DUX4 expression in cancer induces a metastable early embryonic totipotent program.

The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the DUX4-regulated ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages, while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions increased the fraction of cells expressing DUX4 and its downstream programs. Our demonstration that transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and suppresses antigen presentation has implications for cancer growth, progression, and immune evasion.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: implications for preclinical models of FSHD.

Human DUX4 and its mouse ortholog Dux are normally expressed in the early embryo-the 4-cell or 2-cell cleavage stage embryo, respectively-and activate a portion of the first wave of zygotic gene expression. DUX4 is epigenetically suppressed in nearly all somatic tissue, whereas facioscapulohumeral dystrophy (FSHD)-causing mutations result in its aberrant expression in skeletal muscle, transcriptional activation of the early embryonic program and subsequent muscle pathology. Although DUX4 and Dux both activate an early totipotent transcriptional program, divergence of their DNA binding domains limits the use of DUX4 expressed in mice as a preclinical model for FSHD. In this study, we identify the porcine DUXC messenger ribonucleic acid expressed in early development and show that both pig DUXC and human DUX4 robustly activate a highly similar early embryonic program in pig muscle cells. These results support further investigation of pig preclinical models for FSHD.

Cholesterol-rich naked mole-rat brain lipid membranes are susceptible to amyloid beta-induced damage in vitro.

Naked mole-rats are extraordinarily long-lived rodents that offer unique opportunities to study the molecular origins of age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta (Aß) peptide from a young age. Therefore, they represent a particularly favourable organism to study the mechanisms of resistance against Aß neurotoxicity. Here we examine the composition, phase behaviour, and Aß interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with the metabolism of ceramides, sphingomyelins and sphingosine-1-phosphate receptor 1 were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase extending to 80% of the supported lipid bilayer. These observations are consistent with the 'membrane pacemaker' hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. We also found that exposure to Aß disrupts naked mole-rat brain lipid membranes significantly, breaking the membrane into pieces while mouse brain derived lipids remain largely intact upon Aß exposure.

Activating Hair Follicle Stem Cells via R-spondin2 to Stimulate Hair Growth.

Wnt signaling is required for the development of the hair follicle, and for inciting the growth (anagen) phase of the hair cycle. Most strategies to enhance Wnt signaling for hair growth create a state of constitutive Wnt activation, which leads to neoplastic transformation of the epithelial hair matrix. Using Axin2(LacZ/+) and Axin2(Cre/+)R26R(mTmG/+) reporter mice and RNA analyses, we show that Wnt signaling is elevated during anagen, is reduced at the onset of catagen, and can be reamplified in the skin and surrounding hair follicles via intradermal injection of recombinant R-spondin2 protein. Using Lgr5(LacZ/+) reporter mice, we demonstrate that this amplified Wnt environment leads to activation of leucine-rich repeat-containing G-protein coupled receptor 5-positive stem cells in the hair follicle. The onset of catagen is repressed by R-spondin2 injection, and the anagen phase persists. As a consequence, hair shafts grow longer. We conclude that R-spondin2 treatment activates hair follicle stem cells and therefore may have therapeutic potential to promote hair growth.

Linking suckling biomechanics to the development of the palate.

Skulls are amongst the most informative documents of evolutionary history but a complex geometry, coupled with composite material properties and complicated biomechanics, have made it particularly challenging to identify mechanical principles guiding the skull's morphogenesis. Despite this challenge, multiple lines of evidence, for example the relationship between masticatory function and the evolution of jaw shape, nonetheless suggest that mechanobiology plays a major role in skull morphogenesis. To begin to tackle this persistent challenge, cellular, molecular and tissue-level analyses of the developing mouse palate were coupled with finite element modeling to demonstrate that patterns of strain created by mammalian-specific oral behaviors produce complementary patterns of chondrogenic gene expression in an initially homogeneous population of cranial neural crest cells. Neural crest cells change from an osteogenic to a chondrogenic fate, leading to the materialization of cartilaginous growth plate-like structures in the palatal midline. These growth plates contribute to lateral expansion of the head but are transient structures; when the strain patterns associated with suckling dissipate at weaning, the growth plates disappear and the palate ossifies. Thus, mechanical cues such as strain appear to co-regulate cell fate specification and ultimately, help drive large-scale morphogenetic changes in head shape.

Reengineering autologous bone grafts with the stem cell activator WNT3A.

Autologous bone grafting represents the standard of care for treating bone defects but this biomaterial is unreliable in older patients. The efficacy of an autograft can be traced back to multipotent stem cells residing within the bone graft. Aging attenuates the viability and function of these stem cells, leading to inconsistent rates of bony union. We show that age-related changes in autograft efficacy are caused by a loss in endogenous Wnt signaling. Blocking this endogenous Wnt signal using Dkk1 abrogates autograft efficacy whereas providing a Wnt signal in the form of liposome-reconstituted WNT3A protein (L-WNT3A) restores bone forming potential to autografts from aged animals. The bioengineered autograft exhibits significantly better survival in the hosting site. Mesenchymal and skeletal stem cell populations in the autograft are activated by L-WNT3A and mitotic activity and osteogenic differentiation are significantly enhanced. In a spinal fusion model, aged autografts treated with L-WNT3A demonstrate superior bone forming capacity compared to the standard of care. Thus, a brief incubation in L-WNT3A reliably improves autologous bone grafting efficacy, which has the potential to significantly improve patient care in the elderly.

Disrupting the intrinsic growth potential of a suture contributes to midfacial hypoplasia.

Children with unoperated cleft palates have nearly normal growth of their faces whereas patients who have had early surgical repair often exhibit midfacial hypoplasia. Surgical repair is responsible for the underlying bone growth arrest but the mechanisms responsible for these surgical sequelae are poorly understood. We simulated the effect of cleft palate repair by raising a mucoperiosteal flap in the murine palate. Three-dimensional micro-CT reconstructions of the palate along with histomorphometric measurements, finite element (FE) modeling, immunohistochemical analyses, and quantitative RT-PCR were employed to follow the skeletal healing process. Inflammatory bone resorption was observed during the first few days after denudation, which destroyed the midpalatal suture complex. FE modeling was used to predict and map the distribution of strains and their associated stresses in the area of denudation and the magnitude and location of hydrostatic and distortional strains corresponded to sites of skeletal tissue destruction. Once re-epithelialization was complete and wound contracture subsided, the midpalatal suture complex reformed. Despite this, growth at the midpalatal suture was reduced, which led to palatal constriction and a narrowing of the dental arch. Thus the simple act of raising a flap, here mimicked by denuding the mucoperiosteum, was sufficient to cause significant destruction to the midpalatal suture complex. Although the bone and cartilage growth plates were re-established, mediolateral skeletal growth was nonetheless compromised and the injured palate never reached its full growth potential. These data strongly suggest that disruption of suture complexes, which have intrinsic growth potential, should be avoided during surgical correction of congenital anomalies.

The capacity of neural crest-derived stem cells for ocular repair.

Whether it is due to a particular epigenetic signature, or some other component of an embryonic differentiation program, accumulating evidence indicates that the origins of a stem cell has a profound impact on the potential of a tissue to regenerate and repair. Here, we focus on Müller glia, long considered the stem cells of the retina, and their surprising derivation from the neural crest. Whether the multipotent properties of a subset of Müller glia is associated with their neural crest origin remains a tantalizing possibility.

Left-right organizer flow dynamics: how much cilia activity reliably yields laterality?

Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charon expression.

Molecular mechanisms underlying skeletal growth arrest by cutaneous scarring.

In pediatric surgeries, cutaneous scarring is frequently accompanied by an arrest in skeletal growth. The molecular mechanisms responsible for this effect are not understood. Here, we investigated the relationship between scar contracture and osteogenesis. An excisional cutaneous wound was made on the tail of neonatal mice. Finite element (FE) modeling of the wound site was used to predict the distribution and magnitude of contractile forces within soft and hard tissues. Morphogenesis of the bony vertebrae was monitored by micro-CT analyses, and vertebral growth plates were interrogated throughout the healing period using assays for cell proliferation, death, differentiation, as well as matrix deposition and remodeling. Wound contracture was grossly evident on post-injury day 7 and accompanying it was a significant shortening in the tail. FE modeling indicated high compressive strains localized to the dorsal portions of the vertebral growth plates and intervertebral disks. These predicted strain distributions corresponded to sites of increased cell death, a cessation in cell proliferation, and a loss in mineralization within the growth plates and IVD. Although cutaneous contracture resolved and skeletal growth rates returned to normal, vertebrae under the cutaneous wound remained significantly shorter than controls. Thus, localized contractile forces generated by scarring led to spatial alterations in cell proliferation, death, and differentiation that inhibited bone growth in a location-dependent manner. Resolution of cutaneous scarring was not accompanied by compensatory bone growth, which left the bony elements permanently truncated. Therefore, targeting early scar reduction is critical to preserving pediatric bone growth after surgery.

Augmenting endogenous Wnt signaling improves skin wound healing.

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2(LacZ/+) reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2(LacZ/LacZ) mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2(LacZ/LacZ) mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.

Modelling the fluid mechanics of cilia and flagella in reproduction and development.

Cilia and flagella are actively bending slender organelles, performing functions such as motility, feeding and embryonic symmetry breaking. We review the mechanics of viscous-dominated microscale flow, including time-reversal symmetry, drag anisotropy of slender bodies, and wall effects. We focus on the fundamental force singularity, higher-order multipoles, and the method of images, providing physical insight and forming a basis for computational approaches. Two biological problems are then considered in more detail: 1) left-right symmetry breaking flow in the node, a microscopic structure in developing vertebrate embryos, and 2) motility of microswimmers through non-Newtonian fluids. Our model of the embryonic node reveals how particle transport associated with morphogenesis is modulated by the gradual emergence of cilium posterior tilt. Our model of swimming makes use of force distributions within a body-conforming finite-element framework, allowing the solution of nonlinear inertialess Carreau flow. We find that a three-sphere model swimmer and a model sperm are similarly affected by shear-thinning; in both cases swimming due to a prescribed beat is enhanced by shear-thinning, with optimal Deborah number around 0.8. The sperm exhibits an almost perfect linear relationship between velocity and the logarithm of the ratio of zero to infinite shear viscosity, with shear-thickening hindering cell progress.

Wnt signaling and injury repair.

Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. In this review we summarize recent data elucidating the roles that the Wnt pathway plays in the injury repair process. These data provide a foundation for potential Wnt-based therapeutic strategies aimed at stimulating tissue regeneration.

Polynuclear vanadium complexes from thermal decomposition of [V3O(O2CPh)6(H2O)3]Cl.

Solid-state decomposition of [V3O(O2CPh)6(H2O)3]Cl at 300 degrees C followed by alcoholysis of the product gives the new vanadium complexes [V6O6(PhCO2)6(CH3O)6(CH3OH)3] (1), [V6O6(PhCO2)6(C2H5O)6(C2H5OH)3] (2), [V6O6(PhCO2)6(C3H7O)6(C3H7OH)3] (3), [V6O6(PhCO2)6(C4H9O)6(C4H9OH)3] (4) and [V4O4(OCH3)6(O2CPh)2(HOCH3)2] (5). Complexes 2, 3 and 5 have been crystallographically characterised. DC magnetic susceptibility studies on complex shows antiferromagnetic coupling leading to a S = 0 spin ground state.

Hospitalization and emergency department visits among seniors receiving homecare: a pilot study.

BACKGROUND: Despite the recent growth in home health services, data on clinical outcomes and acute health care utilization among older adults receiving homecare services are sparse. Obtaining such data is particularly relevant in Ontario where an increasing number of frail seniors receiving homecare are awaiting placement in long-term care facilities. In order to determine the feasibility of a large-scale study, we conducted a pilot study to assess utilization of acute health care services among seniors receiving homecare to determine associated clinical outcomes. METHODS: This prospective cohort study followed forty-seven seniors admitted to homecare by two homecare agencies in Hamilton, Ontario over a 12-month period. Demographic information and medical history were collected at baseline, and patients were followed until either termination of homecare services, death, or end of study. The primary outcome was hospitalization. Secondary outcomes included emergency department visits that did not result in hospitalization and death. Rates of hospitalization and emergency department visits without admission were calculated, and univariate analyses were performed to test for potential risk factors. Survival curves for accumulative rates of hospitalization and emergency department visits were created. RESULTS: 312 seniors were eligible for the study, of which 123 (39%) agreed to participate initially. After communicating with the research nurse, of the 123 who agreed to participate initially, 47 (38%) were enrolled in the study. Eleven seniors were hospitalized during 3,660 days of follow-up for a rate of 3.0 incident hospitalizations per 1,000 homecare-days. Eleven seniors had emergency department visits that did not result in hospitalization, for a rate of 3.3 incident emergency department visits per 1,000 homecare-days. There were no factors significantly associated with hospitalization or emergency department visits when adjustment was made for multiple comparisons. CONCLUSION: The incidence of hospitalization and visits to the emergency department among seniors receiving homecare services is high. Getting satisfactory levels of enrollment will be a major challenge for larger prospective studies.

Nickel pivalate complexes: structural variations and magnetic susceptibility and inelastic neutron scattering studies.

The synthesis and structural characterisation of three small nickel(II) cages are reported, all stabilised by pivalate ligands. The magnetic properties of the cages have been studied by a combination of susceptibility measurements and inelastic neutron scattering. For a dinuclear cage, [Ni2(mu-OH2)(O2CCMe3)4(HO2CCMe3)4] 1 the ground state is S=2, with a ferromagnetic exchange interaction between the Ni(II) centres of J=0.32 meV and D(S=2)=-0.09 meV in the ground state. For a tetranuclear heterocubane cage, [Ni4(mu(3)-OMe)4(O2CCMe3)4(MeOH)4] 2, two ferromagnetic exchange interactions are found and an S=4 ground state observed. While the zero-field splitting of this state cannot be determined unambigiously the most likely value is DS=4=-0.035 meV. For a tetranuclear nickel butterfly, [Ni4(mu3-OH)2(O2CCMe3)6(EtOH)6] 3, three exchange interactions are required, two anti-ferromagnetic and one weakly ferromagnetic; the resulting ground state is S=0. The data enable us to estimate the zero-field splitting of single Ni(II) ions involved in the cage as Di=+1.0 meV. Both and are therefore expected to be new single molecule magnets.

Synthetic and structural studies of cobalt-pivalate complexes.

The synthesis and characterisation of a range of cobalt pivalate cage complexes are reported. The cages include: a dinuclear Co(II) complex; an oxo-centred Co(III) triangle; tetranuclear Co(II) heterocubanes and butterflies; tetranuclear heterovalent cobalt butterflies and hexanuclear edge-sharing bitetrahedra; heterovalent penta-, hexa- and hepta-nuclear cages based on [M(4)O(4)] heterocubane cores; and a tetradecanuclear cage based on heterocubanes sharing edges and vertices. Spectroscopic studies suggest that some of these cores are retained in solution, but that only in the Co(III) triangle is the structure including ligands retained. A scheme is proposed to account for the many structures observed, which may be applicable to other polymetallic cage complexes.