Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma.

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.

Metabolism, Disposition, Excretion, and Potential Transporter Inhibition of 7-16, an Improving 5-HT2A Receptor Antagonist and Inverse Agonist for Parkinson's Disease.

Compound 7-16 was designed and synthesized in our previous study and was identified as a more potential selective 5-HT2A receptor antagonist and inverse agonist for treating Parkinson's disease psychosis (PDP). Then, the metabolism, disposition, and excretion properties of 7-16 and its potential inhibition on transporters were investigated in this study to highlight advancements in the understanding of its therapeutic mechanisms. The results indicate that a total of 10 metabolites of 7-16/[14C]7-16 were identified and determined in five species of liver microsomes and in rats using UPLC-Q Exactive high-resolution mass spectrometry combined with radioanalysis. Metabolites formed in human liver microsomes could be covered by animal species. 7-16 is mainly metabolized through mono-oxidation (M470-2) and N-demethylation (M440), and the CYP3A4 isozyme was responsible for both metabolic reactions. Based on the excretion data in bile and urine, the absorption rate of 7-16 was at least 74.7%. 7-16 had weak inhibition on P-glycoprotein and no effect on the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 transporters. The comprehensive pharmacokinetic properties indicate that 7-16 deserves further development as a new treatment drug for PDP.

Development, validation and application of an ion-pair reversed-phase liquid chromatography-tandem mass spectrometry method for the quantification of nusinersen.

Background: The fully phosphorothioate-modified oligonucleotide (OGN) nusinersen has low ionization efficiency in the negative ion mode, resulting in a low mass spectrometry response. There have been no relevant reports on developing a LC-MS method for the determination of nusinersen by optimizing mobile phase composition. Materials & methods: Mobile phase additives comprised of 15 mM triethylamine/25 mM 1,1,1,3,3,3-hexafluoro-2-propanol with a pH of 9.6. Nusinersen was extracted from plasma using Oasis® HLB solid-phase extraction (Waters, MA, USA). Results & conclusion: By adjusting the pH of the mobile phase to 9.6 by optimizing the type and concentration of ion-pair reagents, a high mass spectrometry response was obtained. The developed method was applied to nusinersen and met the requirements for the pharmacokinetic study of nusinersen in rabbits.

Direct determination of nosiheptide residue in animal tissues by liquid chromatography-tandem mass spectrometry.

Because only very weak signals of fragment ions of nosiheptide can be obtained, nosiheptide is usually detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) via the determination of its hydrolyzed degradation product named HMIA in previous studies. The indirect method suffers from several problems, such as complicated samplepreparation, unavailable commercial HMIA, and the risk of the false-positive result by HMIA. However, we found that nosiheptide could produce several significant fragment ions under high collision energy (CE). Therefore, we developed a method for the direct determination of nosiheptide by LC-MS/MS in animal tissues. The sample was extracted with ACN, then degreased with n-hexane, and purified by an HLB solid-phase extraction (SPE) cartridge. After being filtered through the PTFE filter, it was analyzed by LC-MS/MS in selected reaction monitoring (SRM) mode. The influencing factors, such as mobile phase, SPE cartridge, filter material, and matrix effect, were investigated. Nosiheptide showed a good linear relationship (R2 ≥ 0.999) within the concentration range from 0.3 µg/L to 20 µg/L under optimized conditions. The limit of detection (LOD) was 0.3 µg/kg, while the limit of quantification (LOQ) was 1.0 µg/kg in chicken, bovine muscle, swine muscle, and swine liver. The average recoveries at spiked levels of 1.0, 2.0, and 10 µg/kg ranged from 83% to 101%, with the relative standard deviations (RSDs) <12%. Compared with the methods previously reported, our newly developed method was more simple, convenient, and sensitive. Moreover, it was successfully applied for the determination of nosiheptide residue in medicated chicken samples.

Immediate acupuncture with GB34 for biliary colic: protocol for a randomised controlled neuroimaging trial.

INTRODUCTION: As the main manifestation of gallstone disease, biliary colic (BC) is an episodic attack that brings patients severe pain in the right upper abdominal quadrant. Although acupuncture has been documented with significance to lead to pain relief, the immediate analgesia of acupuncture for BC still needs to be verified, and the underlying mechanism has yet to be covered. Therefore, this trial aims first to verify the immediate pain-alleviation characteristic of acupuncture for BC, then to explore its influence on the peripheral sensitised acupoint and central brain activity. METHODS AND ANALYSIS: This is a randomised controlled, paralleled clinical trial, with patients and outcome assessors blinded. Seventy-two patients with gallbladder stone disease presenting with BC will be randomised into a verum acupuncture group and the sham acupuncture group. Both groups will receive one session of immediate acupuncture treatment. Improvements in patients' BC will be evaluated by the Numeric Rating Scale, and the pain threshold of acupoints will also be detected before and after treatment. During treatment, brain neural activity will be monitored with functional near-infrared spectroscopy (fNIRS), and the needle sensation will be rated. Clinical and fNIRS data will be analysed, respectively, to validate the acupuncture effect, and correlation analysis will be conducted to investigate the relationship between pain relief and peripheral-cerebral functional changes. ETHICS AND DISSEMINATION: This trial has been approved by the institutional review boards and ethics committees of the First Teaching Hospital of Chengdu University of Traditional Chinese Medicine, with the ethical approval identifier 2019 KL-029, and the institutional review boards and ethics committees of the First People's Hospital of Longquanyi District, with the ethical approval identifier AF-KY-2020071. The results of this trial will be disseminated through peer-reviewed publications and conference abstracts or posters. TRIAL REGISTRATION NUMBER: CTR2000034432.

Effect of Parthenium hysterophorus L. Invasion on Soil Microbial Communities in the Yellow River Delta, China.

Parthenium hysterophorus L., as an invasive plant, has negatively impacted the ecosystem functioning and stability of the terrestrial ecosystem in China. However, little information was available for its effects on microorganisms in the Yellow River Delta (YRD), the biggest newly-formed wetland in China. In the present study, high-throughput sequencing technology was used to obtain the bacterial community in soils and roots of different plant species, including P. hysterophorus and some native ones in the YRD. Our results showed that the Proteobacteria, Acidobacteriota, Gemmatimonadota, and Actinobacteriota were dominant in the rhizosphere soils of P. hysterophorus (84.2%) and Setaria viridis (86.47%), and the bulk soils (80.7%). The Proteobacteria and Actinobacteriota were dominant within the root of P. hysterophorus. A total of 2468 bacterial OTUs were obtained from different groups among which 140 were observed in all the groups; 1019 OTUs were shared by P. hysterophorus non-rhizosphere soil bacteria (YNR) P. hysterophorus rhizosphere soil bacteria (YRR) groups. The indexes of the ACE (823.1), Chao1 (823.19), Simpson (0.9971), and Shannon (9.068) were the highest in the YRR groups, showing the greatest bacterial community diversity. Random forest analysis showed that the Methylomirabilota and Dadabacteria (at the phylum level) and the Sphingomonas, and Woeseia (at the genus level) were identified as the main predictors among different groups. The LEfSe results also showed the essential role of the Acidobacteriota in the YRR group. The SourceTracker analysis of the bacterial community of the YRR group was mainly from GBS groups (average 53.14%) and a small part was from YNR groups (average 6.56%), indicating that the P. hysterophorus invasion had a more significant effect on native plants' rhizosphere microorganisms than soil microorganisms. Our observations could provide valuable information for understanding the bacterial diversity and structure of the soil to the invasion of P. hysterophorus.

Dummy Template-Based Molecularly Imprinted Membrane Coating for Rapid Analysis of Malachite Green and Its Metabolic Intermediates in Shrimp and Fish.

A novel malachite green molecularly imprinted membrane (MG-MIM) with specific selectivity for malachite green (MG) and leucomalachite green (LMG) was prepared using a hydrophobic glass fiber membrane as the polymer substrate, methyl violet as a template analog, 4-vinyl benzoic acid as the functional monomer, and ethyleneglycol dimethacrylate as the crosslinking agent. MG-MIM and non-imprinted membrane (NIM) were structurally characterized using scanning electron microscopy, surface area analyzer, Fourier-transform infrared spectrometer and synchronous thermal analyzer. The results showed that MG-MIM possessed a fluffier surface, porous and looser structure, and had good thermal stability. Adsorption properties of MG-MIM were investigated under optimal conditions, and adsorption equilibrium was reached in 20 min. The saturated adsorption capacities for MG and LMG were 24.25 ng·cm-2 and 13.40 ng·cm-2, and the maximum imprinting factors were 2.41 and 3.20, respectively. Issues such as "template leakage" and "embedding" were resolved. The specific recognition ability for the targets was good and the adsorption capacity was stable even after five cycles. The proposed method was successfully applied for the detection of MG and LMG in real samples, and it showed good linear correlation in the range of 0 to 10.0 µg·L-1 (R2 = 0.9991 and 0.9982), and high detection sensitivity (detection limits of MG and LMG of 0.005 µg/kg and 0.02 µg·kg-1 in shrimp, and 0.005 µg/kg and 0.02 µg/kg in fish sample). The recoveries and relative standard deviations were in the range of 76.31-93.26% and 0.73-3.72%, respectively. The proposed method provides a simple, efficient and promising alternative for monitoring MG and LMG in aquatic products.

Pharmacological Characterization of Toludesvenlafaxine as a Triple Reuptake Inhibitor.

Toludesvenlafaxine hydrochloride dihydrate is a novel chemical entity and a potential triple monoamine reuptake inhibitor. This study characterized the in vitro triple reuptake inhibition activity, antidepressant-like activity in animals, and pharmacokinetic profiles in rats of toludesvenlafaxine. Binding affinity was determined using human serotonin transporter (SERT) protein, norepinephrine transporter (NET) protein and dopamine transporter (DAT) protein, and the reuptake inhibition was determined using Chinese hamster ovary cells expressing human SERT, NET and DAT. The antidepressant-like activity was examined in rat chronic unpredictable mild stress model and olfactory bulbectomized model. In rats, the tissue distribution and pharmacokinetic parameters were determined. Toludesvenlafaxine had high binding affinity on SERT, NET and DAT, and significantly inhibited the reuptake of serotonin (IC50 = 31.4 ± 0.4 nM), norepinephrine (IC50 = 586.7 ± 83.6 nM) and dopamine (IC50 = 733.2 ± 10.3 nM) in vitro. Toludesvenlafaxine demonstrated significant antidepressant-like effects in rat models at 8-16 mg/kg. In addition, toludesvenlafaxine significantly reduced serum corticosterone and significantly increased testosterone levels in rats. Toludesvenlafaxine was quickly absorbed and converted to O-desvenlafaxine (ODV) after oral administration, both of which were selectively distributed into the hypothalamus with high concentration. Plasma ODV exposure was proportionally related to the doses after oral dosing. These results suggest that toludesvenlafaxine is a triple reuptake inhibitor with relatively fast-acting antidepressant-like activity and good therapeutic profile including improvement of anhedonia and sexual function.

Cross-Sectional Study of the Effects of Job Burnout on Immune Function in 105 Female Oncology Nurses at a Tertiary Oncology Hospital.

BACKGROUND Nurses who work in hospitals experience a high level of burnout and the relationship between immune variables and burnout syndrome has yet to be elucidated. The aim of the present study was to investigate the effects of job burnout on immune function in female oncology nurses in a tertiary oncology hospital in Guangxi, China. The aspects of the human immune system evaluated were humoral and cellular immunity and complement components 3 (C3) and 4 (C4). MATERIAL AND METHODS We administered the Maslach Burnout Inventory-General Survey (MBI-GS), which includes scales for emotional exhaustion, depersonalization (DP), and personal accomplishment (PA), to measure variables related to immune function in 105 female nurses in a tertiary oncology hospital in Guangxi, China. Levels of humoral immunity and C3 and C4 were detected with immune turbidimetry. Cellular immunity was assessed with indirect immunofluorescence. RESULTS A Spearman rank correlation analysis revealed that levels of C3, C4, and CD4- and CD8-positive T cells were significantly associated with burnout symptoms (P<0.05, P<0.01, and P<0.05, respectively). Furthermore, there was a correlation between demographic data and humoral and cellular immunity (both P<0.05). Multivariable linear regression analysis showed that C4 levels were closely related to DP (P<0.05) and that CD4 and CD8 levels were closely related to PA (P<0.01). CONCLUSIONS These results suggest that DP and PA have an impact on immune function, and that timely psychological and behavioral interventions can be used to reduce the degree of job burnout among nurses and regulate their immunity, thus enabling them to better serve patients.

Dual labeled mesoporous silica nanospheres based electrochemical immunosensor for ultrasensitive detection of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a well-known cancer biomarker for the detection of several malignancies. The development of ultrasensitive CEA diagnostic tools is crucial for early detection and progression observation of tumors. Herein, a dual signal amplified sandwich-type electrochemical immunoassay was developed based on dual-labeled mesoporous silica nanospheres as a signal amplifier, combined with NiO@Au decorated graphene as a conductive layer for ultrasensitive and rapid determination of CEA. The dual-labeled mesoporous silica (DLMS) nanosphere, which was synthesized by entrapping Au nanorod (Au NR) and horseradish peroxidase (HRP) in the channels of amine-functionalized SBA-15 followed by subordinate antibody (Ab2) conjugation which was denoted as Au NR@SBA-15/Ab2-HRP. The dual signal amplification from Au NR@SBA-15 and HRP enhanced the sensitivity of the proposed immunoassay. Consequently, the developed DLMS based immunosensor displayed ultra-low limits of detection of 5.25 fg/mL and a wide range of linearity (0.1-5 pg/mL), which was extended for CEA determination in real-time samples with improved recoveries of >98%. Therefore, this dual amplification prototype would cater to the clinical requirements for the ultrasensitive detection of CEA biomarkers.

The Combination of Charge and Energy Transfer Processes in MOFs for Efficient Photocatalytic Oxidative Coupling of Amines.

Combining electron and energy transfer processes is very significant for efficient photocatalytic oxidation of organic molecules. The first synthesized MOF, Co2(L)(2,6-NDC)2·xguest (FJI-Y10, L = bis(N-pyridyl) tetrachloroperylene peryleneimide, 2,6-NDC = 2,6-naphthalenedicarboxylic acid, FJI = Fujian Institute), shows a 2-fold interpenetrated pcu net, in which the 2,6-NDC ligand connects typical Co2(COO)4 paddle wheel clusters to form square lattices pillared by new PDI-type ligand L. FJI-Y10 as a heterogeneous and recyclable photocatalyst is applied for photo-oxidation of benzylamine and its derivatives with an excellent yield of 100%, which is much higher than that (59%) of the equivalent L ligand as a homogeneous photocatalyst under the same reaction conditions. Such a high-efficiency photocatalytic activity attributes to the combination of charge and energy transfer processes in catalyst FJI-Y10 during the catalytic process.

Optimizing H2, D2, and C2H2 Sorption Properties by Tuning the Pore Apertures in Metal-Organic Frameworks.

By adjustment of the arm lengths of two triphenylamine-based ligands, two nearly isostructural metal-organic frameworks (MOFs), namely, the reported nanoporous FIR-29 (FIR = Fujian Institute of Research) and the new microporous FJI-Y9 (FJI = Fujian Institute), are obtained, and all exhibit honeycomb lattices of hexagonal channels with Ca-COO chains connected by tris[(4-carboxyl)phenylduryl]amine (H3TCPA) ligands and 4,4',4''-nitrilotribenzoic acid (H3NTB) ligands, respectively. Although the Brunauer-Emmett-Teller (BET) surface area (1117 m2 g-1) and pore size (8.5 Å) of FJI-Y9 are much lower than those (BET surface area of 2061 m2 g-1 and pore size of 16 Å) of the reported FIR-29 because of the shorter arm lengths of H3NTB, the activated FJI-Y9-ht shows high H2 (202.3 cm3 g-1) and D2 (221.9 cm3 g-1) uptake under 77 K and 1 bar and C2H2 uptake of 168.9 cm3 g-1 under 273 K and 1 bar, which are all at least 48% enhancement over those of FIR-29-ht. The above results indicate that small pores in MOFs are beneficial to the uptake of some special gases including H2, D2, C2H2, etc.

Validated LC-MS/MS method for the simultaneous determination of rotigotine and its prodrug in rat plasma and an application to pharmacokinetics and biological conversion in vitro.

Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been synthesized for use in a sustained delivery system. The aim of the present report was to develop and validate a simple, sensitive and reliable LC-MS/MS method for the simultaneous determination of RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-CN chromatography column (2.1mm×100mm, 3.5µm) by isocratic elution using a 0.2% formic acid aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample preparation method employed 50µL of a plasma sample and liquid-liquid extraction with a mixture of diethyl ether-dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and stability. Good linearity was found within the range of 0.1-10.0ng/mL for both analytes (r>0.996). This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation in rats following a single intramuscular injection and biological conversion in vitro.

Rapid determination of 54 pharmaceutical and personal care products in fish samples using microwave-assisted extraction-Hollow fiber-Liquid/solid phase microextraction.

In this paper, a simple, rapid, solvent-less and environmental friendliness microextraction method, microwave-assisted extraction-hollow fiber-liquid/solid phase microextraction (MAE-HF-L/SME), was developed for simultaneous extraction and enrichment of 54 trace hydrophilic/lipophilic pharmaceutical and personal care products (PPCPs) from fish samples. A solid-phase extraction material, solid-phase microextraction (SPME) fiber, was synthesized. The SPME fiber had a homogeneous, loose structure and good mechanical properties, and they exhibited a good adsorption capacity for most PPCPs selected. The material formed the basis for the method of MAE-HF-L/SME. A method of liquid chromatography-high resolution mass spectroscopy (LC-HRMS) for analysis of 54 PPCPs. Under optimal synthesis and extraction conditions, the limits of detection (LODs, n=3) and the limits of quantitation (LOQs, n=10) for the 54 PPCPs were between 0.01-0.50µg·kg-1 and 0.052.00µg·kg-1, respectively. Percent recoveries and the relative standard deviations (RSDs) in spiked fish samples (n=6) were between 56.3%-119.9% and 0.3%-17.1%, respectively. The microextraction process of 54 PPCPs in MAE-HF-L/SME took approximately 12min. The method has a low matrix interference and high enrichment factor and may be applicable for determination of 54 different PPCPs in fish samples.

Determination of a metabolite of nifursol in foodstuffs of animal origin by liquid-liquid extraction and liquid chromatography with tandem mass spectrometry.

An analytical method has been developed for the detection of a metabolite of nifursol, 3,5-dinitrosalicylic acid hydrazide, in foodstuffs of animal origin (chicken liver, pork liver, lobster, shrimp, eel, sausage, and honey). The method combines liquid chromatography and tandem mass spectrometry with liquid-liquid extraction. Samples were hydrolyzed with hydrochloric acid and derivatized with 2-nitrobenzaldehyde at 37°C for 16 h. The solutions of derivatives were adjusted to pH 7.0-7.5, and the metabolite was extracted with ethyl acetate. 3,5-Dinitrosalicylic acid hydrazide determination was performed in the negative electrospray ionization method. Both isotope-labeled internal standard and matrix-matched calibration solutions were used to correct the matrix effects. Limits of quantification were 0.5 µg/kg for all samples. The average recoveries, measured at three concentration levels (0.5, 2.0, and 10 µg/kg) were in the range of 75.8-108.4% with relative standard deviations below 9.8%. The developed method exhibits a high sensitivity and selectivity for the routine determination and confirmation of the presence of a metabolite of nifursol in foodstuffs of animal origin.

[Determination of perfluoroalkyl acids in lamb liver by high performance liquid chromatography- tandem mass spectrometry combined with dispersive solid phase extraction].

A method was developed for the determination of 19 perfluoroalkyl acids (PFAs) in lamb liver by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) combined with dispersive solid phase extraction. The sample was extracted with acidified acetonitrile, and then cleaned-up by a mixture of N-propylethylenediamine (PSA), C18 and graphitized carbon black (GCB) sorbents. The 19 PFAs were analyzed by HPLC-MS/MS with a C18 chromatographic column, adopting the multiple reaction monitoring (MRM) mode with negative electrospray ionization. The effects of the dosages of hydrochloric acid and the sorbents on the recoveries of the 19 PFAs were studied. For accurate quantitative analysis, the isotope internal standard method was used. The calibration curves were linear with the correlation coefficients over 0.998 in the range of 0.05-20 µg/kg for the 19 PFAs. The limits of detection were 0.004-0.111 µg/kg. The limits of quantification were 0.012-0.370 µg/kg. The mean recoveries of the 19 PFAs at spiked levels of 0.5, 1.0, 2.0 µg/kg were in the range from 80% to 128% with the relative standard deviations of 0.31%-11.1%. The developed method is rapid, simple, accurate. It is suitable for the determination of the 19 PFAs in large quantities of lamb liver samples.

Lactoferrin Promotes Early Neurodevelopment and Cognition in Postnatal Piglets by Upregulating the BDNF Signaling Pathway and Polysialylation.

Lactoferrin (Lf) is a sialic acid (Sia)-rich, iron-binding milk glycoprotein that has multifunctional health benefits. Its potential role in neurodevelopment and cognition remains unknown. To test the hypothesis that Lf may function to improve neurodevelopment and cognition, the diet of postnatal piglets was supplemented with Lf from days 3 to 38. Expression levels of selected genes and their cognate protein profiles were quantitatively determined. The importance of our new findings is that Lf (1) upregulated several canonical signaling pathways associated with neurodevelopment and cognition; (2) influenced ~10 genes involved in the brain-derived neurotrophin factor (BDNF) signaling pathway in the hippocampus and upregulated the expression of polysialic acid, a marker of neuroplasticity, cell migration and differentiation of progenitor cells, and the growth and targeting of axons; (3) upregulated transcriptional and translational levels of BDNF and increased phosphorylation of the cyclic adenosine monophosphate (cAMP) response element-binding protein, CREB, a downstream target of the BDNF signaling pathway, and a protein of crucial importance in neurodevelopment and cognition; and (4) enhanced the cognitive function and learning of piglets when tested in an eight-arm radial maze. The finding that Lf can improve neural development and cognition in postnatal piglets has not been previously described.

Salting-out homogeneous liquid-liquid extraction approach applied in sample pre-processing for the quantitative determination of entecavir in human plasma by LC-MS.

A convenient, robust, economical and selective sample preparation method for the quantitative determination of entecavir in human plasma by LC-MS was developed and validated. Entecavir and the internal standard of acyclovir were extracted from 500 µL of human plasma by a salting-out homogeneous liquid-liquid extraction approach (SHLLE) with acetonitrile as the organic extractant and magnesium sulfate as the salting-out reagent. They were analyzed on a Hanbon® Lichrospher RP C18 HPLC column (150 mm×2.0 mm; 5 µm) with gradient elution. The mobile phase comprised 0.1% acetic acid-0.2 mmol ammonium acetate in water (mobile phase A) and acetonitrile (mobile phase B). The flow rate is 0.2 mL/min. The analytes were detected by a LC-MS 2010 single quadrupole mass spectrometer instrument equipped with an electrospray ionization interface using selective ion monitoring positive mode. A "post cut" column switch technique was incorporated into the method to remove interferences of earlier and later eluting matrix components than entecavir and internal standard, including salting-out reagent used in sample pre-processing. The method was validated over the concentration range of 0.05-20 ng/mL. The intra-day and inter-day precision of the assay, as measured by the coefficient of variation (%CV), was within 3.59%, and the intra-day assay accuracy was found to be within 4.88%. The average recovery of entecavir was about 50% and the ion suppression was approximately 44% over the standard curve. Comparison of matrix effect between SHLLE and SPE by continuous post column infusion showed that these two methods got similar, slight ion suppression. The SHLLE method has been successfully utilized for the analysis of entecavir in post-dose samples from a clinical study.

Suppression of matrix metalloproteinase-9 expression by RNA interference inhibits SGC7901 gastric adenocarcinoma cell growth and invasion in vitro and in vivo.

Matrix metalloproteinase-9 (MMP-9) is considered the key enzyme that degrades extracellular matrix (ECM) via breaking down type IV collagens. Up-regulated MMP-9 promotes growth and invasion of gastric adenocarcinomas. The present study is to block MMP-9 expression in gastric adenocarcinoma cells in order to inhibit tumor growth and invasion. The association between MMP-9 expression and tumor pathology was reconfirmed by applying immunohistochemistry on tissue arrays. Small interference RNAs (siRNA) targeted on human MMP-9 were used to suppress gene expression in SGC7901 human gastric adenocarcinoma cells. Cell growth and invasion were significantly inhibited in specific siRNA-targeted cells. In addition, we generate a SGC7901-subcutaneous mice model to observe anti-tumor effects from RNA interference (RNAi). Data showed tumor masses in MMP-9 siRNA-treated mice were significantly smaller than those in control mice. The expression of vascular endothelial growth factor and proliferating cell nuclear antigen were down-regulated in MMP-9 siRNA treated cells. Our results demonstrate that MMP-9 targeted RNAi is able to successfully suppress MMP-9 gene expression and inhibit cell growth and invasion of SGC7901 gastric adenocarcinoma in vitro and in vivo. MMP-9 is a potential therapeutic target for gastric adenocarcinomas.

Suppression of Akt1 expression by small interference RNA inhibits SGC7901 cell growth in vitro and in vivo.

The Akt/PKB kinase family, including Akt1, 2 and 3, plays critical roles in regulating cell growth, proliferation, survival, metabolic and many other cellular activities. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer including gastric cancer. In the present study, we applied immunohistochemistry to tissue microarray to detect the expression of Akt1, followed by Akt1 small interference RNA (siRNA) to examine knock down of the Akt1 gene on the growth inhibition of human gastric cancer SGC7901 cells. Our results indicate that the expression of Akt1 was significantly increased in gastric cancer compared to normal gastric tissue and adjacent non-cancer tissue. The in vitro study shows that cell growth was significantly inhibited and G0/G1 arrest was observed in siRNA-Akt1-treated group. In vivo, the size of tumors was significantly smaller in SGC7901 subcutaneous mice model treated with siRNA-Akt1 than those treated with siRNA-nonsense and PBS. Our studies demonstrated siRNA-Akt1 can inhibit Akt1 expression, exerted growth inhibition effect on SGC7901 cells in vitro and in vivo. Suppression of Akt1 expression by siRNA could be a new strategy in gastric cancer treatment.