Noninvasive isolation of transcervical trophoblast cells for fetal identification.

OBJECTIVE: To identify trophoblastic cells retrieved from the cervix at a gestational age (GA) of 5-9 weeks by a noninvasive modality in fetuses. METHOD: Transcervical cells (TCCs) were noninvasively extracted by a cytobrush using the Papanicolaou sampling method. TCCs were immunostained with antihuman leukocyte antigen (HLA)-G and anticytokeratin (CK)-7 antibodies to identify trophoblastic cells. Maternal finger blood, gestational sacs, and 20 trophoblastic cells collected by a laser-guided microscopic single-cell capture system were examined and compared by short tandem repeat (STR) genotyping. RESULTS: Forty-nine pregnant women with GA of 5-9 weeks and six nonpregnant healthy women were included in the study. Trophoblastic cells were identified in 37 (75.5%) TCC samples, among which 34 (69.4%) were eligible for STR genotyping analysis. No trophoblastic cells were identified in nonpregnant healthy women. The STR genotyping analyses revealed 24 female and 10 male fetuses. TCC trophoblastic cells exhibited the same STR profiles as gestational sac and maternal blood in all samples, which indicated that the TCC trophoblastic cells originated from fetuses. CONCLUSION: This primary study validated that trophoblastic cells from TCCs at GA 5-9 weeks originated from the fetus. Further studies are needed to verify whether this method can be used for early noninvasive prenatal diagnosis and paternity testing.

Drug molecules bridge with small gatekeeper to co-block mesoporous silica nanoparticles for drug delivery.

In this study, a filter-like blocking system based on MSN with small gatekeeper 5- mercapto-2 nitrobenzoic acid (MNBA) has developed. The MNBA grafted nanoparticle MSN-SS-MNBA shows excellent blocking performance with negligible leakage when loaded with doxorubicin (DOX), and the release profiles illustrate stimuli-responsive property when triggered by GSH. Viability experiments indicate that MSN-SS-MNBA has obvious inhibition for both Hela cells and HCT116 cells, while showing good biocompatibility for L929 cells, which suggests that the modified MNBA has a synergistic effect on cancer cells-killing. Since the small grafted molecule MNBA cannot block the channels of MSN via self-assembly, a filter-like blocking model that the loaded drug bridged with modified MNBA to fulfill the blocking process is proposed. The novel blocking strategy provides a new possible way for pore blocking, and the small nanovalve can be used as synergistic molecule for cancer therapy.

An Adjustable pH-Responsive Drug Delivery System Based on Self-Assembly Polypeptide-Modified Mesoporous Silica.

In this study, an adjustable pH-responsive drug delivery system using mesoporous silica nanoparticles (MSNs) as the host materials and the modified polypeptides as the nanovalves is reported. Since the polypeptide can self-assemble via electrostatic interaction at pH 7.4 and be disassembled by pH changes, the modified poly(l-lysine) and poly(l-glutamate) are utilized for pore blocking and opening in the study. Poly(l-lysine)-MSN (PLL-MSN) and poly(l-glutamate)-MSN (PLG-MSN) are synthesized via the ring opening polymerization of N-carboxyanhydrides onto the surface of mesoporous silica nanoparticles. The successful modification of the polypeptide on MSN is proved by Zeta potential change, X-ray photoelectron spectroscopy (XPS), solid state NMR, and MALDI-TOF MS. In vitro simulated dye release studies show that PLL-MSN and PLG-MSN can successfully load the dye molecules. The release study shows that the controlled release can be constructed at different pH by adjusting the ratio of PLL-MSN to PLG-MSN. Cellular uptake study indicates that the drug is detected in both cytoplasm and nucleus, especially in the nucleus. In vitro cytotoxicity assay indicates that DOX loaded mixture nanoparticles (ratio of PLL-MSN to PLG-MSN is 1:1) can be triggered for drug release in HeLa cells, resulting in 88% of cell killing.

Magnetically Stimulated Drug Release Using Nanoparticles Capped by Self-Assembling Peptides.

A novel self-assembling peptide-functionalized core-shell mesoporous silica nanoparticle was developed as a drug carrier. Superparamagnetic manganese- and cobalt-doped iron oxide nanoparticles formed the core for the mesoporous silica shell coating. On the silica outer shell, the peptide Boc-Phe-Phe-Gly-Gly-COOH was covalently conjugated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt coupling. The self-assembling property of the peptide at physiological temperature was utilized to block the pore openings, while the disassembly at elevated local particle temperature released cargo molecules without bulk heating that would cause cell damage. Both conventional heating and heating in an alternating magnetic field were tested for the release of fluorescein and daunorubicin. In vitro experiments showed high cytotoxicity on pancreatic carcinoma cells (PANC-1) when this delivery system was activated by an alternating magnetic field, while control particles without drugs showed no obvious cytotoxicity.

Expression of MMIF, HIF-1α and VEGF in Serum and Endometrial Tissues of Patients with Endometriosis.

The aim of this study was to investigate the expression of macrophage migration inhibitory factor (MMIF), hypoxia-inducible factor-1 a (HIF-1α) and vascular endothelial growth factor (VEGF) in the serum and endometrial tissues of patients with endometriosis (EM) and the clinical significance. Eighty EM patients [American Reproductive Association stage I (n=20), stage II (n=22), stage III (n=21) and stage IV (n=17)] were enrolled and divided into mild (10-14 points, n=28), moderate (16-24 points, n=27) and severe (26-30 points, n=25) dysmenorrhea groups. The control group included 40 healthy women of childbearing age who underwent routine healthcare examinations in the enrolment period. The expression of MMIF, HIF-1α and VEGF in the serum and endometrial tissues was measured by enzyme-linked immunosorbent assay and Western blotting, respectively. Meanwhile, the sensitivity and specificity of serum MMIF, HIF-1α, and VEGF when separately used as single indexes or jointly used as one index were examined as well. The results showed that serum concentrations of MMIF, HIF-1α, and VEGF were significantly higher in EM patients than in controls (P<0.05). The expression of all three proteins in both serum and endometrial tissues increased significantly with the R-AFS stage (P<0.05) and with dysmenorrheal severity (P<0.05). The sensitivity and specificity of the combined detection of serum MMIF, HIF-1α, and VEGF levels were significantly higher than those of single index detection (P<0.05). In conclusion, the expression of MMIF, HIF-1α, and VEGF in the serum and endometrial tissues may be used to assess the stage of EM and the severity of dysmenorrhea. Combined evaluation of MMIF, HIF-1α, and VEGF significantly improves the diagnostic sensitivity and specificity.

[Effect of Xinfeng Capsule on Beclin1/PI3K-AKT-mTOR of Adjuvant Arthritis Rats].

Objective To observe expression levels of autophagy related 5,7,12 mRNA (Atg5, 7,12), microtubule-associated protein 1 light chain 3 (LC3-II), Beclin1, phosphatidylinositol 3-kinase (PI3K) , protein kinase B (AKT) , mammalian target of rapamycin (mTOR) , IL-1 ß, TNF-α, IL-4, and IL- 10 in adjuvant arthritis (AA) rats, and effects of Xinfeng Capsule (XFC) on them. Methods Totally 48 male SD rats were divided into 4 groups, i.e., the control group, the model group, the Western medicine (WM) group (leflunomide, 5. 0 mg/kg) , the Chinese medicine (CM) group (Xinfeng Capsule, 3.0 g/kg) , 12 in each group. Thirty days after medication body weight (BW) , toe swelling degree (E%) , arthritis in- dex (AI) , and pathological changes of ankle joint and ultrastructural changes were observed. mRNA ex- pressions of Atg5, 7, 12, protein expressions of LC3-L , Beclin1 , PI3K, AKT, mTOR, serum contents of IL-1 ß, TNF-α, IL-4, and IL-10 were detected. Results Compared with the normal group, E%, Al, IL-1 ß and TNF-α increased; BW, levels of IL-4 and IL-10, mRNA expressions of Atg5 and Atg12, protein ex- pressions of LC3-ll and Beclin1 decreased (P <0.01, P <0.05), protein expressions of PI3K, AKT, mTOR increased (P<0.01) in the model group. Compared with the model group, E%, Al, mRNA expres- sions of IL-1ß , TNF-α-, and Atg12, protein expressions of PI3K, AKT, and mTOR decreased (P <0.01, P<0.05), IL-4, IL-10, protein expressions of LC3-II and Beclinl increased (P <0.01, P <0.05) in the two medicated groups. Atg5 mRNA expression decreased (P <0.01) , Atg7 mRNA expression increased (P < 0.05) in the WM group. Compared with the WM group,BW, IL-4, mRNA expressions of Atg5 and Atg12, protein expressions of PI3K and mTOR increased in the CM group (P <0.01 , P <0. 05). Conclusions The level of autophagy in AA rats was decreased, leading to excessive proliferation of synovial cells, swollen joints, elevated proinflammatory factors, decreased inflammatory factors, resulting in inflamma- tory reactions of joints. XFC could improve Al, toe swelling degree, and expressions of synovium autoph- agy related genes and proteins.

[Effect of Xinfeng Capsule on AS Patients and Their Serum Immunoglobulin Subtypes and Peripheral Lymphocyte Autophagy].

OBJECTIVE: To observe the effect of Xinfeng Capsule (XFC) on ankylosing spondylitis (AS) patients' symptoms and signs, serum immunoglobulin levels, peripheral blood lymphocyte autophagy protein, autophagy gene, and to explore its mechanism. METHODS: Totally 59 AS patients were assigned to the treatment group (39 cases) and the control group (20 cases) according to random digit table. Patients in the treatment group received XFC, 0.5 g each pill, three pills each time, 3 times per day, while those in the control group received sulfasalazine (SASP), 0.25 g per tablet, 4 tablets each time, twice per day. Three months consisted of one therapeutic course. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) were statistically calculated. Serum immunoglobulins (IgG1, IgG2, IgG3, IgG4, IgA , SIgA, and IgM) were detected using ELISA. Changes of Beclin1, LC3-II, phosphatidylinositol 3-kinase (PI3K), Akt, the mammalian target of rapamycin (mTOR) were detected using Western blot. Serum autophagy related genes such as Atg1, Atg5, Atg12, Atg13, and Atg17 were detected using the polymerase chain reaction (PCR). The correlation between immunoglobulin subtypes and autophagy gene in AS patients using Spearman correlation. RESULTS: Compared with before treatment, BASDAI, IgG1, lgG3, and IgA decreased (P < 0.01); PI3K, Akt, and mTOR protein expressions decreased (P < 0.01); ATG1, ATG12, ATG13, and ATG17 mRNA expressions decreased, ATG5 mRNA expression increased (P < 0.01) in the treatment group. But BASDAI, IgG1, and IgA levels decreased (P < 0.05, P < 0.01); PI3K, Akt, and mTOR protein expressions decreased (P < 0.05); ATG1 and ATG13 mRNA expressions decreased (P < 0.05, P < 0.01) in the control group. Compared with the control group, BASDAI, IgG1, and IgA levels decreased (P < 0.05); PI3K, Akt, mTOR protein expressions decreased (P < 0.01); ATG12 and ATG17 mRNA expression decreased, ATG5 mRNA expression increased (P < 0.01) in the XFC group. Correlation analysis showed AS patients' IgG1, IgG2, IgG3, IgA, SIgA, IgM had negative correlation with ATG17; IgG4 and ATG17 were positively correlated (P < 0.05, P < 0.01). CONCLUSION: XFC could elevate clinical efficacy of AS patients and enhance their autophagy, which might be achieved by acting on PI3K/Akt/mTOR signal, affecting autophagy gene and autophagy protein expression, taking part in the regulation of proliferation and differentiation of lymphocyte B, and strengthen humoral immunity.

[Simultaneous determination of 20 illegally added anti-diabetic chemical components in hypoglycemic and weight-reducing health foods by ultra-high performance liquid chromatography-tandem mass spectrometry].

A rapid method for the simultaneous screening and detection of 20 illegally added anti-diabetic chemical components in hypoglycemic and weight-reducing health foods was developed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After extracted by methanol, the sample was separated on a Poroshell 120 EC C18 column (100 mm x 2.1 mm, 2.7 microm) with the gradient elution of 5 mmol/L ammonium acetate and acetonitrile as mobile phases. The electrospray ionization (ESI) source in positive or negative ion mode was used for multiple reaction monitoring (MRM) mode. The 20 illegally added chemical components showed good linear relationships with the correlation coefficients more than 0.99. The recoveries were in the range of 75.9% - 114.0%, and the relative standard deviations (RSDs) were all not more than 11.3%. The limits of detection (LODs) were all in the range of 0.3 - 1.5 microg/L. This method is rapid, simple, sensitive, accurate and of good specificity for cracking down illegally added anti-diabetic chemical components.

[Role of Notch-Jagged/Delta signaling pathway in arthritis rats of reduced lung function induced by adjuvant].

OBJECTIVE: To observe the changes of pulmonary function and Notch signaling pathway of lung tissues in adjuvant-induced arthritis rats, and to investigate the mechanism of reduced lung function. METHODS: A total of 30 rats were randomly divided into a normal group and a model group. Rats in the model group were induced to establish the adjuvant arthritis AA model by intradermally injecting 0.1 mL Freund's complete adjuvant into the right paw. After 30 days, we observed the paw edema volume, arthritis index, pulmonary function, histomorphology, and Notch receptor/ligand of the lung tissue. RESULTS: Compared with the normal group, the paw edema volume, arthritis index, average expiratory flow within 0.3 s (FEV0.3/FVC), and the level of Notch3, Notch4 and Jagged2 of the lung tissue in the model group was significantly increased, while maximum expiratory flow at 50% of vital capacity (FEF50), maximum expiratory flow at 75% of vital capacity (FEF75), forced expiratory flow (PEF) and the expression of Notch1 of Jagged1 and Delta1 in the lung were significantly decreased (P<0.05, P<0.01). There were significant positive correlations between FEV0.3/FVC and Notch4. FEV0.3/FVC, FEF25, FEF50 and Notch3, Delta1 were negatively correlated, respectively (P<0.05, P<0.01). CONCLUSION: While arthritis occurs in AA rats, pulmonary function declines and significantly correlates with the expression of Notch receptor/ligand. The deposition of immune complex in the lung after the injection of CFA activates the Notch signaling pathway, and results in further decline of pulmonary function by signaling cascades.

[Simultaneous determination of 23 sedative drugs in health foods by high performance liquid chromatography-tandem mass spectrometry].

An analytical method using HPLC-MS/MS was developed for qualitative and quantitative analysis of 23 sedative drugs in health foods. The method was based on the sonication-assisted extraction of the health food samples using 10 mL methanol. The extract was then isolated by centrifugation and the supernatant was separated on an Agilent Eclipse Plus C, column with gradient elution at a flow rate of 300 microL/min. A binary mobile phase was 10 mmol/L ammonium formate (solvent A) and acetonitrile/methanol (1:1, v/v; solvent B). The electrospray ionization (ESI) source in positive ion mode or negative ion mode was used for multiple reaction monitoring (MRM). The external standard method was used for the quantification. The calibration graphs were linear in their concentration ranges with the correlation coefficients more than 0. 990. The limits of detection (LOD, S/N= 3) were between 0. 02 - 1.0 microg/L. The recoveries for all the drugs in health foods were 82. 3% - 114. 8% with the relative standard deviations less than 14. 1% at three spiked levels. Thirteen kinds of health foods were tested, in which meprobamate and oxazepan were found in one sample separately, and zaleplon was found in two samples. The method is specific, sensitive, easy and quick and suitable for the confirmation and quantification of the 23 sedative drugs in health foods.

Designed amphiphilic peptide forms stable nanoweb, slowly releases encapsulated hydrophobic drug, and accelerates animal hemostasis.

How do you design a peptide building block to make 2-dimentional nanowebs and 3-dimensional fibrous mats? This question has not been addressed with peptide self-assembling nanomaterials. This article describes a designed 9-residue peptide, N-Pro-Ser-Phe-Cys-Phe-Lys-Phe-Glu-Pro-C, which creates a strong fishnet-like nanostructure depending on the peptide concentrations and mechanical disruptions. This peptide is intramolecularly amphiphilic because of a single pair of ionic residues, Lys and Glu, at one end and nonionic residues, Phe, Cys, and Phe, at the other end. Circular dichroism and Fourier transform infrared spectroscopy analysis demonstrated that this peptide adopts stable beta-turn and beta-sheet structures and self-assembles into hierarchically arranged supramolecular aggregates in a concentration-dependent fashion, demonstrated by atomic force microscopy and electron microscopy. At high concentrations, the peptide dominantly self-assembled into globular aggregates that were extensively connected with each other to form "beads-on-a-thread" type nanofibers. These long nanofibers were extensively branched and overlapped to form a self-healing peptide hydrogel consisting of >99% water. This peptide can encapsulate the hydrophobic model drug pyrene and slowly release pyrene from coated microcrystals to liposomes. It can effectively stop animal bleeding within 30 s. We proposed a plausible model to interpret the intramolecular amphiphilic self-assembly process and suggest its importance for the future development of new biomaterials for drug delivery and regenerative medicine.

[Improving the solubility of fraxinellone to increase its oral bioavailability and hepatoprotective action against acute liver injury in mice].

Fraxinellone, the major component of Cortex Dictamni, is naturally degraded limonids compound. Fraxinellone has significant anti-inflammatory activity in acute liver injury model. However, the low solubility and permeability of fraxinellone limited its potential application and even therapeutic effects. The aim of the paper is to increase oral bioavailability of fraxinellone, thus improving its hepatoprotection effect in vivo. We evaluated the effects of different pH values and different solubilizer (PEG 6000, PVP K30, HP-beta-CD, F68 and SDS) on the solubility of fraxinellone. The results showed that HP-beta-CD increased solubility of fraxinellone up to 155 times compared to that of water. More than 2. 1 mg mL1 fraxinellone can be resolved when adding 20% HP-beta-CD. Mouse acute liver injury model induced hy CCl4 was used to evaluate in vivo activity of fraxinellone with or without HP-beta-CD. The result shows that the hepatoprotective activity of fraxinellone in 20% HP-beta-CD solution has been significantly improved compared with that of fraxinellone solution without HP-beta-CD: the former inhibited 59 percent the increase of enzyme activity of ALT in liver, while the latter only inhibited 20 percent. A LC-MS/MS method was also developed to determine the oral bioavailability of fraxinellone. Fraxinellone solution with or without HP-betaCD were administered intra-gastrically to rats, and it was found that the bioavailahility of fraxinellone with HP-beta-CD was 23%, while only 5% without HP-beta-CD. The result showed that HP-beta-CD can significantly increase the solubility and permeability of fraxinellone, and improve bioavailability 3. 5 fold in vivo acute liver injury model as well as administration.

Improving the solubility of ampelopsin by solid dispersions and inclusion complexes.

The aim of this study was to increase the solubility of ampelopsin (AMP) in water by two systems: solid dispersions with polyethylene glycol 6000 (PEG 6000) or polyvinylpyrrolidone K-30 (PVP K30) and inclusion complexes with beta-cyclodextrin (BCD) and hydroxypropyl-beta-cyclodextrin (HPBCD). The interaction of AMP with the hydrophilic polymers was evaluated by differential scanning calorimetry (DSC), Fourier transformation-infrared spectroscopy (FTIR), scanning electron microscopy (SEM). The results from DSC, FTIR and SEC analyses of solid dispersions and inclusion complexes showed that AMP might exist as an amorphous state or as a solid solution. On the other hand, the SEM images of the physical mixtures revealed that to some extent the drug was present in a crystalline form. The influence of various factors (pH, temperature, type of polymer, ration of the drug to polymer) on the solubility and dissolution rate of the drug were also evaluated. The solubility and dissolution rates of AMP were significantly increased by solid dispersions and cyclodextrin complexes as well as their physical mixtures. The improvement of solubility using polymers was in the following order: HPBCD approximately BCD>PVP K30>PEG 6000.