High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

OBJECTIVE: The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. METHODS: A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. RESULTS: The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. CONCLUSIONS: This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

Activation of Follicle-Stimulating Hormone Receptor in Adrenal Zona Fasciculata Cells Promotes Cortisol Secretion: Implications for the Development of Menopause-Associated Diseases.

OBJECTIVE: Changes in postmenopausal hormone levels are associated with a variety of disorders. This study elucidated the mechanism by which follicle-stimulating hormone (FSH) increases cortisol production involved in development of menopause-related diseases. METHODS: The expression of FSH receptors (FSHRs) in murine adrenal zona fasciculata (AZF) cells and ATC7 cells was verified by immunofluorescence, western blotting and RT-PCR. The function of FSHR in promoting cortisol production was analyzed by cell culture and molecular biological methods. FSHR signaling pathways in ATC7 cells were analyzed by ELISA, qRT-PCR, and western blotting. Further, a mouse model was established by ovariectomy. Ovariectomized mice were treated with GnRHa. Ovariectomized mice initially received physiological doses of estrogen and were then injected with recombinant FSH. Then serum FSH, luteinizing hormone (LH), estradiol, and cortisol, and bone mineral density (BMD), blood pressure (BP) and heart rate (HR) were determined. RESULTS: FSHRs were expressed in murine AZF cells and ATC7 cells. FSH accelerated cortisol production through activated protein kinase A (PKA), cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), protein kinase B (PKB/AKT) and 5' AMP-activated protein kinase (MAPK) signaling pathways by Gsα-coupled FSHRs in ATC7 cells. Serum FSH levels (P<0.001) were elevated in ovariectomized mice with concurrent increases in cortisol (P<0.01), areal BMD (aBMD) (P<0.05), volumetric BMD (vBMD) (P<0.05), systolic BP (SBP) (P<0.05), diastolic BP (DBP) (P<0.05), and HR (P<0.05). However, the administration of GnRHa suppressed the increase in FSH levels and the elevation of cortisol, aBMD, vBMD, SBP, DBP, and HR induced by ovariectomy, even in the presence of normal serum estradiol levels. CONCLUSION: The study findings indicate that elevated FSH levels stimulate cortisol secretion, through a mechanism related to FSHRs expression in AZF cells.

[Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry].

Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram-negative organisms and some anaerobes. The components of macrolide antibiotics are generally complicated. Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control. Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles. In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16-membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.

Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods.

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

Artesunate inhibits epithelial-mesenchymal transition in non-small-cell lung cancer (NSCLC) cells by down-regulating the expression of BTBD7.

In recent years, more and more studies have shown that antiparasitic drugs can affect a variety of biological processes of tumor cells and exhibit a potential anti-tumor activity. Although artesunate (ART), a strong bioactive derivative of artemisinin and widely used clinically against malaria, was found to have an inhibitory effect on tumor cells, it is still unclear whether ART could regulate the tumor malignancy of non-small-cell lung cancer (NSCLC) cells. In this study, we aimed to investigate the effect of ART on migration capacities in NSCLC cell lines of A549 and H1975. Cell migration capacity was remarkably inhibited by ART treatment. The expression of epithelial marker E-cadherin was upregulated, while mesenchymal markers (N-cadherin, vimentin and FN1) were inhibited by ART in both protein and mRNA levels in A549 and H1975 cells, indicating ART could suppress the epidermal interstitial transformation (EMT) of NSCLC cells. Meanwhile, BTBD7 was found highly expressed in tumor tissues of NSCLC patient and associated with poor prognosis. The anti-migration activity of ART was found to be mediated by the inhibition of BTBD7 mRNA expression and was reversed when the cells were transiently transfected with the BTBD7 overexpression plasmid. Our study demonstrated the potent anti-migratory activity of ART, thereby presenting it as a new candidate for clinical therapy in NSCLC.

[In vivo anti-tumor effect of Oxytropis kansuensis alkaloid fraction and its influence on immune function].

OBJECTIVE: To study the in vivo anti-tumor effect of Oxytropis kansuensis alkaloid fraction (OKAF) in live cancer cell line H22 and its influence on immune function in KM mice. METHODS: Sixty mice with transplanted mouse originated liver cancer cell line H22 were divided randomly into five groups, the three OKAF groups were administrated with OKAF in low (8 mg/kg), moderate (16 mg/kg) and high (32 mg/kg) dosage, the negative control group was administrated with normal saline 20 mL/kg and the positive control with 5-FU 15 mg/kg, respectively, once a day for 10 successive days. Then the average tumor weight (TW), tumor inhibition rate (TIR), spleen index (SI), and thymus index (TI) in them were calculated, and the spleen cell proliferation rate (SCPR) was measured by MTT assay. RESULTS: TIR of low, moderate and high dosage of OKAF was 23%, 29% and 43% respectively. SI and TI in all the three OKAF groups were higher than those in the negative control group, and the two indexes in the moderate and ligh dosage OKAF groups were higher than those in the positive control group (P < 0.05). SCPR in OKAF groups at 24 h, 48 h and 72 h were higher than those in both two control groups at the same time. CONCLUSION: OKAF shows inhibitory action on liver cancer cell line H22 tumor in KM mice, and it could also enhance the immune function of mice.

[Effect of Warm Acupuncture on the Levels of Serum Immunoglobulin E, Interleukin-1 ß and Tumor Necrosis Factor-α in Rats with Allergic Rhinitis].

OBJECTIVE: To observe the effect of warm acupuncture on behavior and contents of serum immunoglobulin E(IgE), interleukin-1 ß(IL-1 ß) and tumor necrosis factor-α(TNF-α) in allergic rhinitis(AR) rats, so as to explore its mechanism underlying improving AR. METHODS: Forty Wistar rats were randomly divided into four groups: control group, model group, medication group and warm acupuncture group(10 rats/group). The AR model was established by intraperitoneal injection of sensitization and nasal drip. The rats in the medication group were given fluticasone propionate nasal spray, daily for 10 days. Warm acupuncture was applied to "Fengchi"(GB 20), "Yintang"(GV 29), "Yingxiang"(LI 20) for 60 seconds, once daily for 10 days. Behavioral scores were used to evaluate behavioral changes in rats. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression levels of serum IgE, IL-1 ß and TNF-α. RESULTS: Behavioral scores of the model group were significantly higher than those of the control group 0, 3, 7 and 10 days after modeling (P<0.05). After treatment, the behavioral scores of medication group and the warm acupuncture group were lower than those of the model group (P<0.05), and the score was more lower in the warm acupuncture group than in the medication group (P<0.05). Compared with the control group, the levels of serum IgE, IL-1 ß and TNF-α in the model group were all increased (P<0.01), while the levels of serum IgE, IL-1 ß and TNF-α were decreased in the medication and warm acupuncture groups after treatment in comparison with the model group (P<0.05, P<0.01). Compared with the medication group, the levels of serum IgE, IL-1 ß and TNF-α were significantly lower in the warm acupuncture group (P<0.05, P<0.01). CONCLUSION: The expression levels of IgE, IL-1 ß and TNF-α were elevated in serum after AR attack. Warm acupuncture can improve the symptoms of AR rats, which may be associated to its effect in inhibiting the expression of serum IgE, IL-1 ß and TNF-α.

A simple method for HPLC retention time prediction: linear calibration using two reference substances.

BACKGROUND: Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately. But the reference substances are costly. Thus, the relative retention (RR) method has been widely adopted in pharmacopoeias and literatures for characterizing HPLC behaviors of those reference substances unavailable. The problem is it is difficult to reproduce the RR on different columns due to the error between measured retention time (tR) and predicted tR in some cases. Therefore, it is useful to develop an alternative and simple method for prediction of tR accurately. METHODS: In the present study, based on the thermodynamic theory of HPLC, a method named linear calibration using two reference substances (LCTRS) was proposed. The method includes three steps, procedure of two points prediction, procedure of validation by multiple points regression and sequential matching. The tR of compounds on a HPLC column can be calculated by standard retention time and linear relationship. RESULTS: The method was validated in two medicines on 30 columns. CONCLUSION: It was demonstrated that, LCTRS method is simple, but more accurate and more robust on different HPLC columns than RR method. Hence quality standards using LCTRS method are easy to reproduce in different laboratories with lower cost of reference substances.

Bioanalytical LC/MS study of potential bacterial transglycosylation inhibitors.

Bacterial transglycosylation is an interesting target in antibiotic drug development. An in vitro transglycosylation assay was developed and used to search for possible inhibitors of Staphylococcus aureus Penicillin Binding Protein 2-mediated transglycosylation. Since the substrate, Lipid II, has no UV-chromophore, the assay relies on LC coupled to MS for analysis of the incubation mixtures. Extracts from Thymus sipyleus, Salvia verticillata, Salvia virgata and Oolong tea were tested, as well as epigallocatechin gallate and ursolic acid, which are chemical compounds derived from plants. Matrix effects hampered Lipid II quantification in samples treated with very high concentrations of extracts. None of these extracts or isolated compounds appeared to have inhibitory activities towards the transglycosylation function of Penicillin Binding Protein 2.

[Analysis of the response factors of different aminoglycoside antibiotics detected by evaporative light-scattering detector].

AIM: To analyze if the response factors of different aminoglycoside antibiotics detected by evaporative light-scattering detector (ELSD) are the same. If they are, then ELSD can be applied to the quality analysis of this class of antibiotics. METHODS: The response factors of five different aminoglycosides (amikacin, sisomicin, netilmicin, etimicin and vertilmicin) detected by ELSD were determined by using a Diamonsil C18 column (150 mm x 4.6 mm, 5 microns) as analytical column and 0.2 mol.L-1 trifluoroacetic acid-methanol (94:6) as mobile phase at a flow rate of 0.6 mL.min-1, the temperature of the drift tube was set at 110 degrees C, and the flow of carrier gas at 2.80 L.min-1. Detector responses (A) and the amount of injection of each substance (m) were fitted to the logarithmic regression: logA = blogm + loga. RESULTS: The linear regression equation obtained were amikacin: Y = 1.46X + 5.07, gamma = 0.9997; sisomicin: Y = 1.51X + 5.03, gamma = 0.9997; netilmicin: Y = 1.52X + 4.88, gamma = 1.000; etimicin: Y = 1.46X + 4.85, gamma = 0.9999; vertilmicin: Y = 1.41X + 4.90, gamma = 0.9998. The differences between them were negligible. CONCLUSION: Different aminoglycosides can give the same responses with ELSD detection. So, the HPLC-ELSD methods can be applied to the analysis of impurities, the control of the ratio of multi-components drug and the determination of new substances by using another substance as reference, etc.

The selection of suitable columns for a reversed-phase liquid chromatographic separation of beta-lactam antibiotics and related substances via chromatographic column parameters.

The selection of RP-LC columns suitable for a particular analysis in official compendia is difficult as only a general description of the stationary phase in the description of a LC method is given. General methods to characterize RP-LC columns often assume that each of the column parameters is equally important. This can cause the user to select columns inappropriate for particular analyses. This paper focuses on the relationship between the critical peak pairs and the column parameters (H, S, A, B, and C) in the Snyder/Dolan column characterization methodology to find the key parameters influencing real separations. Some varieties of ß-lactam antibiotics and their related compounds were used as test compounds. We found column parameter A to be the most important factor affecting their separation. Parameters B and C also played an important role in some separation processes. This indicated that the hydrogen bonding of column and solute can directly affect the separation of ß-lactam antibiotics. Choosing columns for which column parameter A is near 0.1 can facilitate the ideal separations of impurities from ß-lactam antibiotics. The most suitable column for any common pharmaceutical analysis could be selected easily if the key column parameters would be given in the description of the chromatographic method. For these reasons, key column parameters should be listed in the monographs of official compendia.

Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry.

Reversed-phase liquid chromatography coupled with photo-diode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize the components of meleumycin, a 16-membered macrolide antibiotic produced by fermentation. In total 31 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 others that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known components. Their ultraviolet spectra and chromatographic behavior were used to confirm the proposed structures: e.g. λmax shift from 232 nm to 282 nm would indicate the presence of an α-, ß-, γ-, δ-unsaturated ketone instead of a normal α-, ß-, γ-, δ-unsaturated alcohol in the 16-membered ring of the examined components. Compared to other methods, this LC/MS(n) method is particularly advantageous to characterize minor components at trace levels in multi-components antibiotics, in terms of sensitivity and efficiency.

Identification of the components of bitespiramycin by liquid chromatography-mass spectrometry.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, ß-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics.

Impurity profiling of acetylspiramycin by liquid chromatography-ion trap mass spectrometry.

Investigation of acetylspiramycin (ASPM) and its related substances was carried out using a reversed-phase liquid chromatography/tandem mass spectrometry method. The identification of impurities in the ASPM complex was performed with a quadrupole ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. A total of 83 compounds were characterized in commercial samples, among which 31 impurities that had never been reported and 31 partially characterized impurities were deduced using the collision-induced dissociation (CID) spectra of major ASPM components as templates. Most of the major impurities arise from the starting materials and the synthesis process. This work provides very useful information for quality control of ASPM and evaluation of its synthesis process.