[Progress in preparation of hollow nanomaterials and their application to sample pretreatment].

Sample pretreatment technology plays a vital role in the analysis of complex samples and is key to the entire analytical process. Its main purpose is to separate the substance to be measured from the sample matrix or interfering substances in the sample and to achieve a state in which the instrument can be analyzed and detected. Traditional sample pretreatment techniques include liquid-liquid extraction, liquid-solid extraction, precipitation separation, solvent volatilization-rotary evaporation, filtration, and centrifugation. However, the applications of these methods are limited by their low extraction efficiency, complicated operation, long time consumption, unstable recovery, use of large amounts of organic solvents, and large error rates. Several new sample pretreatment techniques, including solid-phase extraction, magnetic solid-phase extraction, solid-phase microextraction, and dispersive solid-phase extraction, have been developed and rapidly applied to various fields to overcome the shortcomings of traditional sample pretreatment methods. However, the development of adsorbent materials with high selectivity and enrichment capability remains a challenge in sample pretreatment technology, in which adsorbents with excellent adsorption performance are crucial. In recent years, various nanomaterials with remarkable properties have been introduced and applied to sample pretreatment, and numerous nano-extraction materials with diverse functions and high selectivity and enrichment capability have been developed. Hollow nanomaterials are nanoparticles with large voids in their solid shells. Owing to their advantageous properties, which include a large effective surface area, abundant internal space, low density, variety of preparation methods, structural and functional tailorability, short mass transmission path, and high carrying capacity, hollow nanomaterials show great application potential in sample pretreatment. The extraction mechanism of these materials is based on the synergistic effects of π-π stacking, electrostatic, hydrogen-bonding, and hydrophobic interactions to achieve the efficient separation and enrichment of the target analytes. Given their noteworthy physicochemical properties, hollow nanomaterials have gained wide attention in various research fields and are considered a research frontier in the field of materials science. Changing the structure or surface properties of the core and shell can lead to various hollow nanomaterials with unique properties. Such changes can create synergy between the physicochemical properties and structural function of the original core-shell material, leading to novel materials with superior performance compared with the starting materials and broad application prospects in sample pretreatment. Nevertheless, only a few hollow nanomaterials with diverse structures and functions are currently used for sample pretreatment, and their adsorption capacity for target analytes is often unsatisfactory. Consequently, enhancing the adsorption selectivity of these materials toward various analytes is the most important step in sample pretreatment. First, hollow nanomaterials with a large specific surface area and suitable pore size can be designed to achieve the specific adsorption of target analytes of varying sizes. The combination of hollow nanomaterials with other materials presenting desirable adsorption properties could also lead to synergistic effects and enhance the performance of composite hollow nanomaterials. In addition, more green methods to prepare hollow nanomaterials with outstanding selectivity can be explored to achieve the superior adsorption of a specific target analyte. Efforts to synthesize hollow nanomaterials have been met with great success, but the available synthesis methods still suffer from complicated steps, high costs, relatively harsh conditions, and the use of highly toxic substances. This paper summarizes the main types of hollow nanomaterials, their synthesis methods, and research progress on sample pretreatment technologies (solid-phase extraction, solid-phase microextraction, magnetic solid-phase extraction, and dispersive solid-phase extraction) and describes the challenges encountered in the synthesis of hollow nanomaterials. The applications and developments of hollow nanomaterials in sample pretreatment are also discussed.

Ansavaricin J, a New Heterocyclic Ring-Fused Streptovaricin from Gene stvP5-Deleted Mutant of Streptomyces spectabilis CCTCC M2017417.

A new ring-fused streptovaricin analogue, named ansavaricin J, was unprecedently isolated from the culture of the genetically modified strains ΔstvP5 which derived from Streptomyces spectabilis CCTCC M2017417. Its structure was elucidated via comprehensive spectroscopic analyses, including 1D- and 2D-NMR tests, and HR-ESI-MS data analysis. Notably, ansavaricin J and E represent the only two reported examples of heterocyclic ring-fused streptovaricins thus far, however, it only showed insignificant antibacterial activities against Staphylococcus aureus.

Multiple microRNAs control ecdysone signaling in the midgut of Spodoptera litura.

Metamorphosis is one of the most important physiological processes in insects. It is regulated by a serial of ecdysone cascade genes. Recently, lots of microRNAs (miRNAs) were investigated in insects; however, their function in metamorphosis is largely unknown. In the present study, the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis. A total of 101 miRNAs were identified, and 75 miRNAs were differentially expressed during the metamorphic process. The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs, and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets. Among them, miR-14-3p and its two targets (EcR and E75) were chosen for further validation. MiR-14-3p had higher expression level in the 6th instar larvae as compared with either the prepupae or pupae, which was opposite to that of both EcR and E75, two ecdysone cascade genes. Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p. Interestingly, the 3' untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes, including EcR, USP, E75, E74, E78, E93, Hr3, Hr4, Hr39, Krh1 and Ftzf1. Thus, different transcript variants of one ecdysone cascade gene could be regulated by the same miRNA. The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA. These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3ß/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

Expression, subcellular localization and protein-protein interaction of four isoforms of EcR/USP in the common cutworm.

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone (20E) induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.

miR-199a-5p inhibits monocyte/macrophage differentiation by targeting the activin A type 1B receptor gene and finally reducing C/EBPα expression.

miRNAs are short, noncoding RNAs that regulate expression of target genes at post-transcriptional levels and function in many important cellular processes, including differentiation, proliferation, etc. In this study, we observed down-regulation of miR-199a-5p during monocyte/macrophage differentiation of HL-60 and THP-1 cells, as well as human CD34(+) HSPCs. This down-regulation of miR-199a-5p resulted from the up-regulation of PU.1 that was demonstrated to regulate transcription of the miR-199a-2 gene negatively. Overexpression of miR-199a-5p by miR-199a-5p mimic transfection or lentivirus-mediated gene transfer significantly inhibited monocyte/macrophage differentiation of the cell lines or HSPCs. The mRNA encoding an ACVR1B was identified as a direct target of miR-199a-5p. Gradually increased ACVR1B expression level was detected during monocyte/macrophage differentiation of the leukemic cell lines and HSPCs, and knockdown of ACVR1B resulted in inhibition of monocyte/macrophage differentiation of HL-60 and THP-1 cells, which suggested that ACVR1B functions as a positive regulator of monocyte/macrophage differentiation. We demonstrated that miR-199a-5p overexpression or ACVR1B knockdown promoted proliferation of THP-1 cells through increasing phosphorylation of Rb. We also demonstrated that the down-regulation of ACVR1B reduced p-Smad2/3, which resulted in decreased expression of C/EBPα, a key regulator of monocyte/macrophage differentiation, and finally, inhibited monocyte/macrophage differentiation.

Cloning, expression and chitin-binding activity of two peritrophin-like protein genes in the common cutworm, Spodoptera litura.

Insect midgut secretes a semi-permeable peritrophic membrane (PM), which plays important roles in protecting the midgut and helping with food digestion. The lepidopteran larvae produce type 1 PM, which is degraded when insects develop into the metamorphic stages. However, the underlying mechanism is unclear. In the present study, two peritrophin-like proteins (peritrophin-57 and 37) were identified from the midgut expression sequence tag library and transcriptome of the common cutworm, Spodoptera litura. The temporal and spatial expression patterns and responses to the induction of 20-hydroxyecdysone (20E) and starvation were examined by real-time quantitative polymerase chain reaction according to their common sequence region. The chitin-binding activity was also studied using a competitor, calcofluor. The open reading frames are 1 554 and 1 020 bp, respectively. They shared four highly conserved peritrophin-A domains and were expressed only in the midgut rather than in the other tissues, including fat body, epidermis, Malpighian tube and hemolymph. Their transcriptional expression could only be detected at the larval stages rather than in eggs, prepupae, pupae and adults. The purified protein of peritrophin-37 bound to chitin in a dose-dependent manner. These results indicate that the two proteins are peritrophins, the structural components of PM. In addition, the messenger RNA levels of the two peritrophins were significantly down-regulated by 20E injection, whereas feeding/starvation had no effect on the expression. These findings suggest that the increase of 20E titer may be an important factor which controls the degradation of PM during metamorphosis.

De novo characterization of transcriptome and gene expression dynamics in epidermis during the larval-pupal metamorphosis of common cutworm.

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, ß-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and ß-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.

Altered diurnal variation and localization of clock proteins in the remnant kidney of 5/6 nephrectomy rats.

AIM: To investigate the localization and diurnal variation of clock proteins (BMAL1, PER2) and clock output protein (DBP) in the remnant kidney of 5/6 nephrectomy rats (STNx). METHODS: Male wistar rats were randomly divided into sham STNx group (Control) and STNx group. Rats were synchronized 12 weeks to the light: dark cycle 12:12 with light on from 07.00 hours (Zeitgeber time ZT 0). Kidneys were collected to detect the localization and expression rhythm of clock proteins (BMAL1, PER2 and DBP) every 4 h throughout the day by immunohistochemistry and Western blotting. RESULTS: Clock proteins showed diurnal rhythm in the kidney of the control. But diurnal rhythm of clock proteins changed in the STNx rats. Acrophase of BMAL1, DBP and PER2 advanced 4 h, respectively; mesor of clock proteins increased in the STNx rats. BMAL1 was located in endothelial cells of glomerulus and tubular interstitial vasculars, and it was also expressed in nucleus of tubular cells in cortex and medulla. PER2 was mainly expressed in proximal tubular cells at the juncture of cortex and medulla. DBP was widely expressed in the kidney. The localization of BMAL1 and PER2 were changed in remnant kidneys of the STNx group. CONCLUSION: The localization and diurnal variation of BMAL1, DBP and PER2 are changed in remnant kidney of 5/6 nephrectomy rats and are involved in diurnal rhythm of renal function.

High EGFR copy number predicts benefits from tyrosine kinase inhibitor treatment for non-small cell lung cancer patients with wild-type EGFR.

BACKGROUND: This study was designed to determine whether advanced non-small-cell lung cancer (NSCLC) patients with high copy number of epidermal growth factor receptor (EGFR) can benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs). METHODS: EGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) and EGFR mutations was tested using Luminex xTAG technology in 502 TKI-treated NSCLC patients. The association between both biomarkers and clinical benefit from EGFR-TKI were analyzed. RESULTS: EGFR FISH+and EGFR mutations were significantly associated with higher response rates (37.2% and 43.7%, respectively), superior progression-free survival (PFS) (FISH+, 11.2 months; hazard ratio [HR], 0.51; 95% CI, 0.42 to 0.62; p<0.001; mutation+, 11.7 months; HR, 0.37; 95% CI, 0.31 to 0.45; p<0.001) and overall survival (OS) (FISH+, 30.2 months; HR, 0.51; 95% CI, 0.40 to 0.65; p<0.001; mutation+, 30.2 months; HR, 0.45; 95% CI, 0.36 to 0.58; p<0.001). In patients with wild-type EGFR, EGFR FISH+correlated with longer PFS than EGFR FISH- status (4.4 months vs. 2.0 months; HR, 0.56; 95% CI, 0.41 to 0.75; p<0.001), so did amplification (5.0 months vs. 2.0 months; HR, 0.43; 95% CI, 0.24 to 0.76; p=0.003). However, FISH+had no association with improved PFS in EGFR-mutated patients (HR, 0.77; 95% CI, 0.57 to 1.03; p=0.076). CONCLUSIONS: A combined analysis of EGFR FISH and mutation is an effective predictor of EGFR-TKI therapy. Specifically, a high EGFR copy number may predict benefit from TKIs treatment for NSCLC patients with wild-type EGFR.

Reduced expression of Krüppel-like factor 17 is related to tumor growth and poor prognosis in lung adenocarcinoma.

Krüppel-like factor 17 (KLF17), a new member of the Krüppel-like factors (KLFs), has been reported to be a negative regulator of epithelial-mesenchymal transition (EMT) and metastasis in breast cancer. However, the biological role and clinical significance of KLF17 in lung adenocarcinoma has been less clear. In the present study, we showed that KLF17 expression was decreased in lung adenocarcinoma. Reduced expression of KLF17 was correlated significantly with a short survival time in patients with lung adenocarcinoma (P<0.0001). Moreover, KLF17 expression was an independent prognostic indicator for patients with lung adenocarcinoma. KLF17 expression level was correlated with the tumor stage (P=0.016) and tumor size (P=0.001) in lung adenocarcinoma. Overexpression of KLF17 inhibited cell growth in A549 and PC-9 cell lines. In conclusion, it is possible that KLF17 inhibits tumor growth in lung adenocarcinoma. The reduced expression of KLF17 is a valuable prognostic indicator for patients with lung adenocarcinoma, and KLF17 could be a novel target for treatment of lung adenocarcinoma.

PINCH protein expression in normal endometrium, atypical endometrial hyperplasia and endometrioid endometrial carcinoma.

BACKGROUND: Particularly interesting new cysteine-histidine rich protein (PINCH), as an adapter protein of the LIM family for signal transduction in the integrin and growth factor pathway, is upregulated in the stroma of several common types of cancers and involved in promoting tumor progression. In the present study, we examined PINCH expression in normal endometrium, atypical endometrial hyperplasia and endometrioid carcinoma, and further studied the relationships of PINCH expression with clinicopathological variables in cancer patients. METHODS: PINCH expression was examined by immunohistochemistry in 23 normal endometrial samples, 18 atypical endometrial hyperplasias and 48 endometrioid endometrial carcinomas. RESULTS: The PINCH expression in the stroma of cancer (71%) was significantly increased compared to either normal endometrium (17%, p < 0.0001) or atypical hyperplasia (39%, p = 0.017), along with 9 cancers that had stronger PINCH expressions at the invasive margin of the cancers compared to the inner cancers. PINCH expression in cancer was higher in the patients with hypertension (p = 0.041) and estrogen exposure time >30 years (p = 0.021). On the other hand, PINCH expression was not related to menopausal status, gravid status, blood sugar/lipid, family background of cancer, histological grade, myometrial invasion, cervical involvement, lymph nodal metastases, growth pattern, estrogen and progestogen receptors (p > 0.05). conclusion: The results suggest that PINCH seems to play a role, presently unknown, in the tumorigenesis and development of endometrial cancer that merits further study.

[Construction of prokaryotic expression vector for Trichomonas vaginalis silent information regulator 2 and its expression].

Total RNA was isolated from Trichomonas vaginalis and Tv-Sir2-like cDNA was amplified by RT-PCR and cloned into pGEM-T Easy plasmid. A fragment of Tv-Sir2-like cDNA was subcloned into the expression vector pET-41b and expressed in E.coli BL21 with induction of IPTG. The full-length of Tv-Sir2-like cDNA was cloned and sequenced. The prokaryotic expression system of pET-41b/Tv-Sir2-like was constructed. The fusion protein of Tv-Sir2-like was expressed in E. coli BL21, occupying 30% of the total bacterial protein after being induced by IPTG for 5 h. SDS-PAGE analysis showed that the fusion protein was about Mr 59000. The recombinant protein of Tv-Sir2-like is efficiently expressed in E. coli BL21.