Rational Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocte (ESC-CM) transplantation, thereby potentially preventing dilative remodelling and progression to heart failure. Objective Assessment of transport stability, long term survival, structural organisation, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction (MI) model. Methods and Results We constructed EHMs from ESC-CMs and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). After ischemia/reperfusion (I/R) injury, EHMs were implanted onto immunocompromised rat hearts at 1 month to simulate chronic ischemia. Bioluminescence imaging (BLI) showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving up to 25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs −6.7±1.4% vs control −10.9±1.5%, n>12, P=0.05), we observed no difference between EHMs containing viable or non-viable human cardiomyocytes in this chronic xenotransplantation model (n>12, P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. Conclusions EHM transplantation led to high engraftment rates, long term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic MI model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

Reactions of 2,6-diphenylphenol (HOdpp) and rare earth metals, in the presence of mercury, at elevated temperatures in sealed tubes followed by extraction with toluene yielded homoleptic tris(aryloxo)lanthanoid() complexes, [Ln(Odpp) 3 ] (Ln = Y (1), La (2), Ce (3), Pr (4), Nd (5), Gd (6), Ho (7), Er (8), Lu ( 9)). Single crystal X-ray diffraction studies of 1-4 and 9 established monomeric structures with the coordination sphere comprising a triangular array of Odpp oxygen atoms with capping phenyl groups above and below. In the isomorphous (triclinic P1 ¯) 1, 3, 4 and 9, there are additional intramolecular π-interactions of the phenyl substituents with the metals (η 6 -Ph-Ln and η 1 -Ph-Ln). The cell parameters for 2, though similar, suggest a different form, and the structure has additional intramolecular η 6 -Ph-La and η 3 -Ph-La interactions with the capping phenyl groups near parallel.

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.