Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

INTRODUCTION: Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. OBJECTIVES: To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. METHODOLOGY: A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. RESULTS: Using rhamnose as an internal reference standard, the contents of the other monosaccharide components in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. CONCLUSIONS: The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

miRNA-548p suppresses hepatitis B virus X protein associated hepatocellular carcinoma by downregulating oncoprotein hepatitis B x-interacting protein.

AIM: miR-548p is a recently identified and poorly characterized miRNA. However, its role of miR-548p in tumorigenesis and progression remains poorly understood. Here, we aimed to investigate the biofunction of miR-548p in hepatocellular carcinogenesis. METHODS: The expression levels of miR-548p were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The role of miR-548p in hepatocellular carcinoma (HCC) was determined by colony formation, flow cytometry assay and nude mice xenograft experiments. miR-548p target genes were analyzed by miRNA target predication programs and verified by qRT-PCR, western blotting assay and dual-luciferase reporter assay. RESULTS: miR-548p is repressed by hepatitis B virus X protein (HBx) in HCC tumor tissues and hepatoma cells, and inhibited cell growth by inhibiting cell proliferation and promoting cell apoptosis. miR-548p directly downregulated the expression of hepatitis B x-interacting protein (HBXIP) by binding to the 3'-untranslated region of HBXIP mRNA. Further study showed that hepatocyte nuclear factor-4a (HNF4A) promoted the expression of miR-548p and inhibited the transcription of HBXIP. HNF4A is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis, and is shown to be repressed by HBx. CONCLUSION: We proposed the model for HBx/HNF4A/miR-548p/HBXIP pathway that controls hepatoma cell growth and tumorigenesis of HCC. miR-548p was identified as a tumor-suppressor in HBx-associated hepatocellular carcinogenesis.

[Mechanism of biological actions of quercetin based on biomolecular network].

The mechanism of biological actions of quercetin was studied by using metabolomic method and biomolecular network. HPLC-MS was used to analyze the serum metabolome in rats of blank group and quercetin administration group rats, and MS data were processed by MATLAB software. With multivariate statistical analysis of serum metabolite profiles, a clear separation among blank group and quercetin administration group was achieved, potential biomarkers were selected according to the parameters of variable importance in the projection (VIP) and identified according to MS information and database retrieval. Four compounds, related enzymes, action targets and metabolic pathways had been confirmed, namely retinoic acid and RARbeta, arachidonate and COX-2, 3, 5-diodotyrosine and TPO, uridine diphosphate glucose and PDEs. The mechanism of quercetin enhancing ability of retinoic acid on the induction of RARbeta, activating TPO, using as COX-2 and PDEs inhibitor was approved by biomolecular network and related literatures. In this study, a mechanism of multiple biological actions of quercetin was evaluated at the level of the biomolecular network, metabolomics and biomolecular network can be used to investigate the biological effects mechanism of quercetin, which provided a new method to further revealing mechanism of drug action.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells.

OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. CONCLUSION: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.

Radiomics Combined with ACR TI-RADS for Thyroid Nodules: Diagnostic Performance, Unnecessary Biopsy Rate, and Nomogram Construction.

RATIONALE AND OBJECTIVES: To develop a radiomics model with enhanced diagnostic performance, reduced unnecessary fine needle aspiration biopsy (FNA) rate, and improved clinical net benefit for thyroid nodules. METHODS: We conducted a retrospective study of 217 thyroid nodules. Lesions were divided into training (n = 152) and verification (n = 65) cohorts. Three radiomics scores were derived from B-mode ultrasound (B-US) and strain elastography (SE) images, alone and in combination. A radiomics nomogram was constructed by combining high-frequency ultrasonic features and the best-performing radiomics score. The area under the receiver operating characteristic curve (AUC), unnecessary FNA rate, and decision curve analysis (DCA) results for the nomogram were compared to those obtained with the American College of Radiology Thyroid Imaging, Reporting and Data System (ACR TI-RADS) score and the combined TI-RADS+SE+ contrast-enhanced ultrasound (CEUS) advanced clinical score. RESULTS: The three radiomics scores (B-US, SE, B-US+SE) achieved training AUCs of 0.753 (0.668-0.825), 0.761 (0.674-0.838), and 0.795 (0.715-0.871), and validation AUCs of 0.732 (0.579-0.867), 0.753 (0.609-0.892), and 0.752 (0.592-0.899) respectively. The AUC of the nomogram for the entire patient cohort was 0.909 (0.864-0.954), which was higher than that of the ACR TI-RADS score (P < 0.001) and equivalent to the TI-RADS+SE+CEUS score (P = 0.753). Similarly, the unnecessary FNA rate of the radiomics nomogram was significantly lower than that of the ACR TI-RADS score (P = 0.007) and equivalent to the TI-RADS+SE+CEUS score (P = 0.457). DCA also showed that the radiomics nomogram brought more net clinical benefit than the ACR TI-RADS score but was similar to that of the TI-RADS+SE+CEUS score. CONCLUSION: The radiomics nomogram developed in this study can be used as an objective, accurate, cost-effective, and noninvasive method for the characterization of thyroid nodules.

A microstructural study of individual nacre tablet of Pinctada maxima.

Nacre tablets from the shell of Pinctada maxima were studied with SEM, TEM and STEM. The systematic nanolath morphology on the (001) surface of nacre tablets was observed after acidic etching and mechanical polishing. The nanolaths were along the [100] crystallographic orientation of aragonite crystal. The (010) and (100) cross section surfaces of the nacre tablets showed nanolath and nanograin morphologies, respectively, which was consistent with [100] crystallographic orientation of nanolath on the (001) surface. Sheet-like defects with low mass density were observed on the (001) plane inside nacre tablets and were considered to be the cause of nanolath morphology revealed on the surfaces by acidic etching and mechanical polishing. On the other hand, large block [110] twins that divide the nacre tablets into two sectors were identified. The implication of these twins on the understanding to the crystallization mechanism of nacre tablets was discussed.

Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

Microwave assisted free radical degradation of Schisandra polysaccharides: Optimization, identification and application.

In order to establish structural-fingerprinting of polysaccharides for improvement of quality assessment, a sample preparation method based on microwave assisted free radical degradation (MFRD) of plant polysaccharides was proposed to produce oligosaccharides and small Mw polysaccharides. As a case study of Schisandra chinensis and S. sphenanthera fruit polysaccharides (SCP and SSP), the MFRD condition (i.e., 100 °C, 30 s and 80 W) was confirmed to be optimal. The potential structures of the MFRD products of SCP and SSP were further discussed by combinations of HILIC-ESI--QTOF-MSE and HILIC-ESI--Q-OT-IT-MS/MS. As followed, multivariable statistical analysis shows a clear separation of SCP and the SSP in PCA and OPLS-DA plots based HILIC-ESI--QTOF-MSE data. The VIP plot unveils several key Q-markers (e.g., peaks 3, 8, 9, 10, 15, 25, 26, 28, 29 and 30) with significant differences and stable emergences. Furthermore, a low-polymerization compositional fingerprinting was successfully constructed for SCP and SSP using a high-performance anion-exchange chromatography with pulsed amperometric detection. Compared to the conventional sample preparation methods, the MFRD took only a few thousandth of the time to accomplish degradations of plant polysaccharides. It significantly improves sample preparations and is generally applicable to various polysaccharide samples.

Characterization of thyroid metastasis from clear cell renal cell carcinoma on ultrasonography: a report of three cases and literature review.

Introduction: Thyroid metastasis from clear cell renal cell carcinoma (ccRCC) is relatively rare, so ultrasound doctors lack experience with the disease, which can easily lead to misdiagnosis. We describe three cases of thyroid metastasis from ccRCC detected 12, 8, and 7 years after nephrectomy. Case presentation: The first patient, a 78-year-old woman, was admitted to our institution for hoarseness and progressive dyspnea. Ultrasonography revealed bilateral thyroid nodules and abnormal cervical lymph nodes. Fine-needle aspiration biopsy (FNAB) and core needle biopsy (CNB) of the thyroid was nondiagnostic. The other two patients, a 54-year-old man and a 65-year-old man, were admitted to our institution for a goiter pressing on the trachea. In each case, ultrasonography revealed a partially cystic nodule of the left lobe of the thyroid gland. Histological examination of three patients after thyroidectomy showed thyroid metastasis from ccRCC. Discussion/Conclusion: For patients with a history of ccRCC, long-term follow-up and routine thyroid ultrasonography should be performed. If a new thyroid nodule is found during the examination, metastases should be highly suspected. FNAB should be performed, even if benign ultrasound features seem to be in evidence. If the diagnosis of FNAB is incorrect and inconclusive, CNB should be performed.

[Effects of simulated insect herbivory on defense traits of Galinsoga parviflora.] / 模拟昆虫取食对牛膝菊防御特征的影响.

To understand mechanisms underlying Galinsoga parviflora invasion and its responses to simulated insect herbivory, individuals of Galinsoga parviflora were treated with different concentrations of methyl jasmonate (MeJA) before blooming. We measued plant height, abundance of leaves and inflorescences, biomass, specific leaf area, trichome density, condensed tannins, total polyphenols, and flavonoids in leaves and inflorescences. The growth and reproduction parameters of G. parviflora treated with 5 mmol·L-1 MeJA were not significantly different from those of control, higher than those of control when treated with 10 mmol·L-1 MeJA, with significant difference except plant height, and declined when treated with 20 mmol·L-1 MeJA. The trichome density of leaf upper epidermis increased and specific leaf area decreased with increasing MeJA concentration, with both being significantly different from that of control. The contents of flavonoids, total polyphenols, and condensed tannins in leaves treated with 5 mmol·L-1MeJA were not significantly different from those of control. These defensive substances in leaves and inflorescences were highest under 10 mmol·L-1MeJA treatment. The contents of flavonoids and total polyphenols in inflorescences being higher than those of leaves, while condensed tannins was opposite. The defensive substances in leaves declined under 20 mmol·L-1MeJA treatment. The results suggested that G. parviflora could use tolerance and resistance strategies comprehensively, and adopted a variety of defense strategies such as compensatory growth, physical defense, and chemical defense, which was conducive to its success in invasion.

Phragmalin- and mexicanolide-type limonoids from the leaves of Trichilia connaroides.

Phytochemical investigation of the leaves of Trichilia connaroides afforded 12 new limonoids with phragmalin- (1-11) and mexicanolide-type skeletons (12). The structures of these limonoids, including the absolute configuration of 3, were determined by spectroscopic analysis. Compounds 6 and 8 showed moderate cytotoxicity against HL-60 cells.

[Study on the effects of gonadotropin-releasing hormone analogues in the inhibition of ovarian cancer transplanted tumors and in the protection of ovarian function after chemotherapy on nude mice].

OBJECTIVE: To investigate the influence of gonadotropin-releasing hormone (GnRH) analogues on ovarian cancer and ovarian function in vivo. METHODS: ES-2 cells were cultured and xenotransplanted into 36 nude mice, which were divided into 6 groups: normal saline (NS) group: NS 0.1 ml/day subcutaneous injection, and then NS 0.2 ml/week peritoneal injection; cisplatin (DDP) group: NS 0.1 ml/day subcutaneous injection, and then DDP 5 mg/kg (diluted to 0.2 ml) per week peritoneal injection; goserelin group: 100 µg goserelin (diluted to 0.1 ml) per day subcutaneous injection, and then NS 0.2 ml/week peritoneal injection; goserelin + DDP group: 100 µg goserelin (diluted to 0.1 ml) per day subcutaneous injection, and DDP 5 mg/kg (diluted to 0.2 ml) per week peritoneal injection; cetrorelix group:100 µg cetrorelix (diluted to 0.1 ml) per day subcutaneous injection and NS 0.2 ml/week peritoneal injection; cetrorelix + DDP group: 100 µg cetrorelix (diluted to 0.1 ml) per day subcutaneous injection and DDP 5 mg/kg (diluted to 0.2 ml) per week peritoneal injection. All the peritoneal injection started from subcutaneous injection one week later. To compare the weight of nude mice, the volumes of transplanted tumors, the expression of Ki-67 antigen in transplanted tumors, the estrus, the ratio of atretic follicles, the ratio of primary and preantral follicles, the levels of serum anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), estradio (E(2)) and progesterone (P) in each group. RESULTS: There were no significant difference in the weight of nude mice among 6 groups (P > 0.05), which on day 29 in NS group was (19.8 ± 2.2) g, DDP group (20.5 ± 1.4) g, gosereline group (19.6 ± 0.9) g, goserelin + DDP group (19.7 ± 1.6) g, cetrorelix group (20.7 ± 2.2) g, and cetrorelix + DDP group (19.0 ± 1.7) g. The tumor volumes of different groups on the 12(th) day: NS group (241 ± 179) mm(3), DDP group (78 ± 20) mm(3), gosereline group (78 ± 55) mm(3), goserelin + DDP group (64 ± 48) mm(3), cetrorelix group (78 ± 64) mm(3), or cetrorelix + DDP group (70 ± 19) mm(3), in which there were significant difference between NS group and the other groups (P < 0.05); and the same result was obtained on day 15, 19, 22, 26 and 29 (P < 0.05). The expression of Ki-67 in NS group was (33 ± 10)%, in which it was higher than those in DDP group 3.5%, goserelin group 8.8%, goserelin + DDP group 1.5%, cetrorelix group (23 ± 11)%, or cetrorelix + DDP group (8 ± 6)% (P < 0.05). The ratio of primary and preantral follicles in goserelin group was (71.5 ± 8.1)%, in goserelin + DDP group was (62.4 ± 4.1)%, in cetrorelix group was (71.2 ± 7.4)%, and in cetrorelix + DDP group was (63.8 ± 3.1)%, in which they were much higher than that in DDP group (47.0 ± 4.8)% (P < 0.05). The level of AMH in goserelin group was (98 ± 27) ng/ml, which was much higher than that in NS group (66.2 ± 17.4) ng/ml (P < 0.05), while there were no difference in the levels of FSH, E(2) or P among different groups (P > 0.05). CONCLUSION: GnRH analogues could inhibit the growth of transplanted tumors in nude mice, meanwhile increase the secretion of AMH, decrease the frequencies and prolong the lasting time of estrus, decrease the ratio of atretic follicles, raise the ratio of primary and preantral follicles, which may be protect the ovarian function of nude mice.

Extraction, purification, characterization, anticoagulant activity, and anticoagulant mechanism of polysaccharides from the heads of Hypomesus olidus.

The aim of this work was to extract, isolate, and purify polysaccharides from the heads of Hypomesus olidus and to evaluate their anticoagulant activities and anticoagulant mechanisms. Response surface methodology was used to optimize the extraction conditions for ultrasonic-assisted extraction of polysaccharides from the heads of Hypomesus olidus. The optimal extraction conditions consisted of ultrasonic power of 275 W, ultrasonic time of 50 min, and solid-liquid ratio of 5 ml/g, giving the yield of crude polysaccharides (GYT) of 7.73 ± 0.042%. Three polysaccharide fractions, GYT-1, GYT-2, and GYT-3 were purified from GYT by using DEAE-cellulose-52 column and Sephadex G-100 column for anticoagulant activities. The results showed that two doses (2 and 4 mg/ml) of GYT-1 and GYT-3 could significantly prolong (p < .01) in partial thromboplastin time (APTT) (2.19 and 2.37 times, 2.22 and 2.44 times, respectively) and thrombin time (TT) (2.39 and 2.46 times, 2.44 and 2.80 times, respectively) compared with normal control. In particular, GYT-3 had stronger anticoagulant activity than GYT-1, and it was composed of arabinose, fructose, glucose, and lactose with molar ratios of 0.595:1: 2.026:0.273. However, GYT-2 had no anticoagulant activity (p > .05). In addition, anticoagulation mechanism of polysaccharides from the heads of Hypomesus olidus (GYT-3) was evaluated. The results showed that the anticoagulant activity of GYT-3 was based on their binding with antithrombin AT-III. And the inhibitory effects of GYT-3 on factor IIa and Xa were related to the concentration of AT-III in plasma. This study may provide a new and promising anticoagulant drug.

12ß-Acetyloxyperforatin, a New Limonoid from Harrisonia Perforata.

A new A, D-seco limonoid, named 12-acetyloxyperforatin (1), along with three known ones, were isolated from the leaves of Harrisonia perforata. Their structures were elucidated on the basis of spectroscopic analysis, including extensive NMR techniques and computational modelling. These compounds showed no inhibitory activity against the 11ß-HSD1 enzyme.

Characterization, hypolipidemic and antioxidant activities of degraded polysaccharides from Ganoderma lucidum.

The aim of this work is to characterize the primary structure and physicochemical properties of natural polysaccharides (GLP) and degraded polysaccharides (GLPUD) from Ganoderma lucidum, and evaluate their hypolipidemic and antioxidant activities. The results of particle size distribution and scanning electron microscopy (SEM) showed that Ganoderma lucidum polysaccharides were effectively degraded by ultrasonic method. GLPUD was composed of the same monosaccharide units as GLP but with different molar ratios. Infrared spectra and NMR showed that the primary structure of polysaccharides had not been changed by ultrasonic degradation. Meanwhile, the thermal stability of polysaccharides increased after ultrasonic treatment. After administration by GLP and GLPUD four weeks, body weight, visceral index, atherosclerosis index (AI) and biochemical indicators in serum and in liver were determined. The results showed that GLPUD had stronger hypolipidemic and antioxidant activities than GLP. GLPUD was more effective than the GLP for reducing AI, total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), raising high density lipoprotein (HDL-C) (p < 0.01), reducing malondialdehyde (MDA) content, as well as increasing the glutathione peroxidase (GSH-Px) in mice serum, increasing superoxide dismutase (SOD) activity and reducing MDA content in liver (p < 0.05 or p < 0.01). In addition, the histopathological observations of mice livers showed that GLPUD could significantly improve lipid metabolism disorder in hepatocytes. Thus, GLPUD might be tested as a more effective hypolipidemic drug.

Ionic Adsorption and Desorption of CNT Nanoropes.

A nanorope is comprised of several carbon nanotubes (CNTs) with different chiralities. A molecular dynamic model is built to investigate the ionic adsorption and desorption of the CNT nanoropes. The charge distribution on the nanorope is obtained by using a modified gradient method based on classical electrostatic theory. The electrostatic interactions among charged carbon atoms are calculated by using the Coulomb law. It was found here that the charged nanorope can adsorb heavy metal ions, and the adsorption and desorption can be realized by controlling the strength of applied electric field. The distance between the ions and the nanorope as well as the amount of ions have an effect on the adsorption capacity of the nanorope. The desorption process takes less time than that of adsorption. The study indicates that the CNT nanorope can be used as a core element of devices for sewage treatment.

16-nor Limonoids from Harrisonia perforata as promising selective 11ß-HSD1 inhibitors.

Two new 16-nor limonoids, harperspinoids A and B (1 and 2), with a unique 7/5/5/6/5 ring system, have been isolated from the plant Harrisonia perforate together with a known one, Harperforin G (3). Their structures were elucidated by NMR spectroscopy, X-ray diffraction analysis and computational modelling. Compound 1 exists as polymorphic crystals. Conformations of 1 in solution were further discussed based on the computational results. These compounds exhibited notable inhibitory activity against the 11ß-HSD1 enzyme. Compound 3 had potencies for the inhibition of human 11ß-HSD1 with high selectivity against 11ß-HSD2 (IC50 0.58 µM, SI > 174). Molecular docking and quantitative structure-activity relationship studies revealed a mixed regulatory mechanism.

[Effect of hypoxia on the viability of rat brain astrocytes in vitro].

OBJECTIVE: To investigate the effect of hypoxia on the viability of rat brain astrocytes in vitro and determine the optimal duration of hypoxic treatment for studying the biological behavior of the in vitro cultured cells in response to hypoxia. METHODS: Rat astrocytes were cultured and identified by glial fibrillary acidic protein (GFAP) immunofluorescent staining. After incubation with 5%; CO(2)+95%; N(2) for different time to induce hypoxia, the cells were harvested and observed microscopically for morphological changes and counting of the dead cells. The culture media of the cells were also collected and pO(2), concentrations of glucose and lactate as well as lactate dehydrogenase (LDH) activity were analyzed. RESULTS: As the hypoxia prolonged, the astrocytes appeared swollen and floating in the medium with some becoming necrotic. Compared with the cells in the control group, the changes in the number of necrotic cells, concentrations of glucose and lactate, and LDH activity in the cells of the hypoxic group were significantly different after 10 h of hypoxia (P<0.05). However, the flat and polygonal morphology of the astrocytes almost remained unchanged after 8 h of hypoxia in spite of significant changes in the concentrations of glucose and lactate and LDH activity. CONCLUSION: Hypoxia induced injury and necrosis of rat brain astrocytes in vitro, and 8 h of hypoxia can be the optimal time point for studying the biological behaviors of the cells in response to hypoxia.

Anti-candidal activity of a novel peptide derived from human chromogranin A and its mechanism of action against Candida krusei.

Candida species (Candida spp.) are important fungal pathogens, which cause numerous clinical diseases associated with significant mortality and morbidity in healthcare settings. In our previous study, we identified a recombinant peptide, chromogranin A (CGA)-N46, corresponding to the N-terminal Pro31-Gln76 sequence of human CGA, that exhibited antifungal activity against Candida albicans. The present study investigated the antifungal activity of CGA-N46, and its underlying mechanism, against numerous Candida spp. CGA-N46 inhibited the growth of all of the tested Candida spp., of which Candida krusei exhibited the greatest sensitivity. CGA-N46 was able to disrupt the stability of the phospholipid monolayer without damaging the integrity and permeability of the outer membrane of C. krusei cells, and induced cytoplasm vacuolization and mitochondrial damage. In addition, treatment of C. krusei with CGA-N46 was associated with decreased levels of intracellular reactive oxygen species, a reduction in the mitochondrial membrane potential, and DNA synthesis inhibition. The results of the present study suggested that CGA-N46 was able to pass through the cell membrane of Candida spp. by temporarily destabilizing the phospholipid membrane, which in turn led to mitochondrial dysfunction and inhibition of DNA synthesis. Therefore, CGA-N46 may be considered a novel antifungal compound for the treatment of patients with C. krusei infections.

The HIF-2alpha dependent induction of PAP and adenosine synthesis regulates glioblastoma stem cell function through the A2B adenosine receptor.

Glioblastomas are lethal tumors characterized by malignant proliferation and recurrence promoted partly by glioblastoma stem cells (GSCs). GSCs are known to be regulated by hypoxia, but the mechanisms involved in this regulation are not fully understood. We now demonstrate that hypoxia-inducible factor HIF2α and prostatic acid phosphatase (PAP) are preferentially expressed in hypoxic GSCs in comparison with non-stem tumor cells and normal neural stem cells and that PAP is regulated by HIF2α. Targeting PAP in hypoxic GSCs inhibits self-renewal and proliferation in vitro and attenuates tumor initiation potential of GSCs in vivo. Using specific adenosine receptor antagonists, we further find that the pro-proliferative role of PAP is stemmed from stimulated A2B adenosine receptors. Moreover, selective blockage of A2B receptor or knockdown of PAP or A2B on hypoxic GSCs results in significant reduction of phosphorylation of Akt and Erk-1/2. Our results demonstrate that PAP may play a pro-proliferative role in hypoxic GSCs with a HIF2α-induction pattern, which may be ascribed to stimulated A2B receptors and activated Akt and Erk-1/2 pathways. Therefore, we propose that these identified molecular regulators of GSCs in the hypoxic niche might represent promising targets for antiglioblastoma therapies.