Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Integrated species delimitation and conservation implications of an endangered weevil Pachyrhynchus sonani (Coleoptera: Curculionidae) in Green and Orchid Islands of Taiwan

Oceanic islands are productive habitats for generating new species and high endemism, which is primarily due to their geographical isolation, smaller population sizes and local adaptation. However, the short divergence times and subtle morphological or ecological divergence of insular organisms may obscure species identity, so the cryptic endemism on islands may be underestimated. The endangered weevil Pachyrhynchus sonani Kôno (Coleoptera: Curculionidae: Entiminae: Pachyrhynchini) is endemic to Green Island and Orchid Island of the Taiwan‐Luzon Archipelago and displays widespread variation in coloration and host range, thus raising questions regarding its species boundaries and degree of cryptic diversity. We tested the species boundaries of P. sonani using an integrated approach that combined morphological (body size and shape, genital shape, coloration and cuticular scale), genetic (four genes and restriction site‐associated DNA sequencing, RAD‐seq) and ecological (host range and distribution) diversity. The results indicated that all the morphological datasets for male P. sonani, except for the colour spectrum, reveal overlapping but statistically significant differences between islands. In contrast, the morphology of the female P. sonani showed minimum divergence between island populations. The populations of P. sonani on the two islands were significantly different in their host ranges, and the genetic clustering and phylogenies of P. sonani established two valid evolutionary species. Integrated species delimitation combining morphological, molecular and ecological characters supported two distinct species of P. sonani from Green Island and Orchid Island. The Green Island population was described as P. jitanasaius sp.n. Chen & Lin, and it is recommended that its threatened conservation status be recognized. Our findings suggest that the inter‐island speciation of endemic organisms inhabiting both islands may be more common than previously thought, and they highlight the possibility that the cryptic diversity of small oceanic islands may still be largely underestimated.