Heart neurons use clock genes to control myocyte proliferation.

Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neuron­deficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.

Chronic Atrial and Ventricular Pacing in the Mouse.

BACKGROUND: The mouse is the most widely used mammal in experimental biology. Although many clinically relevant in vivo cardiac stressors are used, one that has eluded translation is long-term cardiac pacing. Here, we present the first method to chronically simulate and simultaneously record cardiac electrical activity in conscious mobile mice. We then apply it to study right ventricular pacing induced electromechanical dyssynchrony and its reversal (resynchronization). METHODS AND RESULTS: The method includes a custom implantable bipolar stimulation and recording lead and flexible external conduit and electrical micro-commutator linked to a pulse generator/recorder. This achieved continuous pacing for at least 1 month in 77% of implants. Mice were then subjected to cardiac ischemia/reperfusion injury to depress heart function, followed by 4 weeks pacing at the right ventricle (dyssynchrony), right atrium (synchrony), or for 2 weeks right ventricle and then 2 weeks normal sinus (resynchronization). Right ventricular pacing-induced dyssynchrony substantially reduced heart and myocyte function compared with the other groups, increased gene expression heterogeneity (>10 fold) comparing septum to lateral walls, and enhanced growth and metabolic kinase activity in the late-contracting lateral wall. This was ameliorated by restoring contractile synchronization. CONCLUSIONS: The new method to chronically pace conscious mice yields stable atrial and ventricular capture and a means to dissect basic mechanisms of electromechanical physiology and therapy. The data on dyssynchrony and resynchronization in ischemia/reperfusion hearts is the most comprehensive to date in ischemic heart disease, and its similarities to nonischemic canine results support the translational utility of the mouse.

A planarian nidovirus expands the limits of RNA genome size.

RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication (albeit within a permissive host environment). A 33.5 kilobase (kb) nidovirus has been considered close to the upper size limit for such entities; conversely, the minimal cellular DNA genome is in the 100-300 kb range. This large difference presents a daunting gap for the transition from primordial RNP to contemporary DNA-RNP-based life. Whether or not RNA viruses represent transitional steps towards DNA-based life, studies of larger RNA viruses advance our understanding of the size constraints on RNP entities and the role of genome size in virus adaptation. For example, emergence of the largest previously known RNA genomes (20-34 kb in positive-stranded nidoviruses, including coronaviruses) is associated with the acquisition of a proofreading exoribonuclease (ExoN) encoded in the open reading frame 1b (ORF1b) in a monophyletic subset of nidoviruses. However, apparent constraints on the size of ORF1b, which encodes this and other key replicative enzymes, have been hypothesized to limit further expansion of these viral RNA genomes. Here, we characterize a novel nidovirus (planarian secretory cell nidovirus; PSCNV) whose disproportionately large ORF1b-like region including unannotated domains, and overall 41.1-kb genome, substantially extend the presumed limits on RNA genome size. This genome encodes a predicted 13,556-aa polyprotein in an unconventional single ORF, yet retains canonical nidoviral genome organization and expression, as well as key replicative domains. These domains may include functionally relevant substitutions rarely or never before observed in highly conserved sites of RdRp, NiRAN, ExoN and 3CLpro. Our evolutionary analysis suggests that PSCNV diverged early from multi-ORF nidoviruses, and acquired additional genes, including those typical of large DNA viruses or hosts, e.g. Ankyrin and Fibronectin type II, which might modulate virus-host interactions. PSCNV's greatly expanded genome, proteomic complexity, and unique features-impressive in themselves-attest to the likelihood of still-larger RNA genomes awaiting discovery.

Precardiac organoids form two heart fields via Bmp/Wnt signaling.

The discovery of the first heart field (FHF) and the second heart field (SHF) led us to understand how cardiac lineages and structures arise during development. However, it remains unknown how they are specified. Here, we generate precardiac spheroids with pluripotent stem cells (PSCs) harboring GFP/RFP reporters under the control of FHF/SHF markers, respectively. GFP+ cells and RFP+ cells appear from two distinct areas and develop in a complementary fashion. Transcriptome analysis shows a high degree of similarities with embryonic FHF/SHF cells. Bmp and Wnt are among the most differentially regulated pathways, and gain- and loss-of-function studies reveal that Bmp specifies GFP+ cells and RFP+ cells via the Bmp/Smad pathway and Wnt signaling, respectively. FHF/SHF cells can be isolated without reporters by the surface protein Cxcr4. This study provides novel insights into understanding the specification of two cardiac origins, which can be leveraged for PSC-based modeling of heart field/chamber-specific disease.

Marked disparity of microRNA modulation by cGMP-selective PDE5 versus PDE9 inhibitors in heart disease.

MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.

Stem cell heterogeneity drives the parasitic life cycle of Schistosoma mansoni.

Schistosomes are parasitic flatworms infecting hundreds of millions of people. These parasites alternate between asexual reproduction in molluscan hosts and sexual reproduction in mammalian hosts; short-lived, water-borne stages infect each host. Thriving in such disparate environments requires remarkable developmental plasticity, manifested by five body plans deployed throughout the parasite's life cycle. Stem cells in Schistosoma mansoni provide a potential source for such plasticity; however, the relationship between stem cells from different life-cycle stages remains unclear, as does the origin of the germline, required for sexual reproduction. Here, we show that subsets of larvally derived stem cells are likely sources of adult stem cells and the germline. We also identify a novel gene that serves as the earliest marker for the schistosome germline, which emerges inside the mammalian host and is ultimately responsible for disease pathology. This work reveals the stem cell heterogeneity driving the propagation of the schistosome life cycle.

Genetic dissection of the planarian reproductive system through characterization of Schmidtea mediterranea CPEB homologs.

Cytoplasmic polyadenylation is a mechanism of mRNA regulation prevalent in metazoan germ cells; it is largely dependent on Cytoplasmic Polyadenylation Element Binding proteins (CPEBs). Two CPEB homologs were identified in the planarian Schmidtea mediterranea. Smed-CPEB1 is expressed in ovaries and yolk glands of sexually mature planarians, and required for oocyte and yolk gland development. In contrast, Smed-CPEB2 is expressed in the testes and the central nervous system; its function is required for spermatogenesis as well as non-autonomously for development of ovaries and accessory reproductive organs. Transcriptome analysis of CPEB knockdown animals uncovered a comprehensive collection of molecular markers for reproductive structures in S. mediterranea, including ovaries, testes, yolk glands, and the copulatory apparatus. Analysis by RNA interference revealed contributions for a dozen of these genes during oogenesis, spermatogenesis, or capsule formation. We also present evidence suggesting that Smed-CPEB2 promotes translation of Neuropeptide Y-8, a prohormone required for planarian sexual maturation. These findings provide mechanistic insight into potentially conserved processes of germ cell development, as well as events involved in capsule deposition by flatworms.

GPCRs Direct Germline Development and Somatic Gonad Function in Planarians.

Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

Influence of surfactant type and thermal cycling on formation and stability of flavor oil emulsions fabricated by spontaneous emulsification.

Food-grade emulsions can be fabricated using simple and inexpensive low-energy homogenization methods. In this study, we examined the influence of surfactant type (Tween 40, 60, and 80), oil phase composition (limonene-to-medium chain triglyceride ratio), and temperature (25 to 95°C) on the formation and stability of flavor oil-in-water emulsions (10wt% oil, 15wt% surfactant, pH3) fabricated using spontaneous emulsification. Transparent emulsion-based delivery systems containing ultrafine droplets (d<40nm) could be formed at room temperature at certain limonene contents for all three surfactants. When these emulsions were heated and then cooled, appreciable droplet growth occurred at lower limonene levels (<60% limonene) leading to cloudiness, but ultrafine droplets were still present at higher limonene concentrations (80% limonene) leading to optical clarity. These results were attributed to the influence of oil phase composition and surfactant type on the phase inversion behavior of the surfactant-oil-water systems.

Formation of thermally reversible optically transparent emulsion-based delivery systems using spontaneous emulsification.

Transparent emulsion-based delivery systems suitable for encapsulating lipophilic bioactive agents can be fabricated using low-energy spontaneous emulsification methods. These emulsions are typically fabricated from non-ionic surfactants whose hydrophilic head groups are susceptible to dehydration upon heating. This phenomenon may promote emulsion instability due to enhanced droplet coalescence at elevated temperatures. Conversely, the same phenomenon can be used to fabricate optically transparent emulsions through the phase inversion temperature (PIT) method. The purpose of the current study was to examine the influence of oil phase composition and surfactant-to-oil ratio on the thermal behavior of surfactant-oil-water systems containing limonene, medium chain triglycerides (MCT), and Tween 60. Various types of thermal behavior (turbidity versus temperature profiles) were exhibited by these systems depending on their initial composition. For certain compositions, thermoreversible emulsions could be formed that were opaque at high temperatures but transparent at ambient temperatures. These systems may be particularly suitable for the encapsulation of bioactive agents in applications where optical clarity is important.

Tuneable stability of nanoemulsions fabricated using spontaneous emulsification by biopolymer electrostatic deposition.

Nanoemulsions can be formed spontaneously from surfactant-oil-water systems using low energy methods. In this work, we showed that the droplets in oil-in-water nanoemulsions fabricated by spontaneous emulsification could be coated with an anionic biopolymer (beet pectin) using electrostatic deposition. Nanoemulsions were formed by titrating oil (medium chain triglycerides) and surfactant (polyoxyethylene sorbitan monostearate+lauric arginate) mixtures into an aqueous solution (10 mM citrate buffer, pH 4). Lauric arginate was used to generate a positive charge on the droplet surfaces, thereby enabling subsequent electrostatic deposition of anionic pectin. Extensive droplet aggregation occurred when intermediate pectin concentrations were used due to bridging flocculation. However, stable anionic pectin-coated lipid droplets could be formed at high pectin concentrations. These results demonstrate the possibility of tailoring the functionality of lipid nanodroplets produced by spontaneous emulsification.

Thermal reversibility of vitamin E-enriched emulsion-based delivery systems produced using spontaneous emulsification.

The influence of temperature scanning and isothermal storage conditions on turbidity, particle size, and thermal reversibility of vitamin E-enriched emulsions produced by spontaneous emulsification was examined. Initially, the mini-emulsions formed were optically transparent and contained small droplets (d ≈ 44 nm). When heated (20-90 °C), emulsions exhibited a complex turbidity-temperature profile with a phase inversion temperature (PIT) at ≈ 75-80 °C. Temperature scanning rate had a major influence on emulsion thermal reversibility. Slow heating (0.5 °C/min) above the PIT followed by quench cooling (≈ 67 °C min(-1)) to 30 °C did not appreciably increase turbidity or droplet diameter (d ≈ 50 nm), suggesting these systems were thermo-reversible. However, slow heating to temperatures below the PIT followed by rapid cooling appreciably increased droplet size and turbidity (thermo-irreversible). Cooling rate also affected emulsion thermo-reversibility: the turbidity and droplet size after heating above the PIT decreased with increasing cooling rate.

Retention and release of oil-in-water emulsions from filled hydrogel beads composed of calcium alginate: impact of emulsifier type and pH.

Delivery systems based on filled hydrogel particles (microgels) can be fabricated from natural food-grade lipids and biopolymers. The potential for controlling release characteristics by modulating the electrostatic interactions between emulsifier-coated lipid droplets and the biopolymer matrix within hydrogel particles was investigated. A multistage procedure was used to fabricate calcium alginate beads filled with lipid droplets stabilized by non-ionic, cationic, anionic, or zwitterionic emulsifiers. Oil-in-water emulsions stabilized by Tween 60, DTAB, SDS, or whey protein were prepared by microfluidization, mixed with various alginate solutions, and then microgels were formed by simple extrusion into calcium solutions. The microgels were placed into a series of buffer solutions with different pH values (2 to 11). Lipid droplets remained encapsulated under acidic and neutral conditions, but were released under highly basic conditions (pH 11) due to hydrogel swelling when the alginate concentration was sufficiently high. Lipid droplet release increased with decreasing alginate concentration, which could be attributed to an increase in the pore size of the hydrogel matrix. These results have important implications for the design of delivery systems to entrap and control the release of lipophilic bioactive components within filled hydrogel particles.

Formation of vitamin D nanoemulsion-based delivery systems by spontaneous emulsification: factors affecting particle size and stability.

Oil-in-water nanoemulsions are particularly suitable for encapsulation of lipophilic nutraceuticals because of their ability to form stable and transparent delivery systems with high oral bioavailability. In this study, the influence of system composition and preparation conditions on the particle size and stability of vitamin D nanoemulsions prepared by spontaneous emulsification (SE) was investigated. SE relies on the formation of small oil droplets when an oil/surfactant mixture is titrated into an aqueous solution. The influence of oil phase composition (vitamin D and MCT), surfactant-to-oil ratio (SOR), surfactant type (Tween 20, 40, 60, 80 and 85), and stirring conditions on the initial particle size of vitamin D nanoemulsions was studied. Nanoemulsions with small droplet diameters (d<200 nm) could be formed using Tween 80 at SOR⩾1 at high stirring speeds (800 rpm). These systems were relatively stable to droplet growth at ambient temperatures (<10% in diameter after 1 month storage), but unstable to heating (T>80°C). The thermal stability of the nanoemulsions could be improved by adding a cosurfactant (sodium dodecyl sulphate (SDS)). The spontaneous emulsification method is simple and inexpensive to carry out and therefore has great potential for forming nanoemulsion-based delivery systems for food, personal care, and pharmaceutical applications.

Effect of salts on formation and stability of vitamin E-enriched mini-emulsions produced by spontaneous emulsification.

Emulsion-based delivery systems are being utilized to incorporate lipophilic bioactive components into various food, personal care, and pharmaceutical products. This study examined the influence of inorganic salts (NaCl and CaCl2) on the formation, stability, and properties of vitamin E-enriched emulsions prepared by spontaneous emulsification. These emulsions were simply formed by titration of a mixture of vitamin E acetate (VE), carrier oil (MCT), and nonionic surfactant (Tween 80) into an aqueous salt solution with continuous stirring. Salt type and concentration (0-1 N NaCl or 0-0.5 N CaCl2) did not have a significant influence on the initial droplet size of the emulsions. On the other hand, the isothermal and thermal stabilities of the emulsions depended strongly on salt levels. The cloud point of the emulsions decreased with increasing salt concentration, which was attributed to accelerated droplet coalescence in the presence of salts. Dilution (2-6 times) of the emulsions with water appreciably improved their thermal stability by increasing their cloud point, which was mainly attributed to the decrease in aqueous phase salt levels. The isothermal storage stability of the emulsions also depended on salt concentration; however, increasing the salt concentration decreased the rate of droplet growth, which was the opposite of its effect on thermal stability. Potential physicochemical mechanisms for these effects are discussed in terms of the influence of salt ions on van der Waals and electrostatic interactions. This study provides important information about the effect of inorganic salts on the formation and stability of vitamin E emulsions suitable for use in food, personal care, and pharmaceutical products.

Nanoemulsion-based delivery systems for polyunsaturated (ω-3) oils: formation using a spontaneous emulsification method.

Nanoemulsion-based delivery systems are finding increasing utilization to encapsulate lipophilic bioactive components in food, personal care, cosmetic, and pharmaceutical applications. In this study, a spontaneous emulsification method was used to fabricate nanoemulsions from polyunsaturated (ω-3) oils, that is, fish oil. This low-energy method relies on formation of fine oil droplets when an oil/surfactant mixture is added to an aqueous solution. The influence of surfactant-to-oil ratio (SOR), oil composition (lemon oil and MCT), and cosolvent composition (glycerol, ethanol, propylene glycol, and water) on the formation and stability of the systems was determined. Optically transparent nanoemulsions could be formed by controlling SOR, oil composition, and aqueous phase composition. The spontaneous emulsification method therefore has considerable potential for fabricating nanoemulsion-based delivery systems for incorporating polyunsatured oils into clear food, personal care, and pharmaceutical products.

Stabilization of vitamin E-enriched nanoemulsions: influence of post-homogenization cosurfactant addition.

Oil-in-water nanoemulsions are being used in the food, beverage, and pharmaceutical industries to encapsulate, protect, and deliver lipophilic bioactive components, such as drugs, vitamins, and nutraceuticals. However, nanoemulsions are thermodynamically unstable systems that breakdown over time. We investigated the influence of posthomogenization cosurfactant addition on the thermal and storage stability of vitamin E acetate nanoemulsions (VE-nanoemulsions) formed from 10% oil phase (VE), 10% surfactant (Tween 80), 20% cosolvent (ethanol), and 60% buffer solution (pH 3). Addition of a nonionic cosurfactant (0.5% Tween 20) caused little change in droplet charge, whereas addition of anionic (0.5% SDS) or cationic (0.5% lauric arginate) cosurfactants caused droplets to be more negative or positive, respectively. Tween 20 addition had little impact on the cloud point of VE-nanoemulsions, but slightly decreased their isothermal storage stability at elevated temperatures (37 °C). Lauric arginate or SDS addition appreciably increased the cloud point, but did not improve storage stability. Indeed, SDS actually decreased the storage stability of the VE-nanoemulsions at elevated temperatures. We discuss these effects in terms of the influence of surfactants on droplet growth through Ostwald ripening and/or coalescence mechanisms. This study provides important information about the effect of cosurfactants on the stability of VE-nanoemulsions suitable for use in pharmaceutical and food products.

Effect of glycerol on formation, stability, and properties of vitamin-E enriched nanoemulsions produced using spontaneous emulsification.

Oil-in-water nanoemulsions are finding increasing use as delivery systems to encapsulate lipophilic bioactive components in functional food, personal care, and pharmaceutical products. We investigated the influence of a water-soluble cosolvent (glycerol) on the formation, stability, and properties of vitamin E acetate-loaded nanoemulsions (VE-NEs) prepared by spontaneous emulsification. VE-NEs were formed by titration of a mixture of vitamin E acetate, carrier oil (MCT) and non-ionic surfactant (Tween 80) into an aqueous glycerol solution with continuous mixing. Cosolvent concentration had an appreciable effect on the particle size produced, with the smallest mean droplet diameters (d<50 nm) being formed at 40 and 50 wt% glycerol. Nanoemulsions (d<100 nm) containing 10% vitamin E acetate could be produced at relatively low surfactant concentrations (5%) using these high glycerol levels. The turbidity of the NEs decreased at high glycerol concentrations due to the reduction in droplet size and refractive index contrast. The long-term stability of the VE-NEs was strongly influenced by glycerol concentration and storage temperature. VE-NEs containing 40% glycerol were relatively stable to droplet growth when stored at 5 and 20°C, but a rapid increase in droplet size and turbidity occurred during storage at 37°C. Temperature scanning experiments (20-80-20°C) indicated that a steep and irreversible increase in turbidity occurred during heating, which was around 70°C in the absence of glycerol and 60°C in the presence of 40% glycerol. Droplet instability was attributed to an increase in the rate of Ostwald ripening and/or coalescence as the temperature was increased, associated with dehydration of the non-ionic surfactant head-group leading to a reduction in phase inversion temperature. Dilution (100×) of VE-NEs containing glycerol with water considerably improved their stability to droplet growth, especially at high storage temperatures. This study provides important information about the effect of glycerol on the formation, stability and physical properties of VE-enriched NEs suitable for food, personal care, and pharmaceutical products.

Fabrication of vitamin E-enriched nanoemulsions: factors affecting particle size using spontaneous emulsification.

Oil-in-water nanoemulsions are finding increasing use as delivery systems to encapsulate lipophilic bioactive components in functional food, personal care, and pharmaceutical products. We have investigated the influence of system composition and preparation conditions on the particle size of vitamin E acetate (VE)-loaded nanoemulsions prepared by spontaneous emulsification. This method relies on the formation of very fine oil droplets when an oil/surfactant mixture is added to water. The oil-to-emulsion ratio content was kept constant (10 wt.%) while the surfactant-to-emulsion ratio (%SER) was varied (from 2.5 to 10 wt.%). Oil phase composition (vitamin E to medium chain triglyceride ratio) had a major effect on particle size, with the smallest droplets being formed at 8 wt.% VE and 2 wt.% MCT. Surfactant type also had an appreciable impact on particle size, with TWEEN® 80 giving the smallest droplets from a group of food-grade non-ionic surfactants (TWEEN® 20, 40, 60, 80, and 85). Surfactant-to-emulsion ratio also had to be optimized to produce fine droplets, with the smallest droplets being formed at SER=10 wt.%. Particle size could also be reduced by increasing the temperature and stirring speed used when the oil/surfactant mixture was added to water. By optimizing system composition and homogenization conditions we were able to form VE-loaded nanoemulsions with small mean droplet diameters (d<50 nm) and low polydispersity indexes (PDI<0.13). The spontaneous emulsification method therefore has great potential for forming nanoemulsion-based delivery systems for food, personal care, and pharmaceutical applications.