Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma.

Sorafenib is the first-line medication for advanced hepatocellular carcinoma (HCC), but it can only extend limited survival. It is imperative to find a combination strategy to increase sorafenib efficacy. Artesunate is such a preferred candidate, because artesunate is clinically well-tolerated and more importantly both drugs can induce ferroptosis through different mechanisms. In this study we investigated the combined effect of sorafenib and artesunate in inducing ferroptosis of HCC and elucidated the involved molecular mechanisms. We showed that artesunate greatly enhanced the anticancer effects of low dose of sorafenib against Huh7, SNU-449, and SNU-182 HCC cell lines in vitro and against Huh7 cell xenograft model in Balb/c nude mice. The combination index method confirmed that the combined effect of sorafenib and artesunate was synergistic. Compared with the treatment with artesunate or sorafenib alone, combined treatment induced significantly exacerbated lipid peroxidation and ferroptosis, which was blocked by N-acetyl cysteine and ferroptosis inhibitors liproxstatin-1 and deferoxamine mesylate, but not by inhibitors of other types of cell death (z-VAD, necrostatin-1 and belnacasan). In Huh7 cells, we demonstrated that the combined treatment induced oxidative stress and lysosome-mediated ferritinophagy, two essential aspects of ferroptosis. Sorafenib at low dose mainly caused oxidative stress through mitochondrial impairments and SLC7A11-invovled glutathione depletion. Artesunate-induced lysosome activation synergized with sorafenib-mediated pro-oxidative effects by promoting sequential reactions including lysosomal cathepsin B/L activation, ferritin degradation, lipid peroxidation, and consequent ferroptosis. Taken together, artesunate could be repurposed to sensitize sorafenib in HCC treatment. The combined treatment can be easily translated into clinical applications.

ACSL4 is a predictive biomarker of sorafenib sensitivity in hepatocellular carcinoma.

Sorafenib is the first-line treatment of advanced hepatocellular carcinoma (HCC). However, there is a lack of validated biomarkers to predict sorafenib sensitivity. In this study we investigated the role of ACSL4, a positive-activating enzyme of ferroptosis, in sorafenib-induced cell death and HCC patient outcome. We showed that ACSL4 protein expression was negatively associated with IC50 values of sorafenib in a panel of HCC cell lines (R = -0.952, P < 0.001). Knockdown of ACSL4 expression by specific siRNA/sgRNA significantly attenuated sorafenib-induced lipid peroxidation and ferroptosis in Huh7 cells, and also rescued sorafenib-induced inhibition of xenograft tumor growth in vivo. We selected 29 HCC patients with surgery as primary treatment and sorafenib as postoperative adjunct therapy from a hospital-based cohort. A high proportion (66.7%) of HCC patients who had complete or partial responses to sorafenib treatment (according to the revised RECIST guideline) had higher ACSL4 expression in the pretreated HCC tissues, compared with those who had stable or progressed tumor growth (23.5%, P = 0.029). Since ACSL4 expression was independent of sorafenib treatment, it could serve as a useful predictive biomarker. Taken together, this study demonstrates that ACSL4 is essential for sorafenib-induced ferroptosis and useful for predicting sorafenib sensitivity in HCC. This study may have important translational impacts in precise treatment of HCC.

Ursolic acid potentiated oxaliplatin to induce apoptosis in colorectal cancer RKO cells.

Ursolic acid (UA) is found in multiple anticancer herbs and has shown anticancer effects in colorectal cancer (CRC) cells. The present study aimed to observe the effects of a combination of UA and oxaliplatin (Oxa), a frequently used chemotherapeutic drug in CRC, on human CRC RKO cells. The results showed that UA and Oxa synergistically inhibited the proliferation of RKO cells. A combination of UA and Oxa induced apoptosis in RKO cells and increased the activities of caspase-3, caspase-8, and caspase-9. Z-VAD-FMK, a caspase inhibitor, significantly antagonized UA- and Oxa-activated caspase-3, caspase-8, and caspase-9 and induced apoptosis. In addition, UA and Oxa downregulated the expression of X-linked inhibitor of apoptosis (XIAP) and Survivin in RKO cells. These observations suggested that a combination of UA and Oxa elicited synergistically anticancer effects in RKO cells and provided new evidence for potential application of UA and Oxa for CRC treatment.

Histone deacetylases up-regulate C/EBPα expression through reduction of miR-124-3p and miR-25 in hepatocellular carcinoma.

BACKGROUND: CCAAT enhancer binding protein α (C/EBPα), as an important transcription factor involved in cell proliferation, differentiation and metabolism, was up-regulated in primary hepatocellular carcinoma (HCC) and predicted poorer prognosis. In this study, we explored how histone deacetylases (HDACs) up-regulated C/EBPα in HCC. METHODS: The protein expressions of HDAC1, HDAC2 were associated with C/EBPα by immunohistochemistry staining in a HCC tissue microarray. HCC cells were then treated with HDAC inhibitors or siRNAs to determine the roles of miR-124-3p and miR-25 in the regulation of C/EBPα mRNA expression. RESULTS: Both HDAC1 and HDAC2 proteins were significantly associated with C/EBPα. Inhibition of HDAC by either pharmacological inhibitors or siRNAs decreased C/EBPα mRNA expression in dose-dependent manners in HCC cells. HDAC inhibitors reduced C/EBPα mRNA stability as shown by pmiRGLO luciferase reporter assays. HDAC inhibition consistently induced miR-124-3p and miR-25 expression. Conversely, blockage of miR-124-3p and/or miR-25 by treatment with specific synthetic inhibitors abolished C/EBPα reduction. More importantly, C/EBPα mRNA stability could be rescued by site-directed mutations of miR-124-3p or miR-25 recognition sites in the C/EBPα 3'UTR sequence. In summary, HDAC may up-regulate C/EBPα expression through miR-124-3p and miR-25 in HCC.

Traditional Chinese medicine formulation Yanggan Jiedu Sanjie inhibits TGF-ß1-induced epithelial-mesenchymal transition and metastatic potential in human hepatocarcinoma Bel-7402 cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a vital process in cancer progression and metastasis. Yanggan Jiedu Sanjie (YGJDSJ) is Traditional Chinese Medicine formulation for liver cancer treatment. In the present study, we evaluated the effects of YGJDSJ on TGF-ß1-induced EMT in hepatocellular carcinoma Bel-7402 cells. METHODS: Bel-7402 cells were treated with TGF-ß1 and YGJDSJ. EMT was identified by morphological changes and expression of marker proteins. Cell morphology was observed under a microscope. Protein expression and phosphorylation was detected by western blotting. Cell migration was measured by the scratch assay. Cell adhesion and invasion was detected by a commercial kit. RESULTS: YGJDSJ reversed TGF-ß1-induced morphological changes, as well as the expression of the EMT markers E-cadherin and N-cadherin in Bel-7402 cells. YGJDSJ also inhibited TGF-ß1 up-regulated Smad3 phosphorylation and Snail expression in Bel-7402 cells. Moreover, YGJDSJ inhibited TGF-ß1-induced cell adhesion, migration and invasion in Bel-7402 cells. CONCLUSIONS: YGJDSJ inhibited TGF-ß1-induced EMT and mediated metastatic potential of Bel-7402 cells, which may be related to down-regulation of Smad3 phosphorylation and Snail expression. The present study provides a new basis for application of this herbal formula for prevention of liver cancer metastasis.

Monte Carlo simulation on the dynamics of a semi-flexible polymer in the presence of nanoparticles.

The dynamics of a semi-flexible polymer chain in the presence of periodically distributed nanoparticles is simulated by using off-lattice Monte Carlo simulations. For repulsive or weak attractive nanoparticles, the dynamics are slowed down monotonically by increasing the chain stiffness kθ or decreasing the inter-particle distance d. For strong attractive nanoparticles, however, the dynamics show nonmonotonic behaviors with kθ and d. An interesting result is that a stiff polymer may move faster than a flexible one. The underlying mechanism is that the nanoparticle's attraction is weakened by the chain stiffness. The nonmonotonic behavior of the polymer's dynamics with kθ is explained by the competition between the weakening effect of the chain stiffness on the nanoparticle's attraction and the intrinsic effect of chain stiffness which reduces the dynamics of the polymer. In addition, the nonmonotonic behavior of the polymer's dynamics with d is explained by the competition between the nanoparticle-exchange motion of the polymer dominated at small d and the desorption-and-adsorption motion at large d. The excluded volume effect of the nanoparticles plays a more important role for stiffer polymers as the attraction of the nanoparticles is weakened by the chain stiffness.

Herbal formula YGJDSJ inhibits anchorage-independent growth and induces anoikis in hepatocellular carcinoma Bel-7402 cells.

BACKGROUND: Based on clinical medications and related studies, we established a Yang-Gan Jie-Du Sang-Jie (YGJDSJ) herbal formula for hepatocarcinoma treatment. In present study, we evaluated the anti-cancer potential of YGJDSJ on suspension-grown human hepatocellular carcinoma Bel-7402 cells. METHODS: Bel-7402 cells were cultured in poly(2-hydroxyethyl methacrylate) (poly-HEMA) coated plates and treated with YGJDSJ. Anchorage-independent cell growth was detected by cell Counting Kit-8 (CCK-8) assay and soft agar colony formation assay. Anoikis was detected by ethdium homodimer-1 (EthD-1) staining and flow cytometry analysis. Caspases activities were detected by the cleavage of chromogenic substrate. Reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining. Protein expression and phosphorylation was identified by western blot. Protein expression was knocked-down by siRNA. RESULTS: YGJDSJ inhibited the proliferation of Bel-7402 cells in poly-HEMA coated plates and anchorage-independent growth of Bel-7402 cells in soft agar. YGJDSJ also induced anoikis in Bel-7402 cells as indicated by EthD-1 staining and flow cytometry analysis. YGJDSJ activated caspase-3, - 8, and - 9 in suspension-grown Bel-7402 cells. The pan-caspase inhibitor Z-VAD-FMK significantly abrogated the effects of YGJDSJ on anoikis in suspension-grown Bel-7402 cells. In addition, YGJDSJ increased ROS in suspension-grown Bel-7402 cells. The ROS scavenger N-acetyl-L-cysteine (NAC) partially attenuated YGJDSJ-induced activation of caspase-3, - 8 and - 9 and anoikis in suspension-grown Bel-7402 cells. Furthermore, YGJDSJ inhibited expression and phosphorylation of protein tyrosine kinase 2 (PTK2) in suspension-grown Bel-7402 cells. Over-expression of PTK2 significantly abrogated YGJDSJ induced anoikis. CONCLUSIONS: YGJDSJ inhibits anchorage-independent growth and induce caspase-mediated anoikis in Bel-7402 cells, and may relate to ROS generation and PTK2 downregulation.

AKT activation was not essential for hepatocellular carcinoma cell survival under glucose deprivation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with a dismal 5-year survival rate less than 15%. The present study aimed to investigate whether AKT inhibition and glucose deprivation could synergistically kill HCC cells and the molecular mechanisms involved. HCC cells were starved in glucose deprivation, and then the resultant cell death was determined by flow cytometry and mitochondrial oxygen consumption rates using a Seahorse XF-24 Extracellular Flux Analyzer. Glucose deprivation reduced mitochondrial oxygen consumption rates for ATP production, enhanced mitochondrial proton leaks, reduced Mcl-1 expression, and subsequently caused significant cell death in the sensitive HepG2 and HCC-M cells. In the resistant Hep3B and Huh7 cells that survived, glucose starvation induced time-dependent AKT activation. However, blockage of AKT activation using chemical inhibitors (ZSTK474 and LY290042) or specific AKT1-targeting siRNAs could not markedly sensitize glucose deprivation-induced cell death. In contrast, AKT inhibitors or AKT1-targeting siRNAs significantly protected the sensitive HepG2 cells from glucose deprivation-induced cell death. More importantly, AKT inhibition mechanically suppressed mTOR activity and induced the prosurvival autophagy pathway in the sensitive HCC cells. Taken together, these data demonstrated that AKT activity was not essential for HCC cell survival during glucose deprivation. The reduction of mTOR activity and induction of the autophagy pathway may hinder the potential application of AKT inhibitors in the cancer therapy of solid tumors such as HCC.

A study on the diffusivity of polymers in crowded environments with periodically distributed nanoparticles.

The effect of nanoparticles (NPs) on the diffusivity of polymers in crowded environments is complicated. We study the diffusivity of polymers in the environment with periodically distributed immobile NPs by using off-lattice Monte Carlo simulations. Results show that the diffusion coefficient D of polymers at low temperature is dependent on the inter-particle distance d and polymer length N or end-to-end distance of polymers in dilute solution R0. At low temperature, D is large at both small and large d and a minimum is observed at intermediate d. The nonmonotonic behavior of D is due to the reason that there are two kinds of diffusion modes for the polymers at low temperature: NP-exchange motion for R0 > d and adsorption-and-desorption motion for R0 < d. Moreover, we observe the oscillation of D with N at a relatively low temperature. The novel behavior is relevant to the adsorption of polymers on NPs and is explained from the free energy barrier for polymers jumping from the ground state to others.

The relationship between HbA1c and ultrasound plaque textures in atherosclerotic patients.

OBJECTIVE: Diabetes mellitus (DM) is associated to the morphological and componential characteristics of atheromatous plaques. It has proven that plaque textures are related to plaque components and beneficial for atherosclerotic risk stratification. The aim of this study is to compare plaque textures in patients with and without DM, and examine the relationship between HbA1c levels and the ultrasound plaque textures in atherosclerotic patients. METHODS: A total of 136 participants (among them 66 are diabetic and 70 are non-diabetic) suffering from carotid plaques were included. About 300 texture features were extracted from the ultrasound images of plaques using the algorithms of histogram, absolute gradient, run-length matrix, gray-level co-occurrence matrix, autoregressive model and wavelet transform, respectively. Thirty optimal features were selected by the Fisher coefficient and the mutual information measure. The most discriminating feature (MDF) was obtained from the linear discriminant analysis for the optimal features. Linear regression model was performed to investigate the relationship between HbA1c and MDF. The receiver operating characteristics (ROC) curve was further developed to validate the relation between the estimated HbA1c (models output) and diabetes status. RESULTS: A total of 12 texture features showed statistical difference between patients with and without DM. The MDF was significant higher in non-diabetic patients (0.326 ± 0.049) than diabetic patients (-0.346 ± 0.052) (p < 0.001). The optimal regression model (r = 0.348, p < 0.001) for HbA1c included a constant (p < 0.001) and the MDF (p < 0.001). The areas under ROC curve used to estimate HbA1c was 0.828. CONCLUSIONS: The results indicate that there is a quantitative relationship between the HbA1c levels and plaque textures in ultrasonic images of atherosclerotic patients, which may suggest that texture analysis of the ultrasonic image of plaque is a promising method for evaluating the cardiovascular risk caused by DM in patients with plaques.

[Review on study of Dao-di herbs Artemisiae Annuae Herba].

Artemisiae Annuae Herba has used as a medicine for more than 2 000 years. To infer based on the modern study results, Artemisiae Annuae Herba used for the treatment of malaria recorded in Zhou Hou Bei Ji Fang before 1 700 years should come from Artemisia annua. Based on the data of Chinese materia medica, from the field of treatment hotness and preventing attack of malaria etc., the Dao-di producing district of Artemisiae Annuae Herba should at Jingzhou (now Hubei) and surrounding areas in history. From the view of anti-malaria components artemisinin content, the Dao-di growing producing district of Artemisiae Annuae Herba should locate at Chongqing, Guangxi and its surrounding provinces. The study results showed that A. annua was harvested in flower bloom at autumn, and in this time it also had higher artemisinin content. If A. annua was stored exceed six months, artemisinin could be degraded about thirty percent. So it should be stored in a cool and dry place generally. Wild A. annua had a rich genetic diversity. Artemisinin content of A. annua breeding in experimental field could reache to two percent.

Pharmacological approaches for targeting lysosomes to induce ferroptotic cell death in cancer.

Lysosomes are crucial organelles responsible for the degradation of cytosolic materials and bulky organelles, thereby facilitating nutrient recycling and cell survival. However, lysosome also acts as an executioner of cell death, including ferroptosis, a distinctive form of regulated cell death that hinges on iron-dependent phospholipid peroxidation. The initiation of ferroptosis necessitates three key components: substrates (membrane phospholipids enriched with polyunsaturated fatty acids), triggers (redox-active irons), and compromised defence mechanisms (GPX4-dependent and -independent antioxidant systems). Notably, iron assumes a pivotal role in ferroptotic cell death, particularly in the context of cancer, where iron and oncogenic signaling pathways reciprocally reinforce each other. Given the lysosomes' central role in iron metabolism, various strategies have been devised to harness lysosome-mediated iron metabolism to induce ferroptosis. These include the re-mobilization of iron from intracellular storage sites such as ferritin complex and mitochondria through ferritinophagy and mitophagy, respectively. Additionally, transcriptional regulation of lysosomal and autophagy genes by TFEB enhances lysosomal function. Moreover, the induction of lysosomal iron overload can lead to lysosomal membrane permeabilization and subsequent cell death. Extensive screening and individually studies have explored pharmacological interventions using clinically available drugs and phytochemical agents. Furthermore, a drug delivery system involving ferritin-coated nanoparticles has been specifically tailored to target cancer cells overexpressing TFRC. With the rapid advancements in understandings the mechanistic underpinnings of ferroptosis and iron metabolism, it is increasingly evident that lysosomes represent a promising target for inducing ferroptosis and combating cancer.

[Extended-field intensity modulated radiation therapy and intra-cavitary brachytherapy combined with chemotherapy for stage Ib1-IVa cervical cancer with positive para-aortic lymph nodes].

OBJECTIVE: To investigate the treatment effects and toxicities of extended-field intensity modulated radiation therapy (EF-IMRT) and intra-cavitary brachytherapy combined with chemotherapy for stageIb1-IVa cervical cancer with positive para-aortic lymph nodes. METHODS: A total of 46 stage Ib1-IVa cervical cancer patients with positive para-aortic lymph nodes treated at Fudan University Shanghai Cancer Center between 2009 and 2011 were reviewed. Neoadjuvant, concomitant and adjuvant chemotherapy with paclitaxel and carboplatin were administrated for one cycle before radiation therapy, two cycles during radiation therapy or three cycles after radiation therapy. All patients received EF-IMRT and intra-cavitary brachytherapy. The positive lymph nodes received an additional boost dose. RESULTS: All patients received EF-IMRT to 50.4 Gy (1.8 Gy per fraction). Twenty-six patients was treated with boost dose of 6.0-8.0 Gy in 2.0 Gy per fraction to positive para-aortic lymph nodes. Thirty-seven patients received a positive para-aortic lymph nodes boost or (and) parametrial boost. All patient also received a high-dose-rate intra-cavitary brachytherapy at the point "A" dose of 20.0-30.0 Gy in 5.0 Gy per fraction. Total chemotherapy cycles were 189, and the average patient received 4.1 courses. Two cases (4%, 2/46) experienced grade III gastrointestinal toxicities, no patients suffered grade IV gastrointestinal toxicities. Fifteen cases (33%, 15/46) experienced grade III hematological toxicities, and 3(7%, 3/46) experienced grade IV hematological toxicities.Late grade III-IV toxicity was seen in 3 cases (7%, 3/46). The 3 year progression- free survival rate was 46.2%, and the 3 years overall survival rate was 61.2%. CONCLUSION: EF-IMRT and intra-cavitary brachytherapy combined with chemotherapy is safe and effective for stageIb1-IVa cervical cancer with positive para-aortic lymph nodes.

Anoxybacillus tengchongensis sp. nov. and Anoxybacillus eryuanensis sp. nov., facultatively anaerobic, alkalitolerant bacteria from hot springs.

Two novel thermophilic, spore-forming bacterial strains, T-11(T) and E-112(T), were isolated from hot springs in Tengchong and Eryuan counties of Yunnan province in south-west China. The strains were Gram-stain-positive rods, occurring singly or in chains. Growth of strain T-11(T) was observed between 30 and 75 °C (optimum 50 °C) and at pH 7-11 (optimum pH 8.5), while the temperature range for strain E-112(T) was 35-70 °C (optimum 55 °C) and the pH range was 7.0-11.0 (optimum pH 8.0). The DNA G+C contents of strains T-11(T) and E-112(T) were 41.1 and 42.6 mol%, respectively. On the basis of 16S rRNA gene sequence similarity, the two strains were shown to be related most closely to Anoxybacillus species. The chemotaxonomic characteristics [predominant isoprenoid quinone menaquinone 7 (MK-7); major fatty acids iso-C(15 : 0) and iso-C(17 : 0)] also supported the affiliation of strains T-11(T) and E-112(T) to the genus Anoxybacillus. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains T-11(T) and E-112(T) from Anoxybacillus species with validly published names. Strains T-11(T) and E-112(T) therefore represent two novel species, for which the names Anoxybacillus tengchongensis sp. nov. (type strain T-11(T) =CCTCC AB209237(T) =KCTC 13721(T)) and Anoxybacillus eryuanensis sp. nov. (type strain E-112(T) =CCTCC AB209236(T) =KCTC 13720(T)) are proposed.

Isolation of talathermophilins from the thermophilic fungus Talaromyces thermophilus YM3-4.

Six indole alkaloids with various levels of prenylation were isolated from the thermophilic fungus Talaromyces thermophilus strain YM3-4. Their structures were identified by NMR and MS spectroscopic analyses. Compounds 1 and 2 are new analogues of the key versatile precursor notoamide E. Compound 3 is a novel analogue of preechinulin, and compound 4 was reported as a natural occurring cyclo(glycyltryptophyl) for the first time. The metabolite profile of this thermophilic organism displayed a biosynthetic pathway for talathermophilins.

[Effect of sodium para-aminosalicylic on concentrations of amino acid neurotransmitters in basal ganglia of manganese-exposed rats].

OBJECTIVE: To probe the effect of sodium para-aminosalicylate (PAS-Na) on concentration of amino acid neurotransmitters including glutamate (Glu), glutamine (Gln), glycine (Gly) and gamma-aminobutyric acid (GABA) in basal ganglia of subacute manganese (Mn)-exposed rats. METHODS: Forty Sprague-Dawley male rats were randomly divided into the control, Mn-exposed, low dose PAS-Na (L-PAS) and high dose PAS-Na (H-PAS) groups. Rats in experiment groups received daily intraperitoneally injections of manganese chloride (MnCl2 · 4H2O, 15 mg/kg), while rats in control group received daily intraperitoneally injections of normal saline (NS), all at 5 days/week for 4 weeks. Then the rats in PAS groups followed by a daily subcutaneously dose of PAS-Na (100 and 200 mg/kg as the L-PAS and H-PAS groups, respectively) for another 3 and 6 weeks; while the rats in Mn-exposed and control group received NS. The concentrations of Glu, Gln, Gly and GABA in basal ganglia of rat was detected by the high performance liquid chromatography fluorescence detection technique. RESULTS: After treating with PAS-Na for 3 weeks, the concentration of Gly in the Mn-exposed rats decreased to (0.165 ± 0.022) µmol/L (control = (0.271 ± 0.074) µmol/L, Mn vs control, t = 4.65, P < 0.05). After the further 6-week therapy with PAS-Na, the concentrations of Glu, Gln, Gly in the Mn-exposed rats were lower than those of the control rats ((0.942 ± 0.121), (0.377 ± 0.070), (0.142 ± 0.048), (1.590 ± 0.302), (0.563 ± 0.040), (0.247 ± 0.084) µmol/L; t = 7.72, 5.85, 4.30, P < 0.05); and also lower than in L-PAS and H-PAS groups, whose concentrations were separately (1.268 ± 0.124), (1.465 ± 0.196), (0.497 ± 0.050), (0.514 ± 0.103), (0.219 ± 0.034) µmol/L (L-PAS Glu and Gln vs Mn, t = 3.87, 3.77, P < 0.05; H-PAS Glu, Gln and Gly vs Mn, t = 6.78, 4.70, 3.42, P < 0.05). CONCLUSION: The toxic effect of manganese on Glu, Gln and Gly in basal ganglia of Mn-exposed rats is obvious, especially appears earlier on Gly. The toxic effect still continues to develop when relieved from the exposure. PAS-Na may play an antagonism role in toxic effect of manganese on concentration of Glu, Gln and Gly in basal ganglia of Mn-exposed rats.

[Rapid diagnostics of early phosphorus deficiency in mini-cucumber plants under protected cultivation by near infrared spectroscopy].

The morphological symptom of phosphorus deficiency at early stage is similar to the appearance of leaf aging process in preliminary phase, so that visual diagnostics of phosphorus deficiency in mini-cucumber plants at early stage is practically impossible. Near infrared reflectance spectra contain information about differences in compositions of leaf tissues between phosphorus-deficient plants and healthy plants. In the present paper, near infrared reflectance spectroscopy was used to provide diagnostic information on phosphorus deficiency of mini-cucumber plants grown under non-soil conditions. Near infrared spectra was collected from 90 leaves of mini-cucumber plants. Raw cucumber spectra was preprocessed by SNV and divided into 27 intervals. The top 10 principal components (PCs) were extracted as the input of BP-ANN classifiers by principal component analysis (PCA) while the values of nutrient deficient were used as the output variables of BP-ANN and three layers BP-ANN discrimination model was built. The best experiment results were based on the top 3 principal components of No. 7 interval when the spectra was divided into 27 intervals and identification rates of the ANN model are 100% in both training set and the prediction set. The overall results show that NIR spectroscopy combined with BP-ANN can be efficiently utilized for rapid and early diagnostics of phosphorus deficiency in mini-cucumber plants.

Regulation of subtilisin-like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea.

Nematophagous fungi have been used as biological control agents against nematodes parasitic to plants and animals. These fungi can secret subtilisin-like extracellular serine proteases during the infection of nematodes. The expression of these subtilisin-like serine proteases is regulated by nitrogen sources, including nematode cuticle. However, the mechanisms underlying the nitrogen sources-induced expression of these serine proteases is not well understood. In this study, we investigated the effect of nitrogen sources on the expression of a subtilisin-like extracellular protease, prC, in the nematophagous fungus Clonostachys rosea. Disruption of prC attenuated infection of the fungus to nematodes, indicating that this gene functions as a virulence factor. The inhibition of basal expression of prC by the preferred nitrogen sources (glutamine, ammonia) occurred at the transcriptional level. In contrast, nematode cuticle induced the expression of prC at the post-transcriptional level. The inducible expression of prC by nematode cuticle was significantly suppressed by glutamine, ammonia and phenylmethylsulfonyl fluoride (an inhibitor of serine protease). Thus, the existence of active PrC, albeit at a low level in the medium, is probably essential for further induction of this gene by nematode cuticle. Moreover, the low molecule weight (< 3 kD) degradation products of nematode cuticle could significantly induce the expression of prC. Ammonia suppresses the virulence of C. rosea against nematodes, probably by inhibiting prC expression. Thus, the nematophagous fungi probably could not function well as biocontrol agents in fields fertilized with a large amount of ammonium salt.

Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP.

Hydrogen sulfide (H(2)S) is an endogenously generated gaseous transmitter, which has recently been suggested to regulate cardiovascular functions. The present study aims to clarify the mechanisms underlying the cardioprotective effects of H(2)S. Signaling elements were examined in cardiomyocytes cultured under hypoxia/reoxygenation conditions and in a rat model of ischemia-reperfusion. In cultured cardiomyocytes, sodium hydrosulfide (NaHS; 10, 30, and 50 mumol/l) showed concentration-dependent inhibitory effects on cardiomyocyte apoptosis induced by hypoxia/reoxygenation. These effects were associated with an increase in phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) (Ser9) and a decrease in Bax translocation, caspase-3 activation, and mitochondrial permeability transition pore (mPTP) opening. Transfection of a phosphorylation-resistant mutant of GSK-3beta at Ser9 attenuated the effects of NaHS in reducing cardiomyocyte apoptosis, Bax translocation, caspase-3 activation, and mPTP opening. In a rat model of ischemia-reperfusion, NaHS administration reduced myocardial infarct size and increased the phosphorylation of GSK-3beta (Ser9) at a dose of 30 mumol/kg. In conclusion, the H(2)S donor prevents cardiomyocyte apoptosis by inducing phosphorylation of GSK-3beta (Ser9) and subsequent inhibition of mPTP opening.

Peroxynitrite and hemoglobin-mediated nitrative/oxidative modification of human plasma protein: effects of some flavonoids.

Protein tyrosine nitration is a common post-translational modification occurring under conditions of nitrative/oxidative stress in a number of diseases. The major pathways of protein tyrosine nitration in vivo include peroxynitrite (ONOO(- )) and hemoglobin/[image omitted] /H(2)O(2)-dependent reaction. In this paper, several structural diversity flavonoids (quercetin, kaempferol, (+)-catechin, baicalein, apigenin, and naringenin) were chosen, to study their efficiencies against ONOO(- ) or hemoglobin/NaNO(2)/H(2)O(2)-mediated nitrative/oxidative damage to human plasma proteins in vitro. Protein nitration was efficiently inhibited by these flavonoids regardless of nitration pathways, and the inhibitory effects were consistent with their free radical scavenging activities. These flavonoids dose dependently inhibited ONOO(- )-induced protein oxidation, while they ineffectively suppressed hemoglobin/NaNO(2)/H(2)O(2)-triggered protein oxidation. These results mean that ONOO(- ) and hemoglobin/NaNO(2)/H(2)O(2) can cause plasma protein nitrative and oxidative damage in different pathways, and those flavonoids with strong antioxidant activities may contribute their protective effect partly through inhibiting protein nitration.