Extraction, Isolation and Identification of Low Molecular Weight Peptides in Human Milk.

Human milk contains numerous free low molecular weight peptides (LMWPs), which may play an important role in infant health and growth. The bioactivities of LMWPs are determined by their structures, especially the amino acid sequences. In the present study, 81 human milk samples were collected and purified by cation-exchange solid-phase extraction (SPE). Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for the separation and detection of free LMWPs in human milk. A total of 56 LMWPs were identified and quantified. These LMWPs were mainly derived from 3 regions of ß-casein, which were the amino acid fragments of 16-40, 85-110, and 205-226. The predominant LMWPs were RETIESLSSSEESITEYK, RETIESLSSSEESITEYKQKVEKVK, ETIESLSSSEESITEYK, TQPLAPVHNPIS, and QPLAPVHNPISV with molecular weights of 2247.9573, 2860.2437, 2091.8591, 1372.7666, and 1271.7212, respectively. The results indicated that the technique based on SPE and UPLC-QTOF-MS might greatly facilitate the analysis of LMWPs in human milk.

Multiclass Comparative Analysis of Veterinary Drugs, Mycotoxins, and Pesticides in Bovine Milk by Ultrahigh-Performance Liquid Chromatography-Hybrid Quadrupole-Linear Ion Trap Mass Spectrometry.

A multiclass and multiresidue method for simultaneously screening and confirming veterinary drugs, mycotoxins, and pesticides in bovine milk was developed and validated with ultrahigh-performance liquid chromatography-hybrid quadrupole-linear ion trap mass spectrometry (UHPLC-Qtrap-MS). A total of 209 targeted contaminants were effectively extracted using an optimized QuEChERS method. Quantitative and qualitative confirmation were achieved simultaneously by multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scan mode. The validation results exhibited a good sensitivity with the LOQs of 0.05-5 µg/kg, which was satisfactory for their MRLs in China or EU. The recoveries of in-house spiked samples were in the range of 51.20-129.76% with relative standard deviations (RSD) between replicates (n = 3) 0.82% and 19.76%. The test results of 140 milk samples from supermarkets and dairy farms in China showed that cloxacillin, aflatoxin M1, acetamiprid, and fipronil sulfone were found with lower concentrations. Combined with the residue results from the literature, penicillin G and cloxacillin (beta-lactams), enrofloxacin and ciprofloxacin (fluoroquinolones), and sulfamerazine (sulfonamides) were more frequently detected in different countries and need to receive more attention regarding their monitoring and control.

Administration of Tranexamic Acid to Reduce Intra-articular Hemarthrosis in ACL Reconstruction: A Systematic Review.

BACKGROUND: Although tranexamic acid (TXA) has been shown to reduce bleeding in joint replacement procedures, its effectiveness for anterior cruciate ligament reconstruction (ACLR) has not been widely reported. PURPOSE: To evaluate the effectiveness of TXA to reduce postoperative hemarthrosis and improve clinical outcomes after ACLR. STUDY DESIGN: Systematic review; Level of evidence, 2. METHODS: A systematic review of the literature following the PRISMA guidelines (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) was performed; literature retrieval was carried out using the MEDLINE, Embase, and Cochrane Library electronic databases. The inclusion criteria were comparative studies in English that reported the administration of intravenous or intra-articular TXA versus other modalities or placebo in patients undergoing ACLR. RESULTS: Six studies comprising 418 patients who were treated with TXA were included. Heterogeneity among studies did not allow for the pooling of data. Five studies showed decreased drainage volume in the first 24 or 48 hours postoperatively as compared with control (ACLR with no TXA). Four studies showed lower hemarthrosis grades and visual analog scale scores in TXA versus control in the early postoperative period, although this difference was not evident at 4 weeks postoperatively. No studies showed differences in infection, deep venous thrombosis, or adverse events between the TXA and control groups. CONCLUSION: The current best available evidence suggests that TXA administration at the time of ACLR results in decreased intra-articular bleeding (measured using a drainage system), hemarthrosis grade, and pain when compared with control.

Agomelatine might be more appropriate for elderly, depressed, type 2 diabetes mellitus patients than paroxetine/fluoxetine.

Agomelatine was a novel and melatonergic antidepressant. The present study was conducted to find out whether age was an important factor for agomelatine in treating depressed type 2 diabetes mellitus (T2DM) patients. In total, 193 depressed T2DM patients were included. There were 84 patients ranged from 27 years old to 49 years old (age phase I) (n = 44 receiving agomelatine, n = 40 receiving paroxetine or fluoxetine), and 109 patients ranged from 50 years old to 70 years old (age phase II) (n = 56 receiving agomelatine, n = 53 receiving paroxetine or fluoxetine). The Hamilton Depression Rating Scale (HDRS) score, Hamilton Anxiety Rating Scale (HARS) score, fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) level and body mass index (BMI) were assessed after 12 weeks treatment. After treatment, we found that among patients in age phase I, there were no significant differences in final average HDRS score, HARS score, FPG, HbA1c level, BMI, response rate and remission rate between the two groups. However, among patients in age phase II, compared to patients receiving paroxetine or fluoxetine, patients receiving agomelatine had the significantly lower average HDRS score, HARS score, HbA1c level and BMI, and significantly higher response rate and remission rate. The incidence of treatment-related adverse events was similar between the two groups in both age phases. These results suggested that age was an important factor for agomelatine in treating depressed T2DM patients. Compared to paroxetine/fluoxetine, agomelatine might be more appropriate for elderly depressed T2DM patients.

Occurrence of pharmaceuticals and personal care products in bottled water and assessment of the associated risks.

The occurrence of 187 pharmaceuticals and personal care products (PPCPs) was investigated in bottled water samples (35 and 33 from Chinese and foreign brands, respectively). Forty-four compounds belonging to 14 PPCP categories were detected in 56 of the 68 bottled water samples. Further, more than 35% of water samples contained at least three PPCPs, and in one particular sample, 11 different PPCPs were detected. Macrolides constituted the most prevalent PPCP category, and salbutamol, erythromycin, and azithromycin showed the highest detection frequency (17.6%). The thermal stabilities of the 187 PPCPs were determined, and the results obtained showed that only 35 out of the 187 compounds were degraded by more than 50% after boiling for 5 min. Even though the risk quotients (RQs) of detected PPCPs showed low risk levels, the RQs of 13 compounds with RQs ≥ 0.0001 were 2-4 fold higher in infants than in other life stages. Moreover, further studies are necessary to evaluate the toxicity of PPCP mixtures, the effects of PPCPs on human intestinal microbiota, and their risk of induction of drug-resistant bacteria and drug-resistant genes.

Remediation of fluoride contaminated soil with nano-hydroxyapatite amendment: Response of soil fluoride bioavailability and microbial communities.

Nano-hydroxyapatite (NHAP), possessing high defluoridation capacity, has been widely used to remove fluoride (F) from polluted water, but little is known about how it affects the bioavailability and toxicity of soil F towards plants. Here, the impact of NHAP (2% w/w) amendment on immobilization, speciation and accumulation of F was studied in a soil-plant system. The results revealed that the NHAP amendment worked effectively to reduce levels of water-soluble F (37.3%-87.8%) and increase available P (76.6%-147%). X-ray photoelectron spectroscopy analysis indicated that the formation of insoluble CaF2 and the ion exchange of F- with OH- into NHAP might be involved in the mechanism of F immobilization and soil pH elevation. Exposure to NHAP significantly decreased the abundance of Cyanobacteria in tested soils, and Gemmatimonadetes abundance in bulk soil was significantly higher than that in rhizosphere soil at 1,000 mg kg-1 F spiked level. Additionally, NHAP amendment decreased F accumulation in wheat shoots (9.10%-18.7%) and roots (3.88%-22.4%), which could mainly be attributed to the reduction of soil bioavailable F and the supplement of Ca from NHAP. These results suggest that NHAP could be a promising amendment to be applied to acidic soil contaminated with F.

Efficient multiresidue determination method for 168 pharmaceuticals and metabolites: Optimization and application to raw wastewater, wastewater effluent, and surface water in Beijing, China.

New analytical methods are needed to efficiently measure the growing list of priority pharmaceuticals in environmental samples. In this regard, a rapid, sensitive, and robust method was developed for quantitation of 168 pharmaceuticals and pharmaceutical metabolites using solid-phase extraction (SPE) and liquid chromatography with tandem mass spectrometry. The extraction protocol and instrumental efficiency were specifically addressed to increase analytical workload and throughput. The optimized protocols, which are five times more efficient than US EPA Method 1694, enabled analyte recoveries that ranged from 77% to 117% for 162 analytes with method quantitation limits (MQLs) as low as 0.1 ng L-1. To verify the suitability of the improved analytical method for environmental samples, 24-h composite samples of raw wastewater and wastewater effluent, along with downstream surface water, were analyzed. Overall, 143/168 target compounds were identified in at least one of the samples, and 130/168 analytes were present at concentrations above their MQLs. The total mass concentration of the measured analytes decreased by 93% during wastewater treatment. The analyte concentrations in the wastewater effluent were comparable to those measured in surface water 1 km downstream of the wastewater discharge point. Ultimately, the comprehensive method will serve as an important tool to inform the occurrence, fate, transport, and toxicity of a large suite of priority pharmaceuticals and pharmaceutical metabolites in natural and engineered systems.

Efficacy of ginkgo biloba extract as augmentation of venlafaxine in treating post-stroke depression.

BACKGROUND: Post-stroke depression (PSD) is one of the most common psychiatric diseases afflicting stroke survivors. This study was conducted to assess the efficacy of ginkgo biloba extract as augmentation of venlafaxine in treating PSD. METHODS: The included PSD patients were randomly assigned into the experiment group (receiving ginkgo biloba extract plus venlafaxine) and control group (receiving venlafaxine alone). The treatment was continued for eight weeks. The Hamilton Depression Rating Scale (HDRS) and the Self-rating Depression Scale (SDS) were used to assess the depressive symptoms. The National Institutes of Health Stroke Scale (NIHSS) was used to assess the neurological defect, and the Activities of Daily Living (ADL) was used to assess recovery of abilities of patients after stroke. Meanwhile, the levels of serum 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) were measured before and after treatment. The dose of venlafaxine used and adverse events were also recorded. RESULTS: Each group had 40 PSD patients. After treatment, the depressive symptoms, neurological defect and living function were significantly improved in both groups. But the patients receiving ginkgo biloba extract plus venlafaxine had the significantly lower average HDRS score (p=0.0008), SDS score (p<0.00001), NIHSS score (p=0.00001), and higher average ADL score (p=0.0005). Meanwhile, compared to the control group, patients in the experiment group had the significantly higher 5-HT (p<0.00001) level and BDNF level (p<0.00001), needed lower dose of venlafaxine (p=0.007), and experienced fewer adverse events. CONCLUSION: These results demonstrated that the ginkgo biloba extract was a good augmentation of venlafaxine in treating PSD and should be further investigated.

Urinary biomarkers for diagnosing poststroke depression in patients with type 2 diabetes mellitus.

BACKGROUND: Depression can seriously affect the quality of life of type 2 diabetes mellitus (T2DM) patients after stroke. However, there were still no objective methods to diagnose T2DM patients with poststroke depression (PSD). Therefore, we conducted this study to deal with this problem. METHODS: Gas chromatography-mass spectroscopy (GC-MS)-based metabolomics profiling method was used to profile the urinary metabolites from 83 nondepressed T2DM patients after stroke and 101 T2DM patients with PSD. The orthogonal partial least-squares discriminant analysis was conducted to explore the metabolic differences in T2DM patients with PSD. The logistic regression analysis was performed to identify the optimal and simplified biomarker panel for diagnosing T2DM patients with PSD. The receiver operating characteristic curve analysis was used to assess the diagnostic performance of this biomarker panel. RESULTS: In total, 23 differential metabolites (7 decreased and 16 increased in T2DM patients with PSD) were found. A panel consisting of pseudouridine, malic acid, hypoxanthine, 3,4-dihydroxybutyric acid, fructose and inositol was identified. This panel could effectively separate T2DM patients with PSD from nondepressed T2DM patients after stroke. The area under the curve was 0.965 in the training set and 0.909 in the validation set. Meanwhile, we found that the galactose metabolism was significantly affected in T2DM patients with PSD. CONCLUSION: Our results could be helpful for future development of an objective method to diagnose T2DM patients with PSD and provide novel ideas to study the pathogenesis of depression.

Is implantation of autologous chondrocytes superior to microfracture for articular-cartilage defects of the knee? A systematic review of 5-year follow-up data.

BACKGROUND: Autologous chondrocyte implantation (ACI) and microfracture are two of the main surgical treatment options for articular cartilage lesions of the knee. Consensus regarding the best clinical options to repair knee cartilage lesions is lacking. We undertook a systematic review to clarify the clinical efficacy of ACI and microfracture at minimum mean 5-year follow-up. METHODS: A literature search was conducted using the MEDLINE, Embase and Cochrane Library databases up to August 2018. Only comparative clinical studies of ACI and microfracture for the treatment of articular cartilage lesions of the knee with level I/Ⅱ evidence were included. Clinical outcomes and the prevalence of treatment failure from each study were extracted and compared. The methodological quality of the included studies was analyzed by means of the PEDro scale. RESULTS: Five comparative studies (three randomized controlled trials and two prospective cohort studies) met our eligibility criteria. ACI and microfracture elicited significant improvement in clinical outcomes after 5 years. However, better clinical results with significant differences were found with modified versions of ACI (ACI with a modified collagen membrane [ACI-C] or matrix-applied chondrocyte implantation [MACI]) than with microfracture as determined by the Knee Injury and Osteoarthritis Outcome Score, activities of daily living assessment, Tegner Activity Scale score, and the International Knee Documentation Committee objective and subjective scores. No significant difference was observed in the treatment failure rate between these two methods within a particular study. CONCLUSIONS: Currently, the best-available evidence suggests that some clinical outcomes of articular cartilage lesions of the knee treated with modified versions of ACI (ACI-C or MACI) can significantly improve patient outcomes at the mid-term follow-up of 5 years compared with those obtained using microfracture.

Simultaneous determination of 58 pesticides and relevant metabolites in eggs with a multi-functional filter by ultra-high performance liquid chromatography-tandem mass spectrometry.

A sensitive method for the simultaneous determination of 58 pesticides and relevant metabolites in eggs was developed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) after clean-up with a multi-functional filter (MFF) based on quick, easy, cheap, effective, rugged, and safe method (QuEChERS). The egg sample was extracted with 5 ml water and 10 ml 1% acetic acid in acetonitrile and then salt out with sodium chloride. The extracted solution was filtered directly through an MFF containing 50 mg PSA, 50 mg C18, and 150 mg magnesium sulphate before UHPLC-MS/MS analysis. The clean-up and filter procedures were integrated using the MFF to substantially improve the work efficiency. Good linearity was shown for each analyte, and all the correlation coefficients exceeded 0.99. The recoveries in the eggs at the five spiked levels were 74.4%-115.2%, and the relative standard deviations (RSDs) were less than 15.3%. The limit of detection (LOD) and the limit of quantitation (LOQ) of 58 pesticides and 8 metabolites in eggs were 0.1-1.0 µg/kg and 0.2-5.0 µg/kg, respectively. The decision limit (CCα) and detection capacity (CCß) were 3.4-111.1 µg/kg and 6.8-122.1 µg/kg, respectively. This method has also been successfully applied in the determination of actual eggs samples. This developed method is more effective and faster in the monitoring of pesticide residue in eggs compared to the traditional analytical method.

Performance and membrane fouling of a step-fed submerged membrane sequencing batch reactor treating swine biogas digestion slurry.

To identify the performance of step-fed submerged membrane sequencing batch reactor (SMSBR) treating swine biogas digestion slurry and to explore the correlation between microbial metabolites and membrane fouling within this novel reactor, a lab-scale step-fed SMSBR was operated under nitrogen loading rate of 0.026, 0.052 and 0.062 g NH4+-N (gVSS·d)-1. Results show that the total removal efficiencies for NH4+-N, total nitrogen and chemical oxygen demand in the reactor (>94%, >89% and >97%, respectively) were high during the whole experiment. However, the cycle removal efficiency of NH4+-N decreased significantly when the nitrogen loading rate was increased to 0.062 g NH4+-N (gVSS·d)-1. The total removal efficiency of total phosphorus in the step-fed SMSBR was generally higher than 75%, though large fluctuations were observed during the experiments. In addition, the concentrations of microbial metabolites, i.e., soluble microbial products (SMP) and extracellular polymeric substances (EPS) from activated sludge increased as nitrogen loading rate increased, both showing quadratic equation correlations with viscosity of the mixed liquid in the step-fed SMSBR (both R2 > 0.90). EPS content was higher than SMP content, while protein (PN) was detected as the main component in both SMP and EPS. EPS PN was found to be well correlated with transmembrane pressure, membrane flux and the total membrane fouling resistance. Furthermore, the three-dimensional excitation-emission matrix fluorescence spectroscopy results suggested the tryptophan-like protein as one of the main contributors to the membrane fouling. Overall, this study showed that the step-fed SMSBR could be used to treat swine digestion slurry at nitrogen loading rate of 0.052 g NH4+-N (gVSS·d)-1, and the control strategy of membrane fouling should be developed based on reducing the tryptophan-like PN in EPS.

[Rapid screening and identification of food poisonings by ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry].

A method was developed for the screening and detection of food poisonings by ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF MS). After extracted by acetonitrile and cleaned-up by QuEChERS, the extract was separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm) with the gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile. TOF-MS scan-information dependent acquisition (IDA)-product ion scan was performed in positive electrospray ionization (ESI) mode to acquire high resolution MS and MS/MS spectra in one injection, and rapidly screen 581 target compounds by SCIEX OS software, including 546 pesticides, 24 mycotoxins, 11 rodenticides. The target compounds were qualitatively confirmed by mass accuracy of precursor, isotope distribution of precursor, fragment ions of precursor, and library search. Carbofuran was detected in 9 out of 11 samples with the proposed method. The retention time was further confirmed by the standard of carbofuran. The results showed the retention times were coincident between the samples and reference standard, and the deviations of accurate mass numbers were all less than 3.7×10-6. The scope of the relationship was good and the correlation coefficient was 0.998. The instrumental limit of detection (S/N=3) was 0.3 µg/kg, and the limit of quantification (S/N=10) was 1 µg/kg. The recoveries at 10, 50, 200 µg/kg levels were 75.6%-95.9%, and the RSDs (n=6) were 3.6%-6.9%. The method is rapid, simple, accurate and sensitive. It is suitable for the rapid screening and detection of public safety incidents.

Toxicity of vanadium in soil on soybean at different growth stages.

Vanadium(V) is present in trace amounts in most plants and widely distributed in soils. However, the environmental toxicity of V compound in soils is controversial. A greenhouse study with soybean from germination to bean production under exposure to pentavalent V [V(V)] was conducted to elucidate the interaction of plants and V fractions in soils and to evaluate the toxicity of V at different plant growth stages. Soybean growth has no effect on non-specific-bond and specific-bond fractions of V in soils, but V fractionation occurred in more extraction-resistant phases at high V concentrations. High concentrations of V(V) postponed the germination and growth of the soybeans. Bean production was less than half of that of the control at 500 mg kg-1 spiked V(V). For the 0 mg kg-1 spiked V(V) treated plants, the root was not the main location where V was retained. Vanadium in the soils at ≤ 250 mg kg-1 did not significantly affect the V concentration in the shoot and leaf of soybeans. With the increase in V concentration in soil, V concentrations in roots increased, whereas those in beans and pods decreased. From vegetative growth to the reproductive growth, the soybeans adsorbed more V and accumulated more V in the roots, with <20% transported to the aboveground parts. Hence, the analysis of V concentration in vegetative tissues or beans may not be a useful indicator for V pollution in soil. Meanwhile, the ratio of V concentration in cell wall to the total V concentration in the root increased with the increase in V(V) concentration in soils. Our results revealed that high concentrations of V inhibited soybean germination and biomass production. However, plants may produce self-defense systems to endure V toxicity.

Inhibitory effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced femoral head necrosis.

Apoptosis and DNA oxidative damage serve significant roles in the pathogenesis of steroid­induced femoral head necrosis. Vitamin E demonstrates anti­apoptotic and anti­oxidant properties. Therefore, the present study investigated the effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at an early stage of steroid­induced femoral head osteonecrosis. Japanese white rabbits were randomly divided into three groups (steroid, vitamin E­treated, and control groups), each comprising 12 rabbits. Those in the steroid group (group S) were initially injected twice with an intravenous dose of 100 µg/kg Escherichia coli endotoxin, with a 24 h interval between the two injections, and then with an intramuscular dose of 20 mg/kg methylprednisolone, three times at intervals of 24 h in order to establish a rabbit model of osteonecrosis. The vitamin E treated group (group E) received the same treatment as group S, and were administered 0.6 g/kg/d vitamin E daily from the beginning of modeling. The control group (group C) was injected with normal saline at the equivalent dosage and times as the aforementioned two groups. Two time points, weeks 4 and 6 following the completion of modeling, were selected. Osteonecrosis was verified by histopathology with hematoxylin-eosin staining. The apoptosis rate of osteonecrosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The apoptosis expression levels of caspase­3 and B­cell lymphoma 2 (Bcl­2), and DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry. At weeks 4 and 6 following the completion of modeling, the vacant bone lacunae rates of group E were 15.87±1.97 and 25.09±2.67%, respectively, lower than the results of 20.02±2.21 and 27.79±1.39% for group S; and the osteocyte apoptosis indexes of group E were 20.99±2.95 and 33.93±1.62%, respectively, lower than the results of 26.46±3.37 and 39.90±3.74% from group S. In addition, the Bcl-2 expression at week 4 in the femoral head tissues of group E was higher compared with group S; and the proportion of Bcl­2­positive cells of group E was 9.81±1.01%, higher compared with group S at 8.26±1.13%. The caspase­3 staining data at week 4 in femoral head tissues demonstrated that in the 12 femoral heads of group S, four were negative (32%) and eight were positive (68%); in group E, five were negative (45%) and seven were positive (55%); and in group C, 11 were negative (95%) and one was positive (5%). In addition, the DNA oxidative damage rate at week 4 in the bone marrow hemopoietic cells of group E was (7.24±1.44%), lower compared with group S (11.80±1.26%), and higher compared with group C (5.75±1.47%). Vitamin E is effective in intervening in apoptosis through decreasing caspase­3 expression and upregulating Bcl­2 expression, and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid­induced femoral head necrosis in rabbit models.

The effective mode of growth and differentiation factor-5 in promoting the chondrogenic differentiation of adipose-derived stromal cells.

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.

[Rapid screening and confirmation of 205 pesticide residues in rice by QuEChERS and liquid chromatography-mass spectrometry].

A method for rapid screening and confirmation of 205 pesticide residues in rice was developed by combining QuEChERS and high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (LC-Q-TRAP/MS). The rice samples were extracted with acetonitrile, and then cleaned up with primary secondary amine (PSA), anhydrous magnesium sulfate (MgSO4) and C18 adsorbent. Finally, the samples were detected by LC-Q-TRAP/MS in multiple reaction monitoring with information-dependent acquisition of enhanced product ion (MRM-IDA-EPI) mode followed with database searching. A total of 205 pesticide residues were confirmed by retention times, ion pairs and the database searching using EPI library, and quantified by external standard method. All the pesticides showed good linearities with linear correlation coefficients all above 0.995. The limits of quantification (LOQs) for the 205 pesticides were 0.5-10.0 µg/kg. The average recoveries of the 205 pesticides ranged from 62.4% to 127.1% with the relative standard deviations (RSDs) of 1.0% - 20.0% at spiked levels of 10 µg/kg and 50 µg/kg, and only 20 min were needed for the analysis of an actual rice sample. In brief, the method is fast, accurate and highly sensitive, and is suitable for the screening and confirmation of pesticide residues in rice.

Identification of genes associated with the differentiation potential of adipose-derived stem cells to osteocytes or myocytes.

Adipose-derived stem cells (ADSCs) have been considered as the optimal cells for regenerative medicine because ADSCs have the potential of multi-directional differentiation. To study the mechanisms of ADSCs differentiation, we analyzed microarray of GSE37329. GSE37329 was downloaded from Gene Expression Omnibus including 3 ADSCs, 2 ADSCs-derived osteocytes, and 2 ADSCs-derived myocytes samples. The differentially expressed genes (DEGs) were screened using limma package. Their underlying functions were predicted by gene ontology and pathway enrichment analyses. Besides, the interaction relationships of the proteins encoded by DEGs were obtained from STRING database, and protein-protein interaction (PPI) network was constructed using Cytoscape. Furthermore, modules analysis of PPI network was performed using MCODE in Cytoscape. We screened 662 and 484 DEG separately for the ADSCs-derived osteocytes and myocytes compared with ADSCs. There were 205 common up-regulated and 128 common down-regulated DEGs between the two groups. Function enrichment indicated that these common DEGs, especially, VEGFA, FGF2, and EGR1 may be related to cell differentiation. PPI network for common DEGs also suggested that VEGFA (degree = 29), FGF2 (degree = 17), and EGR1 (degree = 12) might be more important because they had higher connectivity degrees, and they might be involved in cell differentiation by interacting with other genes in module A (e.g., EGR1-NGF and EGR1-LEP), and B (e.g., VEGFA-PDGFD). Additionally, the IGF1 and BTG1 may be, respectively, specific for osteocytes and myocytes differentiation. VEGFA, PDGFD, FGF2, EGR1, NGF, LEP, IGF1, and BTG1 might serve as target genes in regulating ADSCs differentiation.

[Expression of human bone morphogenetic protein 7 gene in adipose-derived stem cells and its effects on osteogenic phenotype].

OBJECTIVE: To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phenotype. METHODS: Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, and CD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Lipofectamine 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkaline phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. RESULTS: ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P < 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P < 0.05). CONCLUSION: The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.

An improved on-line solid phase extraction coupled HPLC-MS/MS system for quantification of sifuvirtide in human plasma.

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1-6250 ng/mL, and the correlation coefficients (r(2)) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from -7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC-MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).