Reciprocal inhibition between TP63 and STAT1 regulates anti-tumor immune response through interferon-γ signaling in squamous cancer.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.

Comparative evaluation of four Lycium barbarum cultivars on NaIO3-induced retinal degeneration mice via multivariate statistical analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Lycium barbarum L. (goji berry) is a traditional Chinese medicine and is often used to improve vision. While various goji cultivars may differentially treat retinal degeneration, however their comparative effectiveness remains unclear. AIM OF THE STUDY: To evaluate the protective effects of four goji cultivars on NaIO3-induced retinal degeneration mouse model and identify the most therapeutically potent cultivar. MATERIALS AND METHODS: The principal compounds in the extracts of four goji cultivars were characterized by UPLC-Q-TOF/MS. A retinal degeneration mouse model was established via NaIO3 injection. Dark-light transition and TUNEL assays were used to assess visual function and retinal apoptosis. The levels of antioxidative, inflammatory, and angiogenic markers in serums and eyeballs were measured. Hierarchical cluster analysis, principal component analysis and partial least squares-discriminant analysis were used to objectively compare the treatment responses. RESULTS: Sixteen compounds were identified in goji berry extracts. All goji berry extracts could reverse NaIO3-induced visual impairment, retinal damage and apoptosis. The samples from the cultivar of Ningqi No.1 significantly modulated oxidative stress, inflammation, and vascular endothelial growth factor levels, which are more effectively than the other cultivars based on integrated multivariate profiling. CONCLUSION: Ningqi No.1 demonstrated a stronger protective effect on mouse retina than other goji cultivars, and is a potential variety for further research on the treatment of retinal degeneration.

Establishing mouse and human oral esophageal organoids to investigate the tumor immune response.

Organoid culture systems are very powerful models that recapitulate in vivo organ development and disease pathogenesis, offering great promise in basic research, drug screening and precision medicine. However, the application of organoids derived from patients with cancer to immunotherapeutic research is a relatively untapped area. Esophageal cancer is one of the most lethal malignancies worldwide, including two major pathological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC shares many biological and genomic features with oral squamous cell cancers. Herein, we provide a versatile protocol for the establishment and maintenance of oral and esophageal organoid cultures derived from both murine and human samples. We describe culture conditions for organoids derived from normal tongue, esophagus and gastroesophageal junction, esophageal cancer and Barrett's esophagus. In addition, we establish an ex vivo model by co-culturing patient tumor-derived organoids and autologous CD8+ T lymphocytes to assess CD8+ T cell-mediated tumor killing. Our protocol can also be modified for organoid establishment from other squamous epithelia and carcinomas. The co-culture model can serve as a template for studies of other tumor-immune cell interactions and the efficacy of immune checkpoint blockade therapy.

Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10-5 and 8.7× 10-7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

Characterization of the complete mitochondrial genome of a barklouse, Lepinotus sp. (Psocodea: Trogiomorpha: Trogiidae).

Barklice in the genus Lepinotus (Psocoptera: Trogiidae) are small, soft-bodied stored-product pests that are difficult to control. We sequenced and annotated the mitochondrial (mt) genome of Lepinotus sp. The mt genome of Lepinotus sp. is 16,299 bp in size with 74.4% A + T content. The gene order was highly conserved in some of the Trogimorpha barklice. Two types of tandem repeat units were identified in CR of Lepinotus sp. The phylogenetic analysis showed that Trogiidae species was the sister group to Lepidopsocidae barklice, and the suborder Troctomorpha was polyphyletic.

Antioxidant Enzymes and Heat Shock Protein Genes from Liposcelis bostrychophila Are Involved in Stress Defense upon Heat Shock.

Psocids are a new risk for global food security and safety because they are significant worldwide pests of stored products. Among these psocids, Liposcelis bostrychophila has developed high levels of resistance or tolerance to heat treatment in grain storage systems, and thus has led to investigation of molecular mechanisms underlying heat tolerance in this pest. In this study, the time-related effects of thermal stress treatments at relatively high temperatures on the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST) and malondialdehyde (MDA), of L. bostrychophila were determined. Thermal stress resulted that L. bostrychophila had a significantly higher MDA concentration at 42.5 °C, which indicated that the heat stress increased lipid peroxidation (LPO) contents and oxidative stress in this psocid pest. Heat stress also resulted in significant elevation of SOD, CAT and GST activities but decreased POD activity. Our data indicates that different antioxidant enzymes contribute to defense mechanisms, counteracting oxidative damage in varying levels. POD play minor roles in scavenging deleterious LPO, while enhanced SOD, CAT and GST activities in response to thermal stress likely play a more important role against oxidative damage. Here, we firstly identified five LbHsps (four LbHsp70s and one LbHsp110) from psocids, and most of these LbHsps (except LbHsp70-1) are highly expressed at fourth instar nymph and adults, and LbHsp70-1 likely presents as a cognate form of HSP due to its non-significant changes of expression. Most LbHsp70s (except LbHsp70-4) are significantly induced at moderate high temperatures (<40 °C) and decreased at extreme high temperatures (40-45 °C), but LbHsp110-1 can be significantly induced at all high temperatures. Results of this study suggest that the LbHsp70s and LbHsp110 genes are involved in tolerance to thermal stress in L. bostrychophila, and antioxidant enzymes and heat shock proteins may be coordinately involved in the tolerance to thermal stress in psocids.

IgG plasma cells initiate changes in the protein C system in mouse ulcerative colitis through CD14+CD64+ macrophage activation.

BACKGROUND: Inhibition of the protein C system (PCS) might be one of the mechanisms of ulcerative colitis (UC). OBJECTIVES: The aim of the study was to explore the role of IgG plasma cells in changes in the PCS in UC. MATERIAL AND METHODS: Dextran sulfate sodium (DSS) was chosen to induce mouse UC. Inflammation was assessed using hematoxylin & eosin (H&E) staining and immunofluorescence. The profiling of colonic plasma cells and macrophages from colitis mice was analyzed with flow cytometry. After stimulation of macrophages with IgG type immune complex (IgG-IC), western blot was used to determine tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) protein levels. After co-incubation of colonic mucosa microvascular endothelial cells (MVECs) with TNF-α or IL-6, mitogen-activated protein kinase (MAPK) expression was detected. RESULTS: The DSS-colitis mice showed higher inflammatory indexes (p < 0.05 or p < 0.01), accompanied by greater infiltration of CD38+IgG+ plasma cells (p < 0.01), CD14+CD64+ macrophages (p < 0.01) and IgG-IC than healthy mice. Enhancement of TNF-α and IL-6 protein expression was demonstrated in this subset of macrophages when stimulated by IgG-IC (p < 0.01). After MVECs were incubated with TNF-α or IL-6, the expression of ß-arrestin1, pP38 MAPK and pJNK MAPK exhibited an increase (p < 0.05 or p < 0.01), but downregulation of endothelial protein C receptor (EPCR) expression was observed (p < 0.05 or p < 0.01); this inhibition of EPCR expression was reversed by SB203580, SP600125 or U0126 (p < 0.05 or p < 0.01). In addition, changes in activated protein C (APC) presented results similar to those for EPCR expression (p < 0.05 or p < 0.01). CONCLUSIONS: These results reveal that the PCS is inhibited during UC processing. There is a possibility that the interaction between IgG plasma cells and CD14+CD64+ macrophages, as well as further secretion of cytokines from CD14+CD64+ macrophages by the formation and stimulation of IgG-IC, subsequently influence MVECs through the ß-arrestin-MAPK pathway. Enhancement of PCS activity may represent a novel approach for treating UC.

Authenticity of pulmonary Lophomonas blattarum infection: A case report.

Pulmonary protozoal infections are rare. A 28-year-old woman was admitted to hospital with chief complains of cough, sputum, and dyspnea. The clinical laboratory tests for blood revealed an increased eosinophil percentage of 31.3% and significantly elevated total IgE. The chest computed tomography scan revealed that bilateral bronchial walls were thickening, accompanied with patchy spots scattered throughout bilateral lungs. A suspected multiflagellated protozoan was observed under a light microscope. But some different features were observed by electron microscopy, such as the orientation of flagella and nucleus. Besides, both bronchoalveolar lavage fluid and bronchoscopic brush smears underwent Gram staining and Pap staining, which revealed that numerous respiratory ciliated cells were scattered or accumulated in the sample. Finally, she was diagnosed with eosinophil pneumonia. Metronidazole, bronchodilators, and mucolytics were taken for 5 d and symptoms and pulmonary ventilation function improved. We herein report a case of chronic eosinophilic pneumonia, which was misdiagnosed as multiflagellated protozoan infection, and it is suggested that reliable diagnosis approaches are necessary, rather than clinical symptoms and morphological features.

Phenotypes, antioxidant responses, and gene expression changes accompanying a sugar-only diet in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

BACKGROUND: Diet composition (yeast:carbohydrate ratio) is an important determinant of growth, development, and reproduction. Recent studies have shown that decreased yeast intake elicits numerous transcriptomic changes and enhances somatic maintenance and lifespan, which in turn reduces reproduction in various insects. However, our understanding of the responses leading to a decrease in yeast ratio to 0% is limited. RESULTS: In the present study, we investigated the effects of a sugar-only diet (SD) on the gene expression patterns of the oriental fruit fly, Bactrocera dorsalis (Hendel), one of the most economically important pests in the family Tephritidae. RNA sequencing analyses showed that flies reared on an SD induced significant changes in the expression levels of genes associated with specific metabolic as well as cell growth and death pathways. Moreover, the observed upregulated genes in energy production and downregulated genes associated with reproduction suggested that SD affects somatic maintenance and reproduction in B. dorsalis. As expected, we observed that SD altered B. dorsalis phenotypes by significantly increasing stress (starvation and desiccation) resistance, decreasing reproduction, but did not extend lifespan compared to those that received a normal diet (ND) regime. In addition, administration of an SD resulted in a reduction in antioxidant enzyme activities and an increase in MDA concentrations, thereby suggesting that antioxidants cannot keep up with the increase in oxidative damage induced by SD regime. CONCLUSIONS: The application of an SD diet induces changes in phenotypes, antioxidant responses, and gene expressions in B. dorsalis. Previous studies have associated extended lifespan with reduced fecundity. The current study did not observe a prolongation of lifespan in B. dorsalis, which instead incurred oxidative damage. The findings of the present study improve our understanding of the molecular, biochemical, and phenotypic response of B. dorsalis to an SD diet.

[Role of IgG plasma cells in the change of protein C system in ulcerative colitis].

The present study is designed to explore the role of plasma cells in the change of protein C system (PCS) in ulcerative colitis (UC). Dextran sulfate sodium (DSS, 4% in concentration) was used to induce mouse UC model. The plasma cells and the type of immune complex in colon were observed by immunofluorescence. The amount and type of plasma cells separated from colonic mucosal lamina propria were detected by flow cytometry using anti-CD54+CD38+ and IgA/M/G antibodies, respectively. After stimulation of macrophages by IgG type immune complex, TNF-α and IL-6 levels were evaluated by ELISA. After co-incubation of microvascular endothelial cells with TNF-α or IL-6, the expressions of endothelial protein C receptor (EPCR) and thrombomodulin (TM), and the activity of activated protein C (APC) were examined. As the results showed, the IgG type plasma cells infiltration and the quantity of IgG type immune complex were increased in DSS group in comparison with control group. After incubation with IgG type immune complex, the levels of TNF-α and IL-6 in the supernatant of macrophages were increased (P < 0.01) in a concentration-dependent manner. Meanwhile, after incubation with TNF-α or IL-6, the expressions of EPCR and TM in the microvascular endothelial cells were decreased (P < 0.05 or P < 0.01), while the activity of APC was reduced (P < 0.05 or P < 0.01). These results suggested that the quantity of IgG type plasma cells increases in UC and forms immune complexes, which affect the secretion of cytokines from macrophage, thereby affecting the function of endothelial cells and finally inhibiting PCS in UC. Therefore, plasma cell may be a novel target for the treatment of UC.

The mitochondrial genome of booklouse, Liposcelis sculptilis (Psocoptera: Liposcelididae) and the evolutionary timescale of Liposcelis.

Bilateral animals are featured by an extremely compact mitochondrial (mt) genome with 37 genes on a single circular chromosome. To date, the complete mt genome has only been determined for four species of Liposcelis, a genus with economic importance, including L. entomophila, L. decolor, L. bostrychophila, and L. paeta. They belong to A, B, or D group of Liposcelis, respectively. Unlike most bilateral animals, L. bostrychophila, L. entomophila and L. paeta have a bitipartite mt genome with genes on two chromosomes. However, the mt genome of L. decolor has the typical mt chromosome of bilateral animals. Here, we sequenced the mt genome of L. sculptilis, and identified 35 genes, which were on a single chromosome. The mt genome fragmentation is not shared by the D group of Liposcelis and the single chromosome of L. sculptilis differed from those of booklice known in gene content and gene arrangement. We inferred that different evolutionary patterns and rate existed in Liposcelis. Further, we reconstructed the evolutionary history of 21 psocodean taxa with phylogenetic analyses, which suggested that Liposcelididae and Phthiraptera have evolved 134 Ma and the sucking lice diversified in the Late Cretaceous.

[Study of the change and role of protein C system in ulcerate colitis].

Hypercoagulable state and thrombosis are major lethal causes of ulcerate colitis (UC). The aim of the present study is to explore the change and role of protein C (PC) system in UC thrombosis. 4% dextran sulfate sodium (DSS) was used to induce the UC model, and the body weight, the length of colon, and the weight of spleen were measured after intake of DSS as drinking water for 1 week. The macroscore and microscore were examined. The quantity of macrophage in colon smooth muscle was observed by immunofluorescence, and TNF-α and IL-6 levels in plasma were evaluated by ELISA. Intravital microscopy was applied to observe colonic mucosal microvascular circulation, activities of PC and protein S (PS) were determined by immunoturbidimetry, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) expressions were detected by immunohistochemistry. In vitro, TNF-α and IL-6 levels were tested in supernatant of macrophage separated from colonic tissue. After stimulation of mouse colonic mucosa microvascular endothelial cells by TNF-α and IL-6 respectively, the activities of PC, PS, activated protein C (APC) were evaluated, and the expressions of EPCR and TM were detected by Western blotting. The results revealed that compared with control, the DSS mouse showed weight loss (P < 0.05), a shortened colon (P < 0.05), and swelled spleen (P < 0.05), accompanied by higher histological score (P < 0.05), as well as infiltration of macrophages, elevated TNF-α and IL-6 levels in plasma (P < 0.01). The intravital microscopy results revealed that compared with control, DSS mice showed significantly enhanced adhesion of leukocytes and colonic mucosal microvascular endothelial cells (P < 0.01), meanwhile, decreased activity of PC and PS in plasma (P < 0.01 or P < 0.05), and down-regulated expression of EPCR (P < 0.01). The degree of inflammation was negatively correlated with the PC activity. In vitro, TNF-α and IL-6 levels were increased in the supernatant of macrophages from DSS mice colonic tissue (P < 0.05), and after incubation of TNF-α or IL-6 with colonic mucosal microvascular endothelial cells, the APC activity was decreased (P < 0.05 or P < 0.01), and expression of EPCR was down regulated (P < 0.05). These results suggest that PC system is inhibited in UC mouse. Presumably, the mechanism may be due to the secretion of cytokines from macrophages and subsequential influence on the function of endothelia cells. Furthermore, enhancement of PC system activity may serve as a new strategy for the treatment of UC.

Phenoloxidase and its zymogen are required for the larval-pupal transition in Bactrocera dorsalis (Diptera: Tephritidae).

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and are considered important enzymes in the insect development process. In the present study, we report the developmental stage and tissue-specific expression patterns of BdPPO1 and PO activity from Bactrocera dorsalis. The results showed that the activity of PO and its zymogen expression were closely related to the development of B. dorsalis during the larval-pupal transition, particularly in the integument. Additionally, biochemical characterization showed that PO from different developmental stages and tissues all had maximum activity at pH 7.5 and 37°C. After feeding a metal ion-containing artificial diet, the activity of PO and expression of BdPPO1 were significantly increased, indicating that PO was a metalloprotein and it could be activated by Zn2+, Mg2+, Ca2+, and Cu2+. The functional analysis showed that the expression of BdPPO1 could be regulated by 20-hydroxyecdysone (20E) after injection. Furthermore, injection of the double-stranded RNA of BdPPO1 into the 3rd instar larvae significantly reduced mRNA levels after 24 h and 48 h, and resulted in a lower pupation rate and abnormal phenotype. These results expand the understanding of the important role of PO and its zymogen in the growth of B. dorsalis.

A bird's eye view of anisatin induced convulsive seizures in brain by a (1)H NMR based metabolic approach.

Anisatin is the main convulsant component in plants of the genus Illicium, many of which are important spices or folk medicines. The neurotoxicity of anisatin has been widely investigated, mainly focusing on its action on the γ-amino butyrate (GABA) system; however, little is known about the metabolic alterations that it causes. In this study, a NMR-based metabolomic approach was performed on the extracts of cortexes and cerebellums of mice administered with anisatin to explore the metabolic events associated with its intoxication. Orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed many differential metabolites that indicated metabolic disturbance in neurotransmission and neuromodulation (GABA, glutamate, glutamine, and taurine), stress of reactive oxygen species (ROS) (ascorbate, phosphatidylcholine, choline, and ethanolamine), energy metabolism (NAD(+)i.e., nicotinamide-adenine dinucleotide, lactate, citrate, fumarate, creatine/phosphocreatine, and creatinine), amino acid metabolism (leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, alanine, threonine, and glycine) and nucleic acid metabolism (NAD(+), nicotinamide/niacinamide, adenosine, and guanosine). This pilot metabolomic study on anisatin intoxication should help to develop a holistic view of convulsive seizures induced by anisatin, and provide a better understanding of the mechanisms.

The effect of dietary restriction on longevity, fecundity, and antioxidant responses in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

Recent studies in fruit flies have imposed dietary restriction (DR) by diluting yeast and have reported increased lifespan as the yeast-to-sugar ratio decreased. In this study, the effects of DR on the lifespan of Bactrocera dorsalis were investigated using constant-feeding diets with different yeast:sugar ratios and an intermittent-feeding diet in which flies ate every sixth day. Antioxidant enzyme activities and the malondialdehyde concentration were also measured in virgin females under constant-feeding DR protocols to investigate their relationships with lifespan. The results showed that B. dorsalis lifespan was significantly extended by DR, and carbohydrate-enriched diet may be important for lifespan-extension. Female flies lived significantly longer than males at all dietary levels under both feeding regimes, indicating no interaction between diet and sex in determining lifespan. Antioxidant enzyme activities increased with the amount of yeast increased in the diets (0-4.76%) between starvation and DR treatments, indicating that the antioxidants may have influences in determining lifespan in B. dorsalis under starvation and DR treatments. However, antioxidants cannot keep up with increased oxidative damage induced by the high yeast diet (25%). These results revealed that the extension of lifespan by DR is evolutionarily conserved in B. dorsalis and that yeast:sugar ratios significantly modulate lifespan in this species.

Simonols A and B, two novel sesqui-neolignans from the fruits of Illicium simonsii.

Two new sesqui-neolignans with novel conjugation way, simonol A (1), featuring a unique motif of a 5,5-dihydro-pyran with a hemiketal carbon, while simonol B (2) possessing two dihydronfuran rings in the same direction, were isolated from the ethanol extract of the fruits of Illicium simonii. Their structures were elucidated by spectroscopic methods including 1D and 2D NMR, HRESIMS, and calculation of electronic circular dichroism (ECD) using density functional theory (DFT). The two isolates were evaluated for their inhibitory activities against the growth of four lines of human cancer cells (NCI-H460, SMMC-7721, MCF-7, BGC-823): 1 showed strong activities comparable to 5-Fluorouracil, and 2 to a less content. In addition, plausible biosynthetic routes for the two compounds were also proposed.

Limonoids from the fruits of Aphanamixis polystachya (Meliaceae) and their biological activities.

Seven new prieurianin-type limonoids, aphapolynins C-I (1-7), and a new aphanamolide-type limonoid, aphanamolide B (8), along with seventeen known compounds, were isolated from the fruits of Aphanamixis polystachya. The structures of these compounds were established on the basis of spectroscopic studies. The absolute configurations were determined by combination of electronic circular dichroism (ECD) calculation, CD exciton chirality method, and single crystal X-ray diffraction. All these isolates were evaluated for their cytotoxicities against three human cancer cell lines, for their inhibitory effects on lipopolysaccharide (LPS) induced RAW264.7 murine macrophages, and for their fungicidal, herbicidal, and insecticidal activities. Compounds 1, 14, 16, and 17 exhibited significant fungicidal activities; 1 and 25 in particular showed good insecticidal activities. The α,ß-unsaturated lactone and 14,15-epoxy ring moieties were essential for the insecticidal activity.

Glaucogenin E, a new C21 steroid from Cynanchum stauntonii.

Glaucogenin E (1), a new C(21) steroid sapogenin, along with three known ones (2-4) were isolated from the rhizomes of Cynanchum stauntonii (Decne.) Schltr. ex Levl. Their structures were established mainly by the spectroscopic analysis, including 2D NMR. All the isolated compounds were evaluated for their cytotoxicity against human cancer cell lines HeLa, Bel-7402, SGC-7901 and BGC-823.

Three new phenolic glucosides from the roots of Rheum palmatum.

A novel naphthalene glucoside, rheumone A (1), with an unprecedented skeleton containing a seven-membered lactone, and two new compounds, 1-O-phloroglucinyl-2-O-galloyl-6-O-cinnamoyl-ß-D-glucoside (2) and chrysophanol 1-O-ß-D-(6'-O-malonyl)glucoside (3), together with three known compounds (4-6) were isolated from the roots of Rheum palmatum. Their structures were elucidated mainly by spectroscopic analysis. These compounds were evaluated in vitro for their cytotoxicities towards human hepatocellular cancer cell lines Bel-7402 and Bel-7402/5Fu, and human gastric carcinoma cell line BGC-823. None of them showed cytotoxicity with IC(50) far beyond 50 µM.