Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field‐collected population of Spodoptera exigua. The field‐collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH‐S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH‐S strain by crossing WF with WH‐S, followed by three rounds of backcrossing with WH‐S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH‐S strain. Compared with WH‐S, the near‐isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.     

Different enzyme classes associated with human natural killer cells may mediate disparate functions

Previous studies have shown that degradation of the acute phase reactant serum amyloid A (SAA) is mediated by enzymes on the plasma membrane of lymphocytes and monocytes. The responsible enzymes had properties of neutral elastases. The present investigations were conducted to explore whether human NK cells enriched by Percoll gradient centrifugation have similar activity and if so, whether the same or different enzyme classes are responsible for proteolysis as well as for tumor cell lysis. Accordingly, human NK cells were enriched on discontinuous Percoll gradients after which the cells were incubated either with SAA or with [3H] proline-labeled melanoma cells at various effector to target cell ratios. When SAA degradation was followed by SDS-polyacrylamide gel electrophoresis, NK fractions proved to be as effective in digesting the protein as unfractionated mononuclear leukocytes. To characterize the enzymes that may be involved in cytotoxicity on the one hand, and SAA degradation on the other, the NK fractions were treated with the following inhibitors: diisopropylfluorophosphate (DFP), soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethylketone (TLCK), the elastase inhibitors elastatinal, Ac-Ala-Ala-Pro-Val-CH2Cl, Meo-Suc-Ala-Ala-Pro-Val-CH2Cl, and an inhibitor of aryl sulfatase, Na2SO4. Preincubation of the cells with DFP or elastase inhibitors abolished their ability to hydrolyze SAA but did not affect their ability to kill tumor cells. On the other hand TLCK, a potent inhibitor of cytotoxicity, did not bring about any reduction in the proteolysis of SAA. DFP and Na2SO4 diminished cytotoxicity partially. Elimination of NK cells by sorting after incubation of lymphocytes with the monoclonal antisera Leu-7 and Leu-11 abolished cytotoxicity as well as proteolysis. The observations are compatible with the concept that NK cells carry several enzymes with different substrate specificities that may be involved in disparate cellular functions.     

Complete mitochondrial genome of <Emphasis Type="Italic">Otis tarda</Emphasis> (Gruiformes: Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences

The complete nucleotide sequence of mitochondrial genome of the Great bustard (Otis tarda) was determined by using polymerase chain reaction (PCR) method. The genome is 16,849 bp in size, containing 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. Sequences of the tRNA genes can be folded into canonical cloverleaf secondary structure except for tRNA-Cys and tRNA-Ser (AGY), which lose “DHU” arm. Sequence analysis showed that the O. tarda mitochondrial control region (mtCR) contained many elements in common with other avian mtCRs. A microsatellite repeat was found in the 3′-peripheral domain of the O. tarda mtCR. Based on the mitochondrial DNA sequences of 12S rRNA, 16S rRNA and tRNA-Val, a phylogenetic study of Gruiformes was performed. The result showed that Otididae was a sister group to “core Gruiformes” and Charadriiformes with strong support (97% posterior probability values) in Bayesian analysis. The taxonomic status of Rhynochetidae, Mesitornithidae, Pedionomidae and Turnicidae that traditionally belonged to Gruiformes was also discussed in this paper.     

The subunit composition of Portunus trituberculatus hemocyanin polymers

The subunit composition of isolated polymeric forms of Portunus trituberculatus hemocyanin were analysed by immunological techniques. The dodecamers contain four monomeric subunits corresponding to subunits I, II, III and IV, whereas the hexamers are devoid of subunit IV. These results suggest that subunit IV is required as a joining piece for the assembly of dodecamers.     

Fluid shear stress regulates HepG2 cell migration though time-dependent integrin signaling cascade

Hepatocellular carcinoma (HCC) is a subtype of malignant liver cancer with poor prognosis and limited treatment options. It is noteworthy that mechanical forces in tumor microenvironment play a pivotal role in mediating the behaviors and functions of tumor cells. As an instrumental type of mechanical forces in vivo, fluid shear stress (FSS) has been reported having potent physiologic and pathologic effects on cancer progression. However, the time-dependent mechanochemical transduction in HCC induced by FSS remains unclear. In this study, hepatocellular carcinoma HepG2 cells were exposed to 1.4 dyn/cm² FSS for transient duration (15s and 30s), short duration (5 min, 15 min and 30 min) and long duration (1h, 2h and 4h), respectively. The expression and translocation of Integrins induced FAK-Rho GTPases signaling events were examined. Our results showed that FSS endowed HepG2 cells with higher migration ability via reorganizing cellular F-actin and disrupting intercellular tight junctions. We further demonstrated that FSS regulated the expression and translocation of Integrins and their downstream signaling cascade in time-dependent patterns. The FSS downregulated focal adhesion components (Paxillin, Vinculin and Talin) while upregulated the expression of Rho GTPases (Cdc42, Rac1 and RhoA) in long durations. These results indicated that FSS enhanced tumor cell migration through Integrins-FAK-Rho GTPases signaling pathway in time-dependent manners. Our in vitro findings shed new light on the role of FSS acting in physiologic and pathological processes during tumor progression, which has emerged as a promising clinical strategy for liver carcinoma.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-Forming Toxin Cellular Defenses in Caenorhabditis elegans

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.