New paeonol derivative C302 reduces hypertension in spontaneously hypertensive rats through endothelium-dependent and endothelium-independent vasodilation.

Hypertension is a major risk factor for cardiovascular disease and Chinese herb monomers could provide new structural skeletons for anti-hypertension new drug development. Paeonol is a Chinese herbal monomer extracted from Cortex moutan, exhibited some anti-hypertensive activity. The study focused on the structural optimization of paeonol to provide promising lead compounds for anti-hypertension new drug development. Herein, twelve new paeonol derivatives (PD) were designed and synthesized and their vasodilation activity was evaluated by in vitro vasodilation drug screening platform based on Myograph. Its anti-hypertension activity, PD-C302 (2-hydroxy-4-methoxyvalerophenone) as a representative with the optimal vasodilation activity, was determined by its response to blood pressure in spontaneously hypertensive rats (SHR) in vivo. Moreover, its molecular mechanism was probed by the vasodilation activity of rat superior mesenteric artery rings with or without endothelium pre-contracted by potassium chloride (KCl) or phenylephrine hydrochloride (PE). It was indicated that PD-C302 significantly reduced the blood pressure in SHR, which would involve in PD-C302-induced vasodilation. Furthermore, endothelium-dependent pathways and endothelium-independent pathways both contributed importantly to PD-C302-induced vasodilation at low concentration of PD-C302. Endothelium-independent pathways (vascular smooth muscle cell-mediated vasodilation), were mainly responsible for the PD-C302-induced vasodilation at high concentration of PD-C302, which involved in opening multiple K+ channels to restrain Ca2+ channels, and then triggered vasodilation to reduce blood pressure. PD-C302 has a simple structure and favorable anti-hypertensive activity in vivo, which could be a promising lead compound for anti-hypertension new drug development.

Melanin synthesis genes BgTH and BgDdc affect body color and cuticle permeability in Blattella germanica.

Melanin is involved in cuticle pigmentation and sclerotization of insects, which is critical for maintaining structural integrity and functional completeness of insect cuticle. The 2 key enzymes of tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) predicted in melanin biosynthesis are usually conserved in insects. However, it is unclear whether their function is related to epidermal permeability. In this study, we identified and cloned the gene sequences of BgTH and BgDdc from Blattella germanica, and revealed that they both showed a high expression at the molting, and BgTH was abundant in the head and integument while BgDdc was expressed highest in the fat body. Using RNA interference (RNAi), we found that knockdown of BgTH caused molting obstacles in some cockroaches, with the survivors showing pale color and softer integuments, while knockdown of BgDdc was viable and generated an abnormal light brown body color. Desiccation assay showed that the dsBgTH-injected adults died earlier than control groups under a dry atmosphere, but dsBgDdc-injected cockroaches did not. In contrast, when dsRNA-treated cockroaches were reared under a high humidity condition, almost no cockroaches died in all treatments. Furthermore, with eosin Y staining assay, we found that BgTH-RNAi resulted in a higher cuticular permeability, and BgDdc-RNAi also caused slight dye penetration. These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle, and the reduction of cuticular permeability may be achieved through cuticle melanization.

Two putative fatty acid synthetic genes of BgFas3 and BgElo1 are responsible for respiratory waterproofing in Blattella germanica.

Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica; however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides.

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is involved in the metabolism of endogenous and exogenous substances, and detoxification of insecticides. RNA interference (RNAi) of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides. However, the CPR of Acyrthosiphon pisum has not been characterized, and its function is still not understood. In this study, we investigated the biochemical functions of A. pisum CPR (ApCPR). ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage, and in the gut, head, and abdominal cuticle. After optimizing the dose and silencing duration of RNAi for downregulating ApCPR, we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents, and of cuticular waxy coatings. Deficiency in cuticular hydrocarbons (CHCs) decreased the survival rate of A. pisum under desiccation stress and increased its susceptibility to contact insecticides. Moreover, desiccation stress induced a significant increase in ApCPR mRNA levels. We further confirmed that ApCPR participates in CHC production. These results indicate that ApCPR modulates CHC production, desiccation tolerance, and insecticide susceptibility in A. pisum, and presents a novel target for pest control.

Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness.

Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum.

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle. Lipophorin loads and transports lipids, including hydrocarbons, from one tissue to another. In some insects, the lipophorin receptor (LpR), which binds to lipophorin and accepts its lipid cargo, is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes. However, it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown. We herein present the results of our molecular characterization, phylogenetic analysis, and functional annotation of the pea aphid (Acyrthosiphon pisum) LpR gene (ApLpR). This gene was transcribed throughout the A. pisum life cycle, but especially during the embryonic stage and in the abdominal cuticle. Furthermore, we optimized the RHA interference (RNAi) parameters by determining the ideal dose and duration for gene silencing in the pea aphid. We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity. Additionally, a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress, with decreased survival rates under simulated drought conditions. Moreover, ApLpR expression levels significantly increased in response to the desiccation treatment. These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress. Furthermore, this gene is vital for aphid reproduction. Therefore, the ApLpR gene of A. pisum may be a novel RNAi target relevant for insect pest management.

[TG-fTIR study of the thermal-conversion properties of holo-cellulose derived from woody biomass].

Thermal-conversion properties of cellulose, hemi-cellulose and holo-cellulose derived from woody biomass were studied using TG-FTIR, and also compared to those of avicel cellulose and xylan. 3-D diffusion model was applied to calculate the kinetic parameters of thermal-conversion reaction of biomass materials, such as the activation energy, pre-exponential factors, etc, which showed good regression results. With the analysis of three-dimensional IR spectra of gas products, featured peaks of HzO, CO, CO2, CH4, and oxygenates were obviously observed where showing up with the maximum weight-loss rate in DTG curves. The possible forming routes of major gaseous products were analyzed and discussed. The order of releasing amounts for gaseous productions was approximately as CO2 > H2O > CO CH4. Based on the comprehensive understanding and comparative analysis of the whole results, it is concluded that the thermal conversion process of holo-cellulose was the result of interaction between cellulose and hemi-cellulose under the dominant role of cellulose.