cells was lost in 86% of the animals. In the egl-44 and egl-46 mutant backgrounds, expression pattern was the same as wild-type animals. In the sem-4 mutant background, more than half of the animals (54-69%) had ectopic staining cells in the tail in addition to the wild-type staining pattern. The modes of action of these genetically interacting genes in the differentiation of mechanosensory neurons are proposed.

PELP1: a review of PELP1 interactions, signaling, and biology.

Proline, glutamic acid, and leucine rich protein 1 (PELP1) is a large multi-domain protein that has been shown to modulate an increasing number of pathways and biological processes. The first reports describing the cloning and characterization of PELP1 showed that it was an estrogen receptor coactivator. PELP1 has now been shown to be a coregulator for a growing number of transcription factors. Furthermore, recent reports have shown that PELP1 is a member of chromatin remodeling complexes. In addition to PELP1 nuclear functions, it has been shown to have cytoplasmic signaling functions as well. In the cytoplasm PELP1 acts as a scaffold molecule and mediates rapid signaling from growth factor and hormone receptors. PELP1 signaling ultimately plays a role in cancer biology by increasing proliferation and metastasis, among other cellular processes. Here we will review (1) the cloning and characterization of PELP1 expression, (2) interacting proteins, (3) PELP1 signaling, and (4) PELP1-mediated biology.

DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.