Phthalate exposure and subfecundity in preconception couples: A nested case-control study.

BACKGROUND: Phthalates (PAEs) are endocrine-disrupting chemicals ubiquitously found in the environment. This study aimed to examine the association between exposure of PAEs and subfecundity in preconception couples. METHODS: This is a nested case-control study based on preconception cohort. Preconception couples with intention to conceive were enrolled and followed up until a clinically confirmed pregnancy or 12 menstrual cycles of preparation for conception. A total of 107 couples with subfecundity- time to pregnancy (TTP) more than 12 menstrual cycles, and 144 couples ≤12 cycles were included in the analysis. The levels of PAE metabolites in one spot urine samples were detected and compared between the groups. The weighted quantile sum (WQS) regression model and Bayesian kernel machine regression (BKMR) model were used to examine the joint effects of couples' exposure to PAEs on subfecundity. RESULTS: Using the multivariate binary logistic regression model, compared to the lowest quartile of urinary ∑PAEs concentration group, both preconception females (aOR=2.42, 95% CI: 1.10-5.30, p=0.027) and males (aOR=2.99, 95% CI: 1.36-6.58, p=0.006) in the highest quartile group had an increased risk of subfecundity, and a dose-response relationship was observed between PAEs and the risk of subfecundity. The WQS analyses found that co-exposure to PAE mixture was a risk factor for subfecundity in preconception female (aOR=1.76, 95% CI: 1.38-2.26, p<0.001), male (aOR=1.58, 95% CI: 1.20-2.08, p=0.001), and couple (aOR=2.39, 95% CI: 1.61-3.52, p<0.001). The BKMR model found a positive combined effect of mixed exposure to PAEs on the risk of subfecundity. CONCLUSIONS: PAEs increase the risk of subfecundity in preconception couples. Our research reinforced the need of monitoring PAE exposure for the purpose of improving human reproductive health.

Human Umbilical Vein Endothelial Cells Survive on the Ischemic TCA Cycle under Lethal Ischemic Conditions.

It is generally believed that vascular endothelial cells (VECs) rely on glycolysis instead of the tricarboxylic acid (TCA) cycle under both normoxic and hypoxic conditions. However, the metabolic pattern of human umbilical vein endothelial cells (HUVECs) under extreme ischemia (hypoxia and nutrient deprivation) needs to be elucidated. We initiated a lethal ischemic model of HUVECs, performed proteomics and bioinformatics, and verified the metabolic pattern shift of HUVECs. Ischemic HUVECs displayed extensive aerobic respiration, including upregulation of the TCA cycle and mitochondrial respiratory chain in mitochondria and downregulation of glycolysis in cytoplasm. The TCA cycle was enhanced while the cell viability was decreased through the citrate synthase pathway when substrates of the TCA cycle (acetate and/or pyruvate) were added and vice versa when inhibitors of the TCA cycle (palmitoyl-CoA and/or avidin) were applied. The inconsistency of the TCA cycle level and cell viability suggested that the extensive TCA cycle can keep cells alive yet generate toxic substances that reduce cell viability. The data revealed that HUVECs depend on "ischemic TCA cycle" instead of glycolysis to keep cells alive under lethal ischemic conditions, but consideration must be given to relieve cell injury.

[Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry].

OBJECTIVE: An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. METHODS: The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 µm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 µm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. RESULTS: The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 µg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 µg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). CONCLUSION: The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Long-Term Outcomes of Transarterial Chemoembolization Combined with Radiofrequency Ablation Versus Transarterial Chemoembolization Alone for Recurrent Hepatocellular Carcinoma After Surgical Resection.

BACKGROUND: There is lack of data for identifying optimal local therapy for the management of recurrent hepatocellular carcinoma (HCC) after hepatic resection. AIMS: A retrospective study was performed to compare the effectiveness of transarterial chemoembolization (TACE) plus radiofrequency ablation (RFA) with that of TACE alone for recurrent HCC. METHODS: From 2007 to 2013, patients with recurrent HCC ≤ 5 cm were treated with either TACE plus RFA (n = 96) or TACE (n = 63). Inverse probability of treatment weighting was used to make allowances for imbalances in treatment assignment. The disease-free survival (DFS) and overall survival (OS) were retrospectively analyzed. RESULTS: The TACE group had lower pretreatment Child-Pugh class (P = 0.025) and shorter pretreatment interval of recurrence (P = 0.028). The 1-, 3-, and 5-year DFS rates for the TACE-RFA group were 55.1%, 22.5%, and 9.7%, respectively, and 41.1%, 9.9%, and 4.9%, respectively, for the TACE group. The OS rates at 1, 3, and 5 years were 82.3%, 42.7%, and 16.5%, respectively, in the TACE-RFA group, and 75.9%, 30.7%, and 11.3%, respectively, in the TACE group. Cirrhosis was significantly associated with disease progression (hazard ratio [HR] 1.53; 95% CI 1.09-2.14; P = 0.014). CONCLUSIONS: In patients with recurrent HCC ≤ 5 cm, TACE-RFA shows better DFS than TACE alone as a first-line local therapy.

[High performance liquid chromatography coupled with electrospray tandem mass spectrometry for the analysis of dicyandiamide and melamine residues in milk and milk products].

OBJECTIVE: To establish a sensitive method for the analysis of dicyanodiamide and melamine residue in milk and milk products by, hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry. METHODS: Samples were extracted with 2.5% (V/V) formic acid solution. Acetonitrile was used to precipitate proteins. The separation was carried on Acquity UPLC BEH Amide column (100 mm x 2.1 mm x 1.7 mm) by gradient elution with acetonitrile - water as mobile phase. Identification was achieved by electrospray ionization (ESI) in positive mode using multiple reaction monitoring, and quantified with external standards. RESULTS: The dicyanodiamide and melamine were linear in the range of 5.0 - 1000.0 µg/L with correlation coefficient of 0.9995 for dicyanodiamide and 0.9997 for melamine. The detection limit of the method were 10.0 µg/kg for dicyanodiamide and melamine. The spiked at three levels ranged 86.0% - 100.0% (0.050 mg/kg), 90.0% - 104.0% (1.0 mg/kg) and 90.0% - 100.1% (10.0 mg/kg). And the relative standard derivations were lower than 1.06% -7.77%. The within-day precisions were 2.35% (dicyanodiamide) and 3.44% (melamine), and the inter-day precision 3.87% (dicyanodiamide) and 5.39% (melamine). CONCLUSION: The method was sensitive, accurate and precise. It can be used in monitoring quality of milk production and daily analysis of milk and milk products.

[Efficiency evaluation of capsaicinoids to discriminate bio-waste oils from edible vegetable oils].

OBJECTIVE: To evaluate the efficiency of capsaicinoids to discriminate bio-waste oil from edible vegetable oil. METHODS: 14 raw vegetable oils, 24 fried waste oils, 34 kitchen-waste oils, 32 edible non-peanut vegetable oil, 32 edible peanuts oil, 16 edible oil add flavorand and 11 refined bio-waste oils were prepared and examined for capsaicinoids including capsaicin, dihydrocapsaicin and nonylic acid vanillylamide. The detection results of the above samples were statistically tested based on sample category to assessment identify the effectiveness of the bio-waste oils with capsaicinoids. RESULTS: As a indicator, capsaincin was possessed of high detection sensitivity and has the highest efficiency to discern kitchen-waste oils and refined bio-waste oils samples from edible non-peanut vegetable oil correctly. The accuracy rate of identification were 100% and 90.1% respectively. There is the background in peanut oil. CONCLUSION Capsaicin added in cooking process can be retained in the refining process and hardly be removed in the refining process. In the case of fully eliminating the background interference, capsaicinoids can effectively identify bio-waste oils and edible vegetable oil in combination.

Control of iodine-deficiency disorders following universal salt iodization in Shenzhen, China, 1997-2011.

BACKGROUND: Since universal salt iodization (USI) was implemented in Shenzhen, China, in 1996, evaluation of the time trend of USI to indicate the control of iodine-deficiency disorders has not been performed. OBJECTIVE: To assess the time trend of median urinary iodine and total goiter rates from 1997 to 2011. METHODS: Probability-proportionate-to-size sampling was employed in the surveillance of iodine-deficiency disorders, for which schoolchildren aged 8 to 10 years were randomly selected from five districts of the city during each iodine-deficiency disorders survey. Urinary iodine content and thyroid size were measured by ammonium persulfate oxidation and B ultrasound, respectively. RESULTS: The coverage of iodized salt increased from 73.2% in 1997 to more than 90% in 2011. The median urinary iodine of children aged 8 to 10 years varied between 207.1 and 278.8 microg/L; these levels were above the urinary iodine level in 1995. The proportion of urine samples with iodine content above 300 microg/L was 45.6% in 1997 and decreased to 20.8% in 2011, indicating excessive consumption of iodine by the children. The goiter rate among children dropped from 10.8% in 1997 to 1.3% in 2011; both values were lower than the goiter rate in 1995, indicating that the spread of endemic goiter was under control. CONCLUSIONS: Preliminary elimination of iodine-deficiency disorders was achieved by USI in Shenzhen. Nevertheless, some problems still existed, such as over-iodization. To clarify the causes of excessive urinary iodine content, the various sources of iodine from the diet need to be investigated in the future.

[Simultaneous determination of melamine and cyanuric acid residues in water and urine by solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry method].

OBJECTIVE: We aimed to establish a sensitive quantified method for the simultaneous determination of melamine and cyanuric acid residues in water and urine by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry (HILIC-ESI-MS/MS) with the pretreatment of hydrophilic functional silica gel and cation exchange resin mixed solid phase extraction column(MCT), and to investigate the melamine and cyanuric acid residues in 501 water and 216 urine from several province and city. METHODS: About 100 ml water (or 10 ml urine) was adjusted to pH 3.0 with concentrated hydrochloric acid, and then mixed with the internal standard solution((15)N3-melamine and (15)N3-(13)C3 -cyanuric acid) and 100 ml acetonitrile (10 ml for urine). The solution was cleaned with MCT solid-phase extraction column, and eluted once by 3 ml methanol and twice by 2.5 ml methanol (containing 5% ammonia water). The effluent was collected and dried by N2 flow at 40 °C, and then diluted to 2 mmol/L ammonium acetate containing 90% volume fraction acetonitrile. The completely dissolved solution was then filtered with 0.22 µm organic membrane; and the filtrate was detected by high performance liquid chromatography-tandem mass spectrometry and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. Then we detected the melamine and cyanuric acid residues in 501 water and 216 urine samples collected from several province and city. RESULTS: By the quantification of internal standard (15)N3-melamine and (15)N3-(13)C3-cyanuric acid, the melamine and cyanuric acid were linear in the range of 2.0-1000.0 µg/L with correlation coefficient of 0.9998 and 0.9997. The detection limits of the method were separately 0.4 ng/L (melamine) and 0.3 ng/L (cyanuric acid) for water, and 4.0 ng/L (melamine) and 3.0 ng/L (cyanuric acid) for urine. The average recovery rate was around 95.3%-100.1% with the relative standard deviation (RSD) was <4.02%. Out of the 501 water samples, melamine was detected out in 19.9% (100/501) and cyanuric acid was detected out in 5.2% (26/501). The content was around 0.03-5.00 g/L. Melamine or cyanuric acid was detected out in 24.5% of the urine samples (53/216), with the content around 0.01-1.00 g/L. CONCLUSION: The established method of solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry can satisfy the requirement for detection of melamine and cyanuric acid residues in all sorts of water and urine. Meanwhile, the two substances widely existed in water and Chinese population.

A comparative in vitro and in vivo study of porcine- and bovine-derived non-cross-linked collagen membranes.

The porcine-derived non-cross-linked collagen membrane Bio-gide® (BG) and the bovine-derived non-cross-linked collagen membrane Heal-all® (HA) were compared to better understand their in vitro biophysical characteristics and in vivo degradation patterns as a reference for clinical applications. It was showed that the porosity, specific surface area, pore volume and pore diameter of BG were larger than those of HA (64.5 ± 5.2% vs. 48.6 ± 6.1%; 18.6 ± 2.8 m2 /g vs. 2.3 ± 0.6 m2 /g; 0.114 ± 0.002 cm3 /g vs. 0.003 ± 0.001 cm3 /g; 24.4 ± 3.5 nm vs. 7.3 ± 1.7 nm, respectively); the average swelling ratio of BG was higher than that of HA (412.6 ± 41.2% vs. 270.0 ± 2.7%); the tensile strength of both dry and wet HA was higher than those of BG (18.26 ± 3.27 MPa vs. 4.02 ± 1.35 MPa; 2.24 ± 0.21 MPa vs. 0.16 ± 0.02 MPa, respectively); 73% of HA remained after 72 h in collagenase solution, whereas only 8.2% of BG remained. A subcutaneous rat implantation model revealed that, at 3, 7, 14, 28, and 56 days postmembrane implantation, there were more total inflammatory cells, especially more M1 and M2 polarized macrophages and higher M2/M1 ratio in BG than in HA; in addition, the fibrous capsule around BG was also thicker than that around HA. Moreover, concentrations of dozens of cytokines including interleukin-2(IL-2), IL-7, IL-10 and so forth. in BG were higher than those in HA. It is suggested that BG and HA might be suitable for different clinical applications according to their different characteristics.

[Determination of five microcystins in drinking water by HPLC/MS/MS].

OBJECTIVE: A high performance liquid chromatography-tandem mass spectrometric method was established for determination of five microcystins( MC-LR,MC-LW,MC-RR, MC-LF, MC-YR)in drinking water and source water. METHODS: The five microcystins in water was cleaned by 0.22 microm millipore filter, then detected by high performance liquid chromatography-tandem mass spectrometry. Identification was achieved by electrospray ionization (ESI) in positive mode using multiple reaction monitoring. RESULTS: The calibration curves of five microcystins showed good linearity in the range of 0.5-50 microg/L with correlation coefficient in the range of 0.9994 -1.0000. The detection limit of the method was from 0.06 microg/L to 0.08 microg/L, the recoveries of two spiking levels ranged from 91.2% to 102%, and RSDs of range from 2.11% to 3.26% were obtained. CONCLUSION: The method for determination of five microcystins in drinking water and source water by HPLC-MS/MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements of national health standard method for the determination of microcystins in drinking water.

ARIMA model forecasting analysis of the prices of multiple vegetables under the impact of the COVID-19.

As a large agricultural country, China's vegetable prices affect the increase in production and income of farmers and the daily life of urban and rural residents and influence the healthy development of Chinese agriculture. 51,567 vegetable price data of 2020 are analyzed to determine the factors that influence vegetable price fluctuations in two dimensions (vertical and horizontal) in the special context of the COVID-19, and an ARIMA model of short-term price prediction is then employed and evaluated. Based on the factors affecting vegetable prices, the results of the model are further examined. Finally, pertinent suggestions are made for the development of the local vegetable industry in the post-epidemic era.

[Left maxillary first molar with three mesiobuccal canals: a case report].

A case of unusual root canal morphology was presented to demonstrate anatomic variations in maxillary first molars. This paper reported the case of a maxillary left first molar that presented three root canals in the mesiobuccal root. Root canal therapy and case management were described. This report points out the importance of looking for additional canals.

[Determination of chloroform and carbon tetrachloride in residential air by capillary gas chromatography].

OBJECTIVE: To establish solvent desorption capillary gas chromatography method for determination of chloroform and carbon tetrachloride in residential air. METHODS: The sample was absorbed by active carbon glass tube, then desorbed by Acetone and determined by Capillary Gas Chromatography. RESULTS: Linear range of this method: 5.0 x 10(-4)-1.0 x 10(-1) mg/m3 for chloroform, 1.9 x 10(-4)-1.0 x 10(-2) mg/m3 for carbon tetrachloride. The sample could be stored in the active carbon glass tube for 7 days. The re-occurrence rate of this method is good. RSD is: 0.41%-1.54% for chloroform, 0.31%-1.03% for carbon tetrachloride. Detection limit of this method: 1.5 x 10(-3) ng for chloroform, and 5.8 x 10(-4) ng for carbon tetrachloride. Recovery rate for different concentrations: 88.5%-98.6% For chloroform, and 91.0%-99.0% for carbon tetrachloride. Sampling efficiency: 93.7%-99.2% for chloroform, and 90.6%-99.2% for carbon tetrachloride. Other Volatile halogenated hydrocarbons with similar structures and similar nature in the residential air do not interfere the measurement. CONCLUSION: The method was easy and quick, with high sensibility. It was applicable to monitor the chloroform and carbon tetrachloride in residential air.

[Flow injection analysis method for hygienic examination of volatile phenol compounds in the air of residential area].

OBJECTIVE: To establish a flow injection analysis for hygienic examination of volatile phenol compounds in the air of residential area. METHOD: Volatile phenol compounds in the air was sampled by a fritted glass bubbler with 10 ml 0.1 mol/L sodium hydroxide inside and analyzed by a flow injection analyzer. The samples were reacted with 4-aminoantipyrine after online distillation in the presence of potassium ferricyanide. RESULTS: There was a linear relationship in the range of 0 - 1500 microg/L. The samples could be stored in a 4 degrees C refrigerator at least for 7 days. This method showed a high reproducibility with relative standard deviations of 0.84% - 1.0%. The recoveries of standard addition of the method were 100.3% - 103.3%. The detection limit was 0.0003 mg/m3 and the linear range was 0.0009 -0.25 mg/m3. The absorption efficiencies were 96.1% - 97.2%. CONCLUSION: This method meets the requirements for the analysis of volatile phenol compounds in the air of residential area.

Imbalance and breakout in the post-epidemic era: Research into the spatial patterns of freight demand network in six provinces of central China.

This study aims to explore the freight demand network spatial patterns in six provinces of central China from the perspective of the spread of the epidemic and the freight imbalance and breakout. To achieve this purpose, the big data of "cart search" demand information provided by small and medium freight enterprises on the freight information platform are analyzed. 343,690 pieces of freight demand big data on the freight information platform and Python, ArcGIS, UCINET, and Gephi software are used. The results show that: (1) The choke-point of unbalanced freight demand network is Wuhan, and the secondary choke-points are Hefei and Zhengzhou. (2) In southern China, a chain reaction circle of freight imbalance is formed with Wuhan, Hefei, and Nanchang as the centers. In northern China, a chain reaction circle of freight imbalance is formed with Zhengzhou and Taiyuan as the centers. (3) The freight demand of the six provinces in central China exhibits typical characteristics of long tail distribution with large span and unbalanced distribution. (4) The import and export of freight in different cities vary greatly, and the distribution is unbalanced. This study indicates the imbalance difference, chain reaction, keys and hidden troubles posed by the freight demand network. From the perspectives of freight transfer breakout, freight balance breakout, freight strength breakout, and breakout of freight periphery cities, we propose solutions to breakouts in the freight market in six provinces of central China in the post-epidemic era.

[Determination of mercury by electric heat digestion and atomic fluorescence spectrometry].

OBJECTIVE: To develop a method to determine mercury by electric heat digestion and atomic fluorescence spectrometry. METHODS: After electric heat digestion using aqua regia, the soil samples were determined by atomic fluorescence spectrometry. RESULTS: The linear range of the method was from 0 to 50 microg/L. The correlation coefficient was 0.9994. The detection limit of the method was 0.0051 microg/L. The relative standard deviations ranged from 1.55% to 6.52%. Four soil samples for quality control were determined by this method and all of the results were in the range of the true values. CONCLUSION: The optimized method have many advantages, such as high sensitivity, few interferences, wide linearity. At the same time,it is simple and rapid.

Prevalence and Distribution Characteristics of blaKPC-2 and blaNDM-1 Genes in Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae infections have caused major concern and posed a global threat to public health. As blaKPC-2 and blaNDM-1 genes are the most widely reported carbapenem resistant genes in K. pneumonia, it is crucial to study the prevalence and geographical distribution of these two genes for further understanding of their transmission mode and mechanism. PURPOSE: Here, we investigated the prevalence and distribution of blaKPC-2 and blaNDM-1 genes in carbapenem-resistant K. pneumoniae strains from a tertiary hospital and from 1579 genomes available in the NCBI database, and further analyzed the possible core structure of blaKPC-2 or blaNDM-1 genes among global genome data. MATERIALS AND METHODS: K. pneumoniae strains from a tertiary hospital in China during 2013-2018 were collected and their antimicrobial susceptibility testing for 28 antibiotics was determined. Whole-genome sequencing of carbapenem-resistant K. pneumoniae strains was used to investigate the genetic characterization. The phylogenetic relationships of these strains were investigated through pan-genome analysis. The epidemiology and distribution of blaKPC-2 and blaNDM-1 genes in K. pneumoniae based on 1579 global genomes and carbapenem-resistant K. pneumoniae strains from hospital were analyzed using bioinformatics. The possible core structure carrying blaKPC-2 or blaNDM-1 genes was investigated among global data. RESULTS: A total of 19 carbapenem-resistant K. pneumoniae were isolated in a tertiary hospital. All isolates had a multi-resistant pattern and eight kinds of resistance genes. The phylogenetic analysis showed all isolates in the hospital were dominated by two lineages composed of ST11 and ST25, respectively. ST11 and ST25 were the major ST type carrying blaKPC-2 and blaNDM-1 genes, respectively. Among 1579 global genomes data, 147 known ST types (1195 genomes) have been identified, while ST258 (23.6%) and ST11 (22.1%) were the globally prevalent clones among the known ST types. Genetic environment analysis showed that the ISKpn7-dnaA/ISKpn27 -blaKPC-2-ISkpn6 and blaNDM-1-ble-trpf-nagA may be the core structure in the horizontal transfer of blaKPC-2 and blaNDM-1 , respectively. In addition, DNA transferase (hin) may be involved in the horizontal transfer or the expression of blaNDM-1 . CONCLUSION: There was clonal transmission of carbapenem-resistant K. pneumoniae in the tertiary hospital in China. The prevalence and distribution of blaKPC-2 and blaNDM-1 varied by countries and were driven by different transposons carrying the core structure. This study shed light on the genetic environment of blaKPC-2 and blaNDM-1 and offered basic information about the mechanism of carbapenem-resistant K. pneumoniae dissemination.

[Determination of ivermectin in pig serum by high performance liquid chromatography].

OBJECTIVE: To establish a high performance liquid chromatographic method for the determination of micro-amount of ivermectin in pig serum. METHODS: Ivermectin in pig serum was extracted with ethyl acetate after the serum protein was precipitated with 0.05% metaphosphoric acid-methanol in the ratio of 7:3 (V/V). Then the sample was centrifuged at 4000 r/min for 5 min and the supernate was evaporated to dryness with rotary vacuum evaporator. The residue was dissolved with 0.20 ml of methanol as the sample solution for HPLC analysis. HPLC column used was a phenomenex C18 (5 microm, 250 mm x 4.6 mm) with a same type of guard column. Mobile phase consisted of methanol and water in the ratio of 90:10 (V/V) and the flow rate was 1.0 ml/min. The detection wavelength was 245 nm. RESULTS: The linear range of the method was found to be 0.010-20 mg/L and its detection limit was 0.010 mg/L. The relative standard deviation of the method was 0.78%-3.82% and the recoveries varied from 94.0% to 100.0%, with an average recovery of 97.3%. CONCLUSION: This method is simple, reproducible, accurate and suitable to the determination of micro-amount of ivermectin in pig serum.

Allogeneic mesenchymal stem cell therapy for bisphosphonate-related jaw osteonecrosis in Swine.

Bisphosphonates (BPs), which are used to treat a variety of clinical disorders, have the side effect of jawbone necrosis. Currently, there is no reliable treatment for BP-related osteonecrosis of the jaw (BRONJ) due to a lack of understanding of its pathogenesis. To investigate the pathogenesis of BRONJ and observe the treatment effect of bone marrow mesenchymal stem cell (BMMSC) transplantation, we established a preclinical animal model of BRONJ in miniature pigs (minipigs). After treatment with zoledronic acid, the clinical and radiographic manifestations of BRONJ could be observed in minipigs after first premolar extraction. The biological and immunological properties of BMMSCs were impaired in the BP-treated minipigs. Moreover, the ratio of Foxp3-positive regulatory T-cells (Tregs) in peripheral blood decreased, and interleukin (IL)-17 increased in the serum of BP-treated minipigs. After allogeneic BMMSC transplantation via intravenous infusion, mucosal healing and bone reconstruction were observed; IL-17 levels were reduced; and Tregs were elevated. In summary, we established a clinically relevant BRONJ model in minipigs and tested a promising allogeneic BMMSC-based therapy, which may have potential clinical applications for treating BRONJ.

Effect of irradiation on microvascular endothelial cells of parotid glands in the miniature pig.

PURPOSE: To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands. METHODS AND MATERIALS: A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVD was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg(2+)-dependent SMase (ASMase and NSMase, respectively) was also assayed. RESULTS: Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics. CONCLUSIONS: Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage.