Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

The sexual difference in gene expression in fat body between 8- and 10-day-old male and female Bactrocera dorsalis was examined using suppression subtractive hybridization. A total of 952 clones were sequenced and searched using BLAST from the subtracted cDNA library. About 22% of these clones showed homology with detoxification enzymes including cytochrome P450 monooxygenases (CYPs) and glutathione S-transferase. NADH dehydrogenases, distributed to energy metabolism, constituted about 9% of these clones. About 10% of these clones were cecropin, an antimicrobial peptide. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that four transcripts were expressed at a higher level in fat body of males, compared to females. Bactrocera dorsalis cyp6g2 (Bdcyp6g2) was cloned (accession number KF469179) and the temporal profile of transcriptional expression showed that Bdcyp6g2 mRNA increased with age in males from day 3 after eclosion, but only on days 0-3 in females. Compared to females, the susceptibility of 9-day-old males to three insecticides was significantly less. These results suggested the genes expressed at a higher level in male act in its survival.

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Aeolesthes oenochrous (Fairmaire), a large and colorful longhorn beetle, is an endangered species in Taiwan. Its complete mitogenome, 15,747 bp, shows a typical coleopteran organization, containing 13 protein coding genes, 22 tRNA genes, 2 rRNA genes and one A + T rich region. Two protein coding genes, i.e. COI and ND1, have the atypical start codon of AAT and TTG, respectively. The third nucleotide position of codons shows extremely low guanine content. In the A + T rich region, there were two poly-T stretches with 14 and 13 thymine each. These two poly-T stretches were clarified by the cloning method.

Overexpression of a cytochrome P450 gene, CYP4G19, is known to associate with pyrethroid resistance in the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae). In this study, we investigated the CYP4G19 expression level in 20 field-collected strains of B. germanica in Taiwan. We also examined the level of adult male susceptibility to imidacloprid, fipronil, indoxacarb, and hydramethylnon using single-diagnostic dose bioassays and their susceptibility to corresponding gel baits to determine how the CYP4G19 expression level influences the cockroach gel bait performance. Results showed that the CYP4G19 gene expression level among the field-collected German cockroach was 1.8- to 9.7-fold higher than that of the susceptible strain. It was negatively correlated (P < 0.05) with the % mortality after treatments with imidacloprid and fipronil diagnostic doses. However, no correlation was found between CYP4G19 gene expression with the % mortality after treatment with indoxacarb and hydramethylnon diagnostic doses. Indoxacarb and hydramethylnon baits showed high efficacy against the field strains with a mean mortality of 97.58 ± 1.35% and 90.95 ±1.65%, respectively. This study provided the first evidence of cross-resistance to imidacloprid and fipronil in pyrethroid-resistant German cockroaches due to overexpression of CYP4G19.