Penicillium fungi mediate behavioral responses of the yellow peach moth, Conogethes punctiferalis (Guenée) to apple fruits via altering the emissions of host plant VOCs.

Plant-associated microbes have been reported as important but overlooked drivers of plant-herbivorous insect interactions. Influence of plant-associated microbes on plant-insect interactions is diverse, including beneficial, detrimental, and neutral. Here, we determined the effects of three Penicillium fungi, including Penicillium citrinum, Penicillium sumatrense, and Penicillium digitatum, on the oviposition selection and behavior of the yellow peach moth (YPM), Conogethes punctiferalis (Guenée). Compared with fungi noninfected apples (NIA), mechanically damaged apples (MDA), and P. citrinum in potato dextrose agar medium (PC), the oviposition selection and four-arm olfactometer experiments both showed that mated YPM females preferred to P. citrinum-infected apples (PCA). For P. sumatrense or P. digitatum, we also found that mated YPM females preferred to P. sumatrense-infected apples (PSA) or P. digitatum-infected apples (PDA), respectively. Among three Penicillium fungi-infected apples, the selection rates including oviposition and olfactometer behavior of mated YPM females on PDA were both higher than those on PSA and PCA. Further analyses of host plant volatile organic compounds (VOCs) by GC-MS showed that the absolute contents of ethyl hexanoate and (Z, E)-α-farnesene in PCA, PSA, and PDA were all higher than those in NIA, and a total of 16 novel VOCs were detected in fungi-infected apples (PCA, PSA, and PDA), indicating that fungi infection changed the components and proportions of apple VOCs. Taken together, three Penicillium fungi play significant roles in mediating the host selection of YPMs via altering the emissions of VOCs. These findings will be beneficial for developing formulations for field trapping of YPMs in the future.

Purification and Characterization of a Thermo- and pH-Stable Laccase From the Litter-Decomposing Fungus Gymnopus luxurians and Laccase Mediator Systems for Dye Decolorization.

An extracellular laccase (GLL) was purified from fermentation broth of the litter-decomposing fungus Gymnopus luxurians by four chromatography steps, which resulted in a high specific activity of 118.82 U/mg, purification fold of 41.22, and recovery rate of 42.05%. It is a monomeric protein with a molecular weight of 64 kDa and N-terminal amino acid sequence of AIGPV TDLHI, suggesting that GLL is a typical fungal laccase. GLL demonstrated an optimum temperature range of 55°C-65°C and an optimum pH 2.2 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It displayed considerably high thermostability and pH stability with about 63% activity retained after 24 h at 50°C, and 86% activity retained after 24 h at pH 2.2, respectively. GLL was significantly enhanced in the presence of K+, Na+, and Mg2+ ions. It demonstrated K m of 539 µM and k cat /K m of 140 mM-1⋅s-1 toward ABTS at pH 2.2 and 37°C. Acetosyringone (AS) and syringaldehyde (SA) were the optimal mediators of GLL (0.4 U/ml) for dye decolorization with decolorization rates of about 60%-90% toward 11 of the 14 synthetic dyes. The optimum reaction conditions were determined to be mediator concentration of 0.1 mM, temperature range of 25°C -60°C, and pH 4.0. The purified laccase was the first laccase isolated from genus Gymnopus with high thermostability, pH stability, and effective decolorization toward dyes, suggesting that it has potentials for textile and environmental applications.

An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01.

A novel laccase was purified from fermentation broth of white rot fungus Trametes sp. LAC-01 using an isolation procedure involving three ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and one gel-filtration step. The purified enzyme (TSL) was proved as a monomeric protein with a Mr of 59kDa based on SDS-PAGE and FPLC. Partial amino acid sequences were obtained by LC-MS/MS sharing considerably high sequence similarity with that of other laccases. It possessed optimal pH of 2.6 and temperature of 60°C using ABTS as the substrate. The Km of the laccase toward ABTS was estimated to 30.28µM at pH 2.6 and 40°C. TSL manifested considerably high oxidizing activity toward ABTS, but was avoid of degradative activity toward benzidine, caftaric acid, etc. It was effective in the decolorization of phenolic dyes - Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24h of incubation. Adjunction of Cu(2+) with the final concentration of 2.0mmol/L significantly activated laccase production with a steady high level of 275.8-282.2U/mL in 96-144h. The high yield and short production period makes Trametes sp. LAC-01 and TSL potentially useful for industrial and environmental application and commercialization.

Raltitrexed induces mitochondrial­mediated apoptosis in SGC7901 human gastric cancer cells.

Raltitrexed is a specific inhibitor of thymidylate synthase (TS), which has been considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In the present study, the apoptosis mechanisms of raltitrexed in SGC7901 human gastric cancer cells were investigated. The cytotoxic activity of raltitrexed on SGC7901 cells was determined by cell counting kit-8 (CCK-8) assay. The CCK­8 assay indicated that raltitrexed inhibits SGC7901 cell growth in a dose- and time-dependent manner. The morphological changes were observed by fluorescent microscopy, and characteristic morphological changes, including nuclear shrinkage and apoptotic bodies, were observed following Hoechst 33258 staining. The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species (ROS) were measured by flow cytometry. The analysis revealed that raltitrexed exerted a growth inhibitory effect by inducing time-dependent apoptosis and cell-cycle arrest at the G0/G1 phase. In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Raltitrexed­induced caspase­3­dependent apoptosis was identified using a caspase-3 activity assay and pretreatment with the caspase-3 inhibitor, Ac­DEVD­CHO (sequence, Ac-Asp-Glu-Val-Asp-CHO). The activity of caspase-3 was analyzed with a spectrometer. The protein expression levels of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and TS were examined by western blot and the mRNA expression level of TS was detected by quantitative polymerase chain reaction. The analysis revealed that the protein levels of Bax, cytochrome c and cleaved caspase­3 were significantly increased by raltitrexed, while Bcl-2 expression levels were reduced. Furthermore, raltitrexed increased the expression of the TS protein and mRNA in a time­dependent manner. These results indicate that raltitrexed induces the apoptosis of SGC7901 cells through the caspase­3­dependent mitochondrial signaling pathway and upregulates the expression of the TS protein and mRNA.

Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation.

Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.

Proteomic analysis of peach endocarp and mesocarp during early fruit development.

The development of the stone and formation of peach (Prunus persica) fruit were explored in this work using a proteomic approach. Sixty-eight proteins with different expression patterns were identified in both the endocarp and mesocarp during early fruit development (from 28 to 59 days after flowering) and the majority were involved in primary or secondary metabolism. In contrast to most proteins associated with primary metabolism in the endocarp, whose expression is down-regulated, expression of pyruvate dehydrogenase (PDH) unexpectedly increased exponentially. Moreover, its expression pattern was linearly positively correlated with the exponentially growing lignin content (R = 0.940), which suggests that PDH may play a role in endocarp lignification. Our data also revealed different spatiotemporal expressions of enzymes involved in the lignin and flavonoid pathways that provided proteome-level evidence to support the hypothesis that these two pathways are competitive during endocarp development. In addition, we observed endocarp-specific oxidative stress and propose that it may act as a stimulating factor in activating lignification and subsequent programmed cell death in the endocarp.

Serological thymidine kinase 1 is a biomarker for early detection of tumours--a health screening study on 35,365 people, using a sensitive chemiluminescent dot blot assay.

Serological thymidine kinase 1 (STK1) is a reliable proliferation marker for prognosis, monitoring tumour therapy, and relapse. Here we investigated the use of STK1 in health screening for early detection of pre-malignant and malignant diseases. The investigation was based on 35,365 participants in four independent health screening studies in China between 2005-2011. All participants were clinically examined. The concentration of STK1 was determined by a sensitive chemiluminescent dot blot ECL assay. The ROCvalue of the STK1 assay was 0.96. At a cut-off STK1 value of 2.0 pM, the likelihood (+) value was 236.5, and the sensitivity and the specificity were 0.78 and 0.99, respectively. The relative number of city-dwelling people with elevated STK1 values (≥2.0 pM) was 0.8% (198/26,484), while the corresponding value for the group of oil-field workers was 5.8% (514/8,355). The latter group expressed significantly higher frequency of refractory anaemia, fatty liver, and obesity, compared to the city dwellers, but no cases of breast hyperplasia or prostate hyperplasia. Furthermore, people working in oil drilling/oil transportation showed higher STK1 values and higher frequency of pre-malignancies and benign diseases than people working in the oil-field administration. In the STK1 elevated group of the city-dwelling people, a statistically significantly higher number of people were found to have malignancies, pre-malignancies of all types, moderate/severe type of hyperplasia of breast or prostate, or refractory anaemia, or to be at high risk for hepatitis B, compared to people with normal STK1 values (<2.0 pM). No malignancies were found in the normal STK1 group. In the elevated STK1 group 85.4% showed diseases linked to a higher risk for pre-/early cancerous progression, compared to 52.4% of those with normal STK1 values. Among participants with elevated STK1 values, 8.8% developed new malignancies or progress in their pre-malignancies within 5 to 72 months, compared to 0.2% among people with normal STK1 values. People who showed elevated STK1 values were at about three to five times higher risk to develop malignancies compared to a calculated risk based on a cancer incidence rate of 0.2-0.3%. We conclude that serological TK1 protein concentration is a reliable marker for risk assessment of pre/early cancerous progression.

mRNA expression of chemokine receptors on peripheral blood mononuclear cells and correlation with clinical features in systemic lupus erythematosus patients.

OBJECTIVE: To investigate the expressions of chemokine receptors and interleukin (IL) receptors on the peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and their correlations with clinical features as well as SLE disease activity index (SLEDAI). METHODS: The mRNA expressions of chemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CXCR3, CXCRS, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis. RESULTS: The level of CCR5 mRNA in SLE patients (including active and inactive SLE) was significantly higher than that in healthy controls (P < 0.05), and there was no significant difference between active and inactive patients in this respect (P > 0.05). CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others (except for CCR8, CXCR3, and IL-10R) in active SLE patients were significantly higher than those in both inactive SLE patients and healthy controls (all P < 0.05). There were positive correlations between SLEDAI and CCR2 (r = 0.424, t = 4.313, P < 0.001), CCR3 (r = 0.518, t = 5.410, P < 0.001), CCR4 (r = 0.376, t = 3.851, P < 0.001), CCR6 (r = 0.457, t = 4.513, P < 0.001), CXCR5 (r = 0.455, t = 4.629, P < 0.001), CX3CR1 (r = 0.445, t = 4.523, P < 0.001), as well as XCR1 (r = 0.540, t = 5.445, P < 0.001). And CCR5 mRNA expression level was positively correlated with IL-4R mRNA (r = 0.313, t = 2.353, P < 0.05). The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CR1, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity (r = 0.426, t = -2.155, P < 0.05). CONCLUSION: Increased expressions of CCR5 and CX3CR1 on PBMCs may be indicators in clinical survey for SLE.

[Effects of exogenous betaine on physiological responses of peach tree under water stress].

With four-year-old potted Prunus persica L. cv. Qingfeng as test material, this paper studied the change pattern of its leaf betaine content under water stress, and its physiological responses under effects of foliage-spraying exogenous betaine. The results showed that under normal water supply, the betaine content in Qingfeng' s leaf was 75.9-80.5 microg x g(-1) FM, which was increased with increasing water stress, and up to 278.9 microg x g(-1) FM on the 16th day after cutting off the water supply. The leaf plasma membrane permeability was 8.06% - 8.61% under normal water supply, but increased to 28.62% under water stress. When 100 and 500 mg x L(-1) of betaine were applied exogenously, the plasma membrane permeability was 26.25% and 21.79% after 16 days, respectively. The hydrogen peroxide (H2O2) content increased from 27.2-32.5 micromol g(-1) FM to 76.4 micromol x g(-1) FM in the course of water stress, and decreased to 73.2 and 68.5 micromol x g(-1) FM after spraying 100 and 500 mg betaine x L(-1), respectively. During the period of intensified water stress, the peak value of ascorbate peroxidase (AsA-POD) activity was 0.435 mg x g(-1) FM, and up to 0.490 mg x g(-1) FM when treated with exogenous betaine. When the peach tree was subjected to water stress, the contents of free proline and soluble sugar accumulated dramatically, but produced on approximately decrease in 500 mg x L(-1) endogenous betaine application on the 16th day which was slightly less than that of control and 100 mg x L(-1) betaine application. There was a gradual decline in the content of soluble protein under water stress, and an increment of 20. 3% was observed when betaine was applied exogenously. These results strongly suggested that foliage-spraying exogenous betaine could increase the drought resistance of peach tree through decreasing its leaf plasma membrane permeability and H2O2, free proline and soluble sugar contents and increasing its leaf AsA-POD activity and soluble protein content.

[Phenomena and pathological significances of the methylated p16 promotor in DNA derived from plasma and blood cells of patients with systemic lupus erythematosus].

OBJECTIVE: To detect the methylation status of p16 gene promotor in DNA derived from plasma and blood cells of patients with systemic lupus erythematosus (SLE) , and it's relationship with clinical symptoms. METHODS: p16 promotor methylation in plasma and peripheral blood cells (PBCs) DNA were simultaneously detected with the methylation specific PCR (MSP) method in 24 active SLE patients, 21 inactive SLE patients, as well as 20 healthy controls. RESULTS: In the plasma DNA, p16 gene methylation ratio (MP%) was higher in SLE patients than in the healthy controls (64.4% vs. 5.0%, P < 0.05). MP% in the active SLE patients was significantly higher than that in the inactive SLE patients (83.3% vs. 42.9%, P < 0.05). In the PBCs, p16 gene methylation ratio (MC%) in the healthy controls was significantly higher than that in SLE (80.0% vs. 48.9%, P < 0.05). MC% in the active SLE patients (29.2%) was the lowest among three groups. There was no significant difference between the inactive SLE patients and healthy controls (71.4% vs. 80.0%, P > 0.05). Each patient could be judged as one of the four methylation patterns: MP/MC, UP/MC (UP: unmethylated plasma p16) , MP/UC (UC: unmethylated PBCs p16) , and UP/UC. The ratios of MP/ MC and UP/UC were similar between the active and inactive SLE patients. However, different distributions of other two patterns were found in the active and inactive SLE patients as UP/MC 4.2% vs. 42.9% (P <0.05) and MP/UC 58.3% vs. 14.3% (P < 0.05), respectively. The active SLE patients with MP/UC and the inactive SLE patients with UP/MC showed different clinical symptoms and laboratory examinations. Significant correlation was found between the disease activity index for lupus patients (SLEDAI) scores and MP% (r = 0.93), between the SLEDAI scores and MC% (r = - 0.96) also between MC% and MP% (r = - 0.79). CONCLUSION: The p16 methylation assay provides available information for the diagnosis, judgment of disease activity, as well as novel insights into the pathogenesis underlying this disease.