Genome editing of human induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of approximately 1–5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with gRNA, donor DNA, and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in seven weeks it is possible to efficiently obtain genome edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40–50% and homology-directed repair frequencies of 10–20%.

ML-1,785,713 is a novel, potent COX-2 inhibitor that is the most selective COX-2 inhibitor described for use in dogs to date. ML-1,785,713 has oral bioavailability and low systemic clearance that is comparable to other non-steroidal anti-inflammatory drugs. It is effective after prophylactic and therapeutic administration in attenuating lameness in dogs with urate crystal-induced synovitis. Drugs that specifically inhibit COX-2 and not COX-1 at therapeutic doses may have an improved tolerability profile, compared with nonselective non-steroidal anti-inflammatory drugs.

Heart attack remains the leading cause of death in both men and women worldwide. Stem cell-based therapies, including the use of engineered cardiac tissues, have the potential to treat the massive cell loss and pathological remodeling resulting from heart attack. Specifically, embryonic and induced pluripotent stem cells are a promising source for generation of therapeutically relevant numbers of functional cardiomyocytes and engineering of cardiac tissues in vitro. This review will describe methodologies for successful differentiation of pluripotent stem cells towards the cardiovascular cell lineages as they pertain to the field of cardiac tissue engineering. The emphasis will be placed on comparing the functional maturation in engineered cardiac tissues and developing heart and on methods to quantify cardiac electrical and mechanical function at different spatial scales.

Background: Modeling of human arrhythmias using induced pluripotent stem cell-derived cardiomyocytes has focused on single cell phenotypes. However, arrhythmias are the emergent properties of cells assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here we tested the hypothesis that CaMKII inhibition using a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. Methods:We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide (AIP), is fused to GFP and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated using invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells (iPSC) derived from CPVT patients with different disease-causing mutations to determine the effectiveness of our proposed therapy on human iPSC-derived cardiomyocytes (iPSC-CMs) and different pathogenic genotypes.Results: AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extra-cardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2 R176Q/+ ) effectively suppressed ventricular arrhythmias induced by either β-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ~50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. iPSC-CMs derived from two different CPVT patients with different pathogenic mutations demonstrated

Insecticide resistance has been a major public health challenge. It is impendent to study the mechanism on insecticide resistance. In our previous study, 14 differentially accumulated insect cuticle proteins (ICPs) based on insecticide resistance proteomes and transcriptomes were found in the deltamethrin-resistant (DR) and -susceptible (DS) strains of Culex pipiens pallens. To investigate if these ICPs are associated with deltamethrin resistance, different transcriptional levels of the 14 ICPs were detected in the DS and DR strains from laboratory and field populations by using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of the 14 ICPs were also measured after short-term exposure of the DS strain to deltamethrin. The full-length complementary DNA (cDNA) of CpCPLCG5 gene, which encodes one of the 14 ICPs, was cloned from Cx. pipiens pallens. Homology analysis and phylogenetic analysis were carried out with some other insects. Furthermore, small interfering RNA (siRNA) was used to knockdown the expression level of CpCPLCG5 gene for characterizing its contribution to deltamethrin resistance. The results showed that the expression level of CpCPLCG5 gene was higher in DR strain than in DS strain both in laboratory and field populations while the other 13 ICPs were downregulated. The full-length cDNA of CpCPLCG5 gene was 732 bp, with the ORF of 390 bp and deduced 129 amino acids (GenBank/KF723314,2013). Knockdown of CpCPLCG5 gene increased the susceptibility of the DR strain while the expression level of the other 13 ICPs elevated. Our findings indicate that the cuticle proteins are associated with deltamethrin resistance in Cx. pipiens pallens.

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MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection in many species. Emerging evidence indicates that miRNAs are involved in drug resistance. However, little is known about the relationship between the miRNAs and insecticide resistance in mosquitos. Here, we reported that conserved miR-278-3p and its target gene are critical for pyrethroid resistance in Culex pipiens pallens. We found that CYP6AG11 is the target of miR-278-3p, through bioinformatic analysis and experimental verification. The expression level of miR-278-3p was lower, whereas the level of CYP6AG11 was higher in deltamethrin-resistant strain, which were detected using qRT-PCR. We also found that CYP6AG11 was regulated by miR-278-3p via a specific target site with the 3′UTR by luciferase reporter assay. In addition, overexpression of CYP6AG11 in the mosquito C6/36 cells showed better prolification than the cells with empty vector when treated by deltamethrin at different concentrations. Moreover, the overexpression of miR-278-3p through microinjection led to a significant reduction in the survival rate, and the level of CYP6AG11 was simultaneously reduced. These results indicated that miR-278-3p could regulate the pyrethroid resistance through CYP6AG11.