Epidermal growth factor receptor-dependent DNA repair promotes murine and human hematopoietic regeneration.

Chemotherapy and irradiation cause DNA damage to hematopoietic stem cells (HSCs), leading to HSC depletion and dysfunction and the risk of malignant transformation over time. Extrinsic regulation of HSC DNA repair is not well understood, and therapies to augment HSC DNA repair following myelosuppression remain undeveloped. We report that epidermal growth factor receptor (EGFR) regulates DNA repair in HSCs following irradiation via activation of the DNA-dependent protein kinase-catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ). We show that hematopoietic regeneration in vivo following total body irradiation is dependent upon EGFR-mediated repair of DNA damage via activation of DNA-PKcs. Conditional deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing increased HSC DNA damage and depressed HSC recovery over time. Systemic administration of epidermal growth factor (EGF) promoted HSC DNA repair and rapid hematologic recovery in chemotherapy-treated mice and had no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but increased intergenic copy number variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA repair and hematopoietic regeneration in vivo via augmentation of NHEJ. EGF has therapeutic potential to promote human hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects.

BMP signaling is required for cell cleavage in preimplantation-mouse embryos.

The mechanisms regulating cell division during development of the mouse pre-implantation embryo are poorly understood. We have investigated whether bone morphogenetic protein (BMP) signaling is involved in controlling cell cycle during mouse pre-implantation development. We mapped and quantitated the dynamic activities of BMP signaling through high-resolution immunofluorescence imaging combined with a 3D segmentation method. Immunostaining for phosphorylated Smad1/5/8 shows that BMP signaling is activated in mouse embryos as early as the 4-cell stage, and becomes spatially restricted by late blastocyst stage. Perturbation of BMP signaling in preimplantation mouse embryos, whether by treatment with a small molecule inhibitor, with Noggin protein, or by overexpression of a dominant-negative BMP receptor, indicates that BMPs regulate cell cleavage up to the morula stage. These results indicate that BMP signaling is active during mouse pre-implantation development and is required for cell cleavage in preimplantation mouse embryos.

Morphology and thermodynamic properties of a copolymer with an electronically conducting block: poly(3-ethylhexylthiophene)-block-poly(ethylene oxide).

We report on the synthesis and morphology of a block copolymer, poly(3-(2'-ethylhexyl)thiophene)-b-poly(ethylene oxide) (P3EHT-b-PEO), that conducts both electrons and ions. We show that in the melt state the P3EHT-b-PEO chains self-assemble to produce traditional nanoscale morphologies such as lamellae and gyroid. This is in contrast to a majority of previous studies on copolymers with electronically conducting blocks wherein a nanofibrillar morphology is obtained. Our approach enables estimation of the Flory-Huggins interaction parameter, χ. The segregation strength between the two blocks is controlled through the addition of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI). For the salt-free sample, the gyroid morphology, obtained in the melt state, is transformed into lamellae below the melting temperature of the P3EHT block. This is due to the "breaking out" of the crystalline phase. For the salt-containing sample, P3EHT-b-PEO has a lamellar morphology in both melt and crystalline states (confined crystallization).

Drosophila variable nurse cells encodes arrest defective 1 (ARD1), the catalytic subunit of the major N-terminal acetyltransferase complex.

Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion, and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Furthermore, we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes.

Simultaneous electronic and ionic conduction in a block copolymer: application in lithium battery electrodes.

Charging ahead: separate values for the simultaneous electronic and ionic conductivity of a conjugated polymer containing poly(3-hexylthiophene) and poly(ethylene oxide) (P3HT-PEO) were determined by using ac impedance and dc techniques. P3HT-PEO was used as binder, and transporter of electronic charge and Li(+) ions in a LiFePO(4) cathode, which was incorporated into solid-state lithium batteries.

Relationship between Mobility and Lattice Strain in Electrochemically Doped Poly(3-hexylthiophene).

Conjugated semiconducting polymers, such as poly(3-hexylthiophene) (P3HT), are poised to play an integral role in the development of organic electronic devices; however, their performance is governed by factors that are intrinsically coupled: dopant concentration, carrier mobility, crystal structure, and mesoscale morphology. We utilize synchrotron X-ray scattering and electrochemical impedance spectroscopy to probe the crystal structure and electronic properties of P3HT in situ during electrochemical doping. We show that doping strains the crystalline domains, coincident with an exponential increase in hole mobility. We believe these observations provide guidance for the development of improved theoretical models for charge transport in semiconducting polymers.

Electrochemically oxidized electronic and ionic conducting nanostructured block copolymers for lithium battery electrodes.

Block copolymers that can simultaneously conduct electronic and ionic charges on the nanometer length scale can serve as innovative conductive binder material for solid-state battery electrodes. The purpose of this work is to study the electronic charge transport of poly(3-hexylthiophene)-b-poly(ethylene oxide) (P3HT-PEO) copolymers electrochemically oxidized with lithium bis(trifluoromethanesulfonyl) imide (LiTFSI) salt in the context of a lithium battery charge/discharge cycle. We use a solid-state three-terminal electrochemical cell that enables simultaneous conductivity measurements and control over electrochemical doping of P3HT. At low oxidation levels (ratio of moles of electrons removed to moles of 3-hexylthiophene moieties in the electrode), the electronic conductivity (σe,ox) increases from 10(-7) S/cm to 10(-4) S/cm. At high oxidation levels, σe,ox approaches 10(-2) S/cm. When P3HT-PEO is used as a conductive binder in a positive electrode with LiFePO4 active material, P3HT is electrochemically active within the voltage window of a charge/discharge cycle. The electronic conductivity of the P3HT-PEO binder is in the 10(-4) to 10(-2) S/cm range over most of the potential window of the charge/discharge cycle. This allows for efficient electronic conduction, and observed charge/discharge capacities approach the theoretical limit of LiFePO4. However, at the end of the discharge cycle, the electronic conductivity decreases sharply to 10(-7) S/cm, which means the "conductive" binder is now electronically insulating. The ability of our conductive binder to switch between electronically conducting and insulating states in the positive electrode provides an unprecedented route for automatic overdischarge protection in rechargeable batteries.

Bmp indicator mice reveal dynamic regulation of transcriptional response.

Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

A Bmp reporter with ultrasensitive characteristics reveals that high Bmp signaling is not required for cortical hem fate.

Insights into Bone morphogenetic protein (Bmp) functions during forebrain development have been limited by a lack of Bmp signaling readouts. Here we used a novel Bmp signaling reporter ("BRE-gal" mice) to study Bmp signaling in the dorsal telencephalon. At early stages, BRE-gal expression was restricted to the dorsal telencephalic midline. At later stages, strong BRE-gal expression occurred in neurons of the marginal zone and dentate gyrus. Comparisons to nuclear phospho-Smad1/5/8 (pSmad) and Msx1 indicated that BRE-gal expression occurred exclusively in neural cells with high-level Bmp signaling. BRE-gal responsiveness to Bmps was confirmed in reporter-negative cortical cells cultured with Bmp4, and both in vivo and in vitro, BRE-gal expression was switch-like, or ultrasensitive. In the early dorsal telencephalon, BRE-gal expression negatively correlated with the cortical selector gene Lhx2, indicating a BRE-gal expression border that coincides with the cortex-hem boundary. However, in Lhx2 null chimeras, neither BRE-gal nor nuclear pSmad increases were observed in ectopic hem cells. These findings establish BRE-gal as an ultrasensitive reporter of Bmp signaling in the dorsal telencephalon, imply that hem fate can be specified at different Bmp signaling intensities, and suggest that Lhx2 primarily regulates the responses to--rather than the intensity of--Bmp signaling in dorsal telencephalic cells.

Simultaneous conduction of electronic charge and lithium ions in block copolymers.

The main objective of this work is to study charge transport in mixtures of poly(3-hexylthiophene)-b-poly(ethylene oxide) (P3HT-PEO) block copolymers and lithium bis(trifluoromethanesulfonyl) imide salt (LiTFSI). The P3HT-rich microphase conducts electronic charge, while the PEO-rich microphase conducts ionic charge. The nearly symmetric P3HT-PEO copolymer used in this study self-assembles into a lamellar phase. In contrast, the morphologies of asymmetric copolymers with P3HT as the major component are dominated by nanofibrils. A combination of ac and dc impedance measurements was used to determine the electronic and ionic conductivities of our samples. The ionic conductivities of P3HT-PEO/LiTFSI mixtures are lower than those of mixtures of PEO homopolymer and LiTFSI, in agreement with published data obtained from other block copolymer/salt mixtures. In contrast, the electronic conductivities of the asymmetric P3HT-PEO copolymers are significantly higher than those of the P3HT homopolymer. This is unexpected because of the presence of the nonelectronically conducting PEO microphase. This implies that the intrinsic electronic conductivity of the P3HT microphase in P3HT-PEO copolymers is significantly higher than that of P3HT homopolymers.

Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution.

Transcription factor AP2 (Tfap2) genes play essential roles in development of the epidermis and migratory cells of the neural crest (NC) in vertebrate embryos. These transcriptional activators are among the earliest genes expressed in the ectoderm and specify fates within the epidermis/crest through both direct and indirect mechanisms. The Tfap2 family arose from a single ancestral gene in a chordate ancestor that underwent gene duplication to give up to five family members in living vertebrates. This coincided with the acquisition of important roles in NC development by Tfap2 genes suggesting that this gene family was important in ectodermal evolution and possibly in the origin of NC. Here, we show that a zebrafish tfap2c is expressed in the nonneural ectoderm during early development and functions redundantly with tfap2a in NC specification. In zebrafish embryos depleted of both tfap2a and tfap2c, NC cells are virtually eliminated. Cell transplantation experiments indicate that tfap2c functions cell-autonomously in NC specification. Cells of the enveloping layer, which forms a temporary skin layer surrounding the ectoderm, also fail to differentiate or to express appropriate keratins in tfap2c deficient embryos. The role of Tfap2 genes in epidermal and NC development is considered here in the broader context of ectodermal evolution. Distinct, tissue-specific functions for Tfap2 genes in different vertebrates may reflect subfunctionalisation of an ancestral gene that consequently led to the gain of novel roles for different subfamily members in patterning the epidermis and NC.