Cuticular proteins in codling moth (Cydia pomonella) respond to insecticide and temperature stress.

The insect cuticle consists of chitin and cuticular proteins (CPs), which stabilize the body shape and provide an effective physical barrier against the external environment. They are also potential target sites for developing environmentally friendly insect management through the utilization of physiology-based methods. The codling moth, Cydia pomonella, is a pest afflicting fruit orchards worldwide. This study used a comparative genomic approach, whole-genome resequencing, and transcriptome data to understand the role that CPs played in the environmental adaptation of the codling moth. A total of 182 putative CPs were identified in the codling moth genome, which were classified into 12 CP families. 119 CPR genes, including 54 RR-1, 60 RR-2, and 5 RR-3 genes were identified and accounted for 65.4% of the total CPs. Eight and seven gene clusters are formed in RR1 and RR2 subfamily and the ancestor-descendant relationship was explained. Five CPAP genes were highly expressed during the egg stage and exposed to high temperature, which indicated their potential role in aiding codling moth eggs in acclimating to varying external heat conditions. Moreover, six CPs belonging to the CPR and CPLCP families were identified in association with insecticide resistance by population resequencing. Their expression levels increased after exposure to insecticides, suggesting they might be involved in codling moth resistance to the insecticides azinphos-methyl or deltamethrin. Our results provide insight into the evolution of codling moth CPs and their association with high temperature adaptation and insecticide resistance, and provide an additional information required for further analysis of CPs in environmental adaptation.

Histone deacetylase inhibitor pracinostat suppresses colorectal cancer by inducing CDK5-Drp1 signaling-mediated peripheral mitofission.

Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells. Pharmacological induction of excessively asymmetric mitofission-associated cell death (MFAD) by switching the scission position from the mitochondrial midzone to the periphery represents a promising strategy for anticancer therapy. By screening a series of pan-inhibitors, we identified pracinostat, a pan-histone deacetylase (HDAC) inhibitor, as a novel MFAD inducer, that exhibited a significant anticancer effect on colorectal cancer (CRC) in vivo and in vitro. Pracinostat increased the expression of cyclin-dependent kinase 5 (CDK5) and induced its acetylation at residue lysine 33, accelerating the formation of complex CDK5/CDK5 regulatory subunit 1 and dynamin-related protein 1 (Drp1)-mediated mitochondrial peripheral fission. CRC cells with high level of CDK5 (CDK5-high) displayed midzone mitochondrial division that was associated with oncogenic phenotype, but treatment with pracinostat led to a lethal increase in the already-elevated level of CDK5 in the CRC cells. Mechanistically, pracinostat switched the scission position from the mitochondrial midzone to the periphery by improving the binding of Drp1 from mitochondrial fission factor (MFF) to mitochondrial fission 1 protein (FIS1). Thus, our results revealed the anticancer mechanism of HDACi pracinostat in CRC via activating CDK5-Drp1 signaling to cause selective MFAD of those CDK5-high tumor cells, which implicates a new paradigm to develop potential therapeutic strategies for CRC treatment.

Polyphyllin D punctures hypertrophic lysosomes to reverse drug resistance of hepatocellular carcinoma by targeting acid sphingomyelinase.

Hypertrophic lysosomes are critical for tumor progression and drug resistance; however, effective and specific lysosome-targeting compounds for cancer therapy are lacking. Here we conducted a lysosomotropic pharmacophore-based in silico screen in a natural product library (2,212 compounds), and identified polyphyllin D (PD) as a novel lysosome-targeted compound. PD treatment was found to cause lysosomal damage, as evidenced by the blockade of autophagic flux, loss of lysophagy, and the release of lysosomal contents, thus exhibiting anticancer effects on hepatocellular carcinoma (HCC) cell both in vitro and in vivo. Closer mechanistic examination revealed that PD suppressed the activity of acid sphingomyelinase (SMPD1), a lysosomal phosphodieserase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine, by directly occupying its surface groove, with Trp148 in SMPD1 acting as a major binding residue; this suppression of SMPD1 activity irreversibly triggers lysosomal injury and initiates lysosome-dependent cell death. Furthermore, PD-enhanced lysosomal membrane permeabilization to release sorafenib, augmenting the anticancer effect of sorafenib both in vivo and in vitro. Overall, our study suggests that PD can potentially be further developed as a novel autophagy inhibitor, and a combination of PD with classical chemotherapeutic anticancer drugs could represent a novel therapeutic strategy for HCC intervention.

Inhibition of nuclear deacetylase Sirtuin-1 induces mitochondrial acetylation and calcium overload leading to cell death.

Sirtuin-1 (SIRT1) is a critical nuclear deacetylase that participates in a wide range of biological processes. We hereby employed quantitative acetyl-proteomics to globally reveal the landscape of SIRT1-dependent acetylation in colorectal cancer (CRC) cells stimulated by specific SIRT1 inhibitor Inauhzin (INZ). We strikingly observed that SIRT1 inhibition enhances protein acetylation levels, with the multisite-acetylated proteins (acetyl sites >4/protein) mainly enriched in mitochondria. INZ treatment increases mitochondrial fission and depolarization in CRC cells. The acetylation of mitochondrial proteins promoted by SIRT1 inhibition prevents the recruitment of ubiquitin and LC3 for mitophagic degradation. We then found that, SIRT1 inhibition increases the acetylation of mitochondrial calcium uniporter (MCU) at residue K332, resulting in mitochondrial Ca2+ overload and depolarization, and ultimately CRC apoptosis. Arginine substitution of the K332 (K332R) dramatically decreases the mitochondrial Ca2+ influx, mitochondrial membrane potential loss and ROS burst induced by INZ. This finding uncovers a non-canonical role of SIRT1 in regulating mitochondrial function and implicates a possible way for anticancer intervention through SIRT1 inhibition.

Laboratory Investigation of Carbon Black/Bio-Oil Composite Modified Asphalt.

As environmentally friendly materials, carbon black and bio-oil can be used as modifiers to effectively enhance the poor high-temperature and low-temperature performance of base asphalt and its mixture. Different carbon black and bio-oil contents and shear time were selected as the test influencing factors in this work. Based on the Box-Behnken design (BBD), carbon black/bio-oil composite modified asphalt was prepared to perform the softening point, penetration, multiple stress creep and recovery (MSCR), and bending beam rheometer (BBR) tests. The response surface method (RSM) was used to analyze the test results. In addition, the base asphalt mixtures and the optimal performance carbon black/bio-oil composite modified asphalt mixtures were formed for rutting and low-temperature splitting tests. The results show that incorporating carbon black can enhance the asphalt's high-temperature performance by the test results of irrecoverable creep compliance (Jnr) and strain recovery rate (R). By contrast, the stiffness modulus (S) and creep rate (M) test results show that bio-oil can enhance the asphalt's low-temperature performance. The quadratic function models between the performance indicators of carbon black/bio-oil composite modified asphalt and the test influencing factors were established based on the RSM. The optimal performance modified asphalt mixture's carbon black and bio-oil content was 15.05% and 9.631%, and the shear time was 62.667 min. It was revealed that the high-temperature stability and low-temperature crack resistance of the carbon black/bio-oil composite modified asphalt mixture were better than that of the base asphalt mixture because of its higher dynamic stability (DS) and toughness. Therefore, carbon black/bio-oil composite modified asphalt mixture can be used as a new type of choice for road construction materials, which is in line with green development.

Hsa-miR-335 enhances cell migration and invasion in lung adenocarcinoma through targeting Copine-1.

Lung adenocarcinoma (LAC) is one of the most common pulmonary adenocarcinomas with a high peak of mortality, and metastasis is the main culprit of LAC deaths. microRNAs play important role in cancer metastasis, and thus are regarded as potential diagnostic and prognostic markers for human cancers. However, many miRNAs exhibit dual roles in diverse cellular contexts. Here, we revealed that hsa-miR-335, a previously reported tumor suppressor, exhibited an oncogenic role in LAC. Overexpression of miR-335 enhanced the abilities of A549 and H1299 cells to invade and migrate by regulating epithelial-mesenchymal transition, while inhibition of miR-335 exhibited an opposite effect in vitro and in vivo. Mechanically, miR-335 inhibited the expression of Copine-1 (CPNE1), an NF-κB suppressor, through interacting with its mRNA 3'UTR, while mutating the binding sites abolished this inhibitory effect. This finding not only highlights the suppressive effect of CPNE1 on cell motility, but also provides new insight into miR-335 in promoting LAC metastasis.

Structure-based discovery of neoandrographolide as a novel inhibitor of Rab5 to suppress cancer growth.

Rab5 is a small GTPase that plays a crucial role in oncogenic signal transduction, which was considered as an attractive target for cancer therapy. Rapid GDP/GTP exchange in the packet of Rab5 sustains its high activity for promoting cancer progression. However, Rab5 currently remains undruggable due to the lack of specific inhibitor. Herein, we reported the discovery of a novel Rab5 inhibitor, neoandrographolide (NAP), by using high-throughput virtual screening with a natural product library containing 7459 compounds, which can occupy the surface groove of Rab5, competing with GDP/GTP for the binding. Ser34 is the most important residue in the groove of Rab5, as it forms most hydrogen-bond interactions with GDP/GTP or NAP, and in silico mutation of Ser34 decreased the stabilization of Rab5. Moreover, fluorescence titration experiment and isothermal titration calorimetry (ITC) assay revealed a direct binding between NAP and Rab5. Biochemical and cell-based assays showed that NAP treatment not only diminished the activity of Rab5, but also suppressed cell growth of cancer cell. This finding firstly identifies NAP as a novel inhibitor of Rab5, which directly binds with Rab5 by occupying the GDP/GTP binding groove to suppress its functions, highlighting a great potential of NAP to be developed as a chemotherapeutic agent in cancer therapy.

Validated UHPLC-MS/MS method for simultaneous determination of four triterpene saponins from Akebia trifoliata extract in rat plasma and its application to a pharmacokinetic study.

Saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, the main bioactive triterpene saponins of Chinese traditional medicine Akebia trifoliata, contribute to its diuretic pharmacological activity. Because of interactions of the multiple ingredients in vivo, pharmacokinetic studies of multiple triterpenes after administration of A. trifoliata extract are essential to clarify their pharmacological effects. The purpose of this study was to develop an efficient and sensitive UHPLC-MS/MS method for simultaneous determination of these four triterpene saponins in rat plasma. The biosamples were prepared by liquid-liquid extraction with n-butanol. The chromatographic separation was performed on a Phenomenex Luna® C18 (150 × 2 mm, 3 µm) with a mobile phase consisting of acetonitrile and water at a flow rate of 0.5 mL/min. The MS/MS system was operated in a negative multiple reaction monitoring mode, and the precursor-product ion transitions were optimized as m/z 941.6 → 471.1 for saponin PH, 941.7 → 471.2 for akemisaponins E, 1089.7 → 601.1 for saponin PJ1 , 957.6 → 487.4 for scheffoleoside A and 799.5 → 637.3 for ginsenoside Rg1 (Rg1 , internal standard). Method validation parameters (calibration curve linearity, lower limit of detection, recovery, matrix effect, intra- and inter-day precision) were within the acceptable ranges. This is the first reported on the UHPLC-MS/MS detection of saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, and applied to a preclinical pharmacokinetic study after oral administration of A. trifoliata extract in rats. This study provides a basis for clinical application and further development of A. trifoliata extract.

An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.

Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/µL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins.