Background Knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids is known to link amino acid substitutions in the voltage-gated sodium channel (VGSC) in Aedes aegypti . Dengue fever primarily transmitted by Ae . aegypti is an annual public health issue in Taiwan. Accordingly, pyrethroid insecticides have been heavily used for decades to control mosquito populations in the summer and autumn. In Taiwan, an Ae . aegypti population with two VGSC mutations, V1016G and D1763Y, was described previously. Methodology/Principal finding Aedes aegypti (G0) were collected in Tainan and Kaohsiung in southern Taiwan. The VGSC gene polymorphisms of the kdr mutations and the intron flanked by exons 20 and 21 were verified. The first generation offspring (G1) were used to measure the resistance level to cypermethrin, a pyrethroid insecticide currently used in Taiwan. In addition to V1016G and D1763Y, we describe two new mutations, S989P and F1534C, which have not been reported in Taiwan. Moreover, we also identify two types (groups A and B) of introns between exons 20 and 21. Intriguingly, the kdr mutations S989P, V1016G and D1763Y are strictly located on the haplotype harboring the group A intron, whereas F1534C links to the group B intron. When those data were taken together, we proposed the following six haplotypes for VGSC genes in Taiwan today: (i)S989-intron A-V1016-F1534-D1763, (ii)S989-intron A-V1016G-F1534-D1763, (iii)S989P-intron A-V1016G-F1534-D1763, (iv)S989-intron A-V1016G-F1534-D1763Y, (v)S989-intron B-V1016-F1534-D1763 and (vi)S989-intron B-V1016-F1534C-D1763. Triple heterozygous mutations of either S989P/V1016G/F1534C or V1016G/F1534C/D1763Y can be found in one single Ae . aegypti mosquito. The proportions of the VGSC mutations were relevant to cypermethrin resistance. Notably, the presence of S989P and V1016G in the population could be a helpful reference to predict the resistance level to cypermethrin. This is the first study to demonstrate the coexistence of four kdr mutations in a population of Ae . aegypti . Conclusions/Significance Four kdr mutations (S989P, V1016G, F1534C and D1763Y) and two intron forms (Group A and B) were commonly found in local Ae . aegypti populations in Taiwan.

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Replication forks are vulnerable to wayward nuclease activities. We report here our discovery of a new member in guarding genome stability at replication forks. We previously isolated a Drosophila mutation, wuho (wh, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in Drosophila, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with mWh knockout are early embryonic lethal and display DNA damage. We identify that the flap endonuclease 1 (FEN1) is one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. We show that WH is able to modulate FEN1’s endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1’s flap versus gap endonuclease activities are consistent with the proposed WH’s functions in protecting the integrity of replication fork. These results suggest that wh is a new member of the guardians of genome stability because it regulates FEN1’s potential DNA cleavage threat near the site of replication.

Recent advances in gene therapy have brought novel treatment options to multiple fields of medicine, including cancer. However, safety concerns and limited payload capacity in commonly-utilized viral vectors prevent researchers from unlocking the full potential of gene therapy. Virus-free DNA transposons, including piggyBac, have been shown to obviate these shortcomings. We have previously demonstrated the superior transposition efficiency of a modified piggyBac system in HEK293 cells. Here, we further advanced and broadened the therapeutic application of this modified piggyBac system. We demonstrated that the internal domain sequence (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. We showed that a plasmid-free donor vector having IDS-free terminal inverted repeats in conjunction with a helper plasmid expressing Quantum pBase™ v2 form the most optimal piggyBac system, Quantum pBac™ (qPB), in T cells. We further demonstrated that T cells transfected with qPB expressing CD20/CD19 CAR outperformed cells transfected with the same donor vector but with plasmid expressing hyPB transposase in CAR-T cell production. Importantly, we showed that qPB produced mainly CD8+ CAR-T cells that are also highly represented by TSCM. These CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells in vitro and in Raji-bearing immunodeficient mice. Our findings confirm that qPB is a promising virus-free vector system that is safer, and highly efficient in mediating transgene integration with the payload capacity to incorporate multiple genes.