The aim of this study was to construct a lentiviral vector of CXCR4-siRNA (Lenti-CXCR4-siRNA) and investigate whether the vector can inhibit the growth, migration, invasion and hepatic metastasis of colorectal cancer (CRC). RT-PCR and western blotting were employed to identify the ideal RNA interference sequence. Lenti-CXCR4-siRNA was constructed and transfected into the SW480 cell line. We used RT-PCR and western blotting to measure the expression of CXCR4 RNA and protein, respectively; the MTS assay to assess the proliferation of SW480 cells; transwell chambers to estimate the inhibitory effect on migration and invasion; and the Balb/c nude mouse model of CRC to examine the inhibition of hepatic metastasis. The relative expression of the CXCR4 gene and protein was 5.4 and 18.95%, respectively, in the siCXCR4 group. The genes in the expression plasmid pLenti-CXCR4-siRNA were in the correct order. In the SW480, nonsense control (NC) and the Lenti-CXCR4-siRNA groups CXCR4 RNA levels were, respectively, 0.54±0.06, 1.00±0.03 and 0.11±0.04 (P=0.0001); CXCR4 protein levels were 0.60±0.03, 0.72±0.03 and 0.18±0.02 (P=0.0001); the OD value was 1.38±0.04 (P=0.0050), 1.28±0.05 (P=0.0256) and 0.92±0.06; SW480 cell number in migration test was 32±6.85, 32.63±1.69 and 0.75±0.71 (P=0.0000); SW480 cell number in the invasion test was 29.13±10.3, 30.38±6.09 and 0.63±0.74 (P=0.0000); hepatic metastasis number was 7.10±3.98 (P=0.034), 7.50±4.09 (P=0.019) and (3.50±2.51); hepatic metastasis mean weight (in g) was 2.25±2.51 (P=0.000), 2.11±2.38 (P=0.000) and 1.45±2.07. Lenti-CXCR4-siRNA constructs were correctly constructed and effectively inhibit the expression of CXCR4 RNA and protein, reducing the proliferation, migration, invasion capacity of SW480 cells and hepatic metastasis of CRC. Colorectal cancer (CRC) is a common malignant disease. Five-year survival rate and cancer-specific 5-year survival rate of patients with CRC (independent of stage and cause of death) are 56 and 64%, respectively. Overall survival is 96% in stage 1, 92% in stage 2, 58% in stage 3, and 0% for patients with metastatic disease at the time of diagnosis. Cancer-specific survival rates range from 100% for patients in stage 1 to 0% for patients with metastatic disease. Local and distant metastasis are the main risk factors reducing survival of patients with CRC  BALB/c mice , Lenti-X Bicistronic Expression System , Lipofectamine 2000, Lipofectamine™ RNAiMAX transfection reagents , Opti-MEM medium , CellTiter96AQ cell proliferation detection kit , Transwell plates (BD), SYBR Green PCR Master Mix , and thermal cycler  were used in the present study. This study was approved by the ethics board at the First Affiliated Hospital of Sun Yat-sen University.Mouse monoclonal anti-human CXCR4 antibody, GAPDH, RPMI-1640, fetal bovine serum (FBS), penicillin, streptomycin, Polybrene, phosphate-buffered saline , Matrigel , RNA extraction kit, reagents for reverse transcription, western blotting kit , human colon cancer cell line (SW480 cells), nude (CXCR4 gene (NM-004363.2)] were synthesized by Qiagen: sequence A (siCXCR4-A): 5’-UAAAAUCUUCCUGC CCAC CdTdT-3’ (sense) and 3’-dTdTAUUUUAGAAGGACGG GUGG-5’ (antisense); sequence B (siCXCR4-B): 5’-CAAGGAA GCUGUUGGCUGAdTdT-3’ (sense) and 3’-dTdTGUUCCUU CGACAACCGACU-5’ (antisense); sequence C (siCXCR4-C): 5’-CUGUCCUGCUAUUGCAUUAdTdT-3’ (sense) and 3’-dTd TGACAGGACGACGAUAACGUAAU-5’ (antisense). In addition, negative control siCXCR4 was synthesized by Guangzhou RiboBio Co.  for monitoring the influence of exogenous genes.Three pairs of siRNAs targeting CXCR4  and tube B (5 μl of Lipofectamine 2000 mixed in 500 μl of Opti-MEM and kept at room temperature for ≤5 min) were mixed, kept at room temperature for 20 min, added to each well, and incubated 4-6 h. Then, the medium was removed, the cells were washed in PBS twice, and 2 ml of complete medium was added. At 24 h after transfection, the medium was removed, 1 ml of TRIzol reagent was added, and quantitative PCR was performed to detect mRNA levels.SW480 cells were routinely cultured and seeded on 6-well plates at a density of 5×10μg of RNA in distilled water (dH2O) was diluted to a final volume of 12 μl with dH2O, was heated to and kept at 65°C for 5 min for RNA denaturation, kept on ice to avoid RNA renaturation, and incubated with 0.5 μl of Oligo(dT) (Promega) random primer (Promega), 2.0 μl of 10 mM dNTP, 0.5 μl of RNase inhibitor, 4.0 μl of 5X buffer and 0.5 μl of M-MLV at 30°C for 10 min, 42°C for 60 min and 80°C for 10 min. The primers were as follows: 18S rRNA-112 bp . CXCR4-159 bp: . The reaction mixture  was kept at 95°C for 5 min, and PCR conditions were as follows: 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 32 sec. The melting temperature was 60–95°C. All experiments were performed in triplicate. Cells transfected with 25 nM siCXCR4-C had the lowest expression of CXCR4 and therefore it was used in the following experiment.Total RNA was extracted using a kit according to the manufacturer’s instructions and the quality of RNA determined. In brief, a solution of 1.0 SW480 cells were passaged 24 h prior to transfection. When 30–50% confluent, cells were transfected with 25 nM siCXCR4-C or NCsiCXCR4 in the presence of Lipofectamine™ RNAiMAX in Opti-MEM for 4 h, and then the medium was replaced with RPMI-1640 containing 10% FBS. Protein extraction, SDS-PAGE, and protein transfer were done according to the manufacturer’s instructions. The membrane was washed with Tris-buffered saline with Tween-20 , incubated with 5% non-fat milk at room temperature overnight to block nonspecific binding, washed with TBST , treated with primary antibody (anti-CXCR4 or anti-GAPDH) at 37°C for 2 h, washed with TBST , treated with secondary antibody at 37°C for 1 h, washed with TBST (3X) and then with distilled water , placed on a plastic board, incubated with chemiluminescence substrate for 5 min, and visualized by an imaging system in the dark.AgeI/EcoRI by incubating 3 μl of vector GV115 with 0.5 μl of EcoRI, 0.5 μl of AgeI, 1 μl of 10X D buffer, and 5 μl of deionized water or distilled water at 37°C overnight, and the products were separated by 1% gel electrophoresis.Vector GV115 was digested with μl), recycled vector GV115 (2 μl), 10X ligase buffer (1 μl), T4 DNA ligase (1 μl), and deionized water (3 μl). Ligation was done at 16°C for 2 h. The lentiviral plasmid product was pLenti-CXCR4-siRNA. In the control group, water was added instead of PCR products.The following reagents were added to a 0.2-ml EP tube: PCR products . The plate was incubated at room temperature until the solution was absorbed, and inverted and placed in a 37°C incubator overnight.On ice, 5 XhoI restriction site in the pLenti-CXCR4-siRNA sequence, and thus XhoI was used for identification. Once the sequence was cleaved, these plasmids were either positive or negative for pLenti-CXCR4-siRNA. The plasmid was cut with restriction enzyme XhoI at 37°C overnight in the following solution: 3.6 μl of ddH2O, 1 μl of 10X H buffer, 1 μl of 10X BSA, 4 μl of plasmid and 0.4 μl of XhoI.One colony was collected from the plate, transferred to a 3-ml LB tube, and incubated overnight with continuous shaking for plasmid extraction. The sequencing of pLenti-CXCR4-siRNA was performed by the BGI Gene Tech Co., Ltd. . There was a 5 cells/ml into flasks (10 ml/flask). At 30 min before transfection, the medium was replaced with complete medium (5 ml/flask). The packaging of lentiviral vector was done according to the instructions provided with the Lenti-X Bicistronic Expression System. In brief, 1.5 μg of packaging plasmid, 0.5 μg of expression plasmid, and 250 μl of serum-free medium in a 1.5-ml sterilized EP tube were incubated at room temperature for 5 min, while 9 μl of Lipofectamine 2000 and 250 μl of serum-free medium in a separate 1.5-ml sterilized EP tube were incubated at room temperature for 5 min. The DNA solution and Lipofectamine were mixed and incubated at room temperature. To each well of a 6-well plate, the following was added with overnight incubation at 37°C: 1 ml of growth medium containing serum and DNA-Lipofectamine mixture and 1 ml of re-suspended 293T cells (1×106 cells/ml). The medium containing DNA-Lipofectamine was removed and replaced by DMEM. At 48–72 h after transfection, the supernatant was collected.293T cells in the logarithmic phase (cell confluence: 70–80%) were washed with 5 ml of PBS, treated with 2–3 ml of trypsin until these cells became round, switched to complete medium [90% DMEM (high glucose: 4.5g/l) + Glu Max + 10% FBS + penicillin + streptomycin (100X)] to stop the cutting, and dispersed into a single cell suspension, counted to determine cell density, and seeded at 3×10NaCl (5 mol/l) was added to the above supernatant to a final concentration of 0.15 mol/l. A solution of 20% (w/v) PEG8000 was added and the final mixture was incubated at 4°C overnight with continuous shaking, then centrifuged at 4°C for 30 min at 10,000 g. The supernatant was collected with a vacuum pump in a biological safety cabinet and the sediment was diluted in an appropriate volume of OptiMEM, covered with Parafilm membrane, and stored at 4°C. The Lenti-CXCR4-siRNA was titered using a kit according to the manufacturer’s instructions.3 cells/ml (90 μl/well).SW480 cells were seeded into 20 wells of a 96-well plate at a density of 3–5×10μl per tube). Then, 5 μl of 1×108 TU/ml Lenti-CXCR4-siRNA was serially diluted to 1×107 TU/ml and 1×106 TU/ml by addition to the first tube followed by gentle vortexing to avoid foaming and subsequent transfer of 5 μl of solution in the first tube to the second tube followed by vortexing. In another tube, 2 μl of Polybrene was diluted to 400 and 10 μl added to each well. The volume of Polybrene per well of 100 μl was diluted 2,000 times to a final concentration of 5 μg/ml. Then, 10 μl of lentivirus solution at three different concentrations , was added to the corresponding wells. The cell count per well was ∼1×104. Thus, the multiplicity of infection (MOI) per well was 100, 10 and 1, respectively. In the Polybrene well, 10 μl of diluted Polybrene was added. The plates were gently vortexed and placed in an incubator for 8–12 h. The cells were observed and the supernatant was removed. The medium was refreshed. At 3–4 days after transfection, fluorescence was detectable under a microscope.Conventional medium was added to 2 sterilized 1.5-ml EP tubes  as fluorescent colonies on the plate under a fluorescence microscope on the third day. The marked cell colonies were digested with 0.25% trypsin, transferred to a 24-well plate, and observed again under a fluorescence microscope. Those with fluorescence were selected for passaging.The expression of CXCR4 mRNA and protein was assayed by real-time reverse transcription (qRT)-PCR and western blotting as described above.5 cells/ml, seeded into a 96-well plate , allowed to adhere, collected at 24, 48, 72 and 96 h, incubated 4 h with MTS solution (ratio: 1:10), and assessed for cell proliferation by optical density (OD) measurement at 490 nm with a micro-plate reader.Cells were divided into the SW480 group, negative control (NC) group, and Lenti-CXCR4-siRNA group. Cells were digested with trypsin, adjusted to a density of 1×105 cells with Lenti-CXCR4-siRNA, the cells were re-suspended in 100 μl of serum-free RPMI-1640, transferred to the upper chamber of a Transwell system with 600 μl of complete medium added to the lower chamber, and incubated at 37°C in an atmosphere containing 5% CO2 for 24 and 48 h. The cells in the upper chamber were removed with a swab, fixed in 4% paraformaldehyde for 15 min, and washed in PBS once. The upper chamber was stained with crystal violet for 10 min and washed in PBS once. The cells migrating from the upper chamber were counted under a microscope.Cells were grouped as described above. Two days after transfection of 1×10μl of Matrigel solution (Matrigel kept at 4°C overnight and then mixed with serum-free medium at a ratio of 1:3) was added to a pre-cooled Transwell system and incubated at 37°C for 2 h. After excess solution was removed from the chamber, serum-free medium  was added to the upper and lower chambers, and the Transwell plate was incubated at 37°C overnight. The procedures described above were performed.Cells were divided into 3 groups as described above. Approximately 40 nu/nu mice aged 4–6 weeks and weighing 14–23 g were housed in a specific pathogen-free (SPF) environment. These animals were divided into three groups as described above (n=10). Cells in the logarithmic growth phase were digested in 0.25% trypsin to prepare a single cell suspension. The cell viability was confirmed to be ≥95% by trypan blue exclusion staining. The cell density was adjusted to 1×107/ml. The procedures were done under aseptic conditions. Mice were intraperitoneally anesthetized with 1% pentobarbital sodium (35 mg/kg). A midline incision was made in the upper abdomen, and the spleen was exposed and slowly injected with cell suspension . The injection site was compressed with a tampon soaked in 95% alcohol and the wound was closed. These mice were returned to SPF housing conditions and monitored for activity, food intake, and change in body weight. At 4 weeks after surgery, laparotomy and subsequent exploration were done to observe liver metastasis. The number of metastatic sites and hepatic metastasis mean weight (in g) were determined.A total of 30 BALB/c Statistical analysis was done using SPSS version 11.0 for Windows. Comparisons were made by t-test. A value of P<0.05 was considered statistically significant.The mRNA expression was the lowest in the sequence C group and ∼5.4% of that in the NCsiCXCR4 group. Thus, cells with sequence C were used in the following experiments and designated the siCXCR4 group.The SW480, NCsiCXCR4, and siCXCR4 groups had a GAPDH optical density (OD) of 367.67, 3593.84 and 3190.51, respectively and a CXCR4 OD of 820.54, 1106.66 and 604.48, respectively. Thus, the relative expression of CXCR4 was 22.34, 30.80 and 18.95%, respectively. These findings demonstrated that siCXCR4 could effectively inhibit expression of CXCR4 protein .XhoI restriction site. However, the siRNA sequence had a XhoI site that could be cut by XhoI. Blank GV115 was circular and therefore migrated more rapidly. Thus, blank GV115 migrated ahead of the cut GV115 (linear sequence), suggesting successful cloning (The vector GV115 (7.5 kb in size) had no  cloning .All the bases in siRNA were correct. BLAST analysis showed successful cloning and that there was no mutation. Thus, pLenti-CXCR4-siRNA could be used in the experiments that followed .All 293T cells transfected with pLenti-CXCR4-siRNA were shown to exhibit green fluorescence under a fluorescence microscope .All infected cells showed green fluorescence under a fluorescence microscope, suggesting successful transfection .In the SW480, NC, and Lenti-CXCR4-siRNA groups, the expression of CXCR4 mRNA was 0.54±0.06, 1.00±0.03 and 0.11±0.04, respectively. The expression of CXCR4 mRNA was the lowest in the CXCR4 RNA group and differed markedly from the SW480 and NC groups (P=0.0001).μg/μl) was 4.61, 3.58 and 3.08, respectively. Western blot assays showed that the expression of CXCR4 protein was the lowest in the Lenti-CXCR4-siRNA group. The relative expression of CXCR4 protein was 0.18±0.02, 0.6±0.03 and 0.72±0.03, respectively, in the Lenti-CXCR4-siRNA, SW480, and NC groups  was markedly lower in the Lenti-CXCR4-siRNA group than in the SW480 group  and NC group . This suggests that RNA interference (RNAi) significantly inhibits the growth of SW480 cells.The number of migrated cells was significantly lower in the Lenti-CXCR4-siRNA group than in the SW480 group  and NC group . This suggests that RNAi significantly suppresses the migration of SW480 cells .The number of migrated cells was dramatically lower in the Lenti-CXCR4-siRNA group than in the SW480 group (0.63±0.74 vs 29.13±10.30) and NC group . This suggests that CXCR4 RNAi markedly inhibits the invasiveness of SW480 cells.The number of metastatic lesions was significantly lower in the Lenti-CXCR4-siRNA group than in the SW480 group  and NC group . The mean weight of metastatic tumors (in g) was dramatically lower in the Lenti-CXCR4-siRNA group than in the SW480 group  and NC group . These findings demonstrate that Lenti-CXCR4-siRNA transfection markedly inhibits the liver metastasis of SW480 cells.As chemoattractant cytokines, chemokines can facilitate cell activation, differentiation, and trafficking. Several organs including lung, lymph nodes, liver, skeletal muscle, brain, kidney, heart, skin, and bone marrow, express CXCL12.CXCR4 is expressed in most cancer cells, and hypoxia and injury stimulate its production. The binding of CXCL12 to CXCR4 initiates several signaling pathways and promotes chemotaxis, cell proliferation, increase in intracellular calcium concentration and gene transcription . CXCR4-p9 TU/ml. In the present study, Lenti-CXCR4-siRNA effectively inhibited the expression of CXCR4 mRNA and reduced the level of CXCR4 protein. Lentiviral vector-mediated Met oncogene-specific, stable RNA interference impaired spontaneous motility and invasiveness of canine osteosarcoma cells (RNAi is a process in which double-stranded RNA is used to enhance the degradation of cognate mRNA. Synthetic 21–23 nucleotide (siRNA) has been demonstrated to induce transient and efficient RNAi . SiRNA-pma cells . Lentivima cells . Thus, iThe increase of tumor cell proliferation and the decrease of apoptosis are essential characteristics of malignant lesions. Our data show that Lenti-CXCR4-siRNA reduces SW480 cell proliferation, which is evidence that the CXCR4/SDF-1 axis stimulates cell multiplication and that inhibition of CXCR4 was able to reduce SW480 cell growth. One study showed that CXCR4 protein expression on the CD34(+) cell surface is lower in the low-grade myelodysplastic syndrome than in high-grade myelodysplastic syndrome; CD34(+) cell apoptosis is significantly higher in low-grade myelodysplastic syndrome, and apoptosis is negatively correlated with CXCR4 expression . In anotin vitro, and reduced the number and mean weight of hepatic metastasis in the liver metastasis model in Balb/c mice. Previously, recombinant CXCR4-RNAi plasmids were found to reduce CXCR4, inhibit cell growth, invasiveness, and migration, and induce cell apoptosis in renal cell carcinoma in vitro  tumor cells, significant inhibition of the proliferation of both cell lines as shown . Lentivias shown . In a leas shown . Knockdo(in vivo ,19. CXCL(in vivo .In conclusion, hepatic metastasis of CRC is a prevalent and serious problem, and efficacious therapy is required to deal with it. The recombinant lentivirus Lenti-CXCR4-siRNA was correctly constructed in the present study and potently inhibited CXCR4 expression and growth, migration, invasion, and liver metastasis of CRC. Lentiviral mediated CXCR4 RNAi is a potential treatment for CRC.


Triclocarban  in the environment has been well documented. Methods have been developed to monitor TCC levels from various matrices including water, sediment, biosolids, plants, blood and urine; however, no method has been developed to document the concentration of TCC in fecal content after oral exposure in animal studies. In the present study, we developed and validated a method that uses liquid extraction coupled with HPLC-MS/MS determination to measure TCC in feces. The limit of detection and limit of quantitation in control rats without TCC exposure was 69.0 ng/g and 92.9 ng/g of feces, respectively. The base levels of TCC in feces were lower than LOD. At 12 days of treatment, the fecal TCC concentration increased to 2220 µg/g among 0.2% w/w exposed animals. The concentration in fecal samples decreased over the washout period in 0.2% w/w treated animals to 0.399 µ/g feces after exposure was removed for 28 days. This method required a small amount of sample (0.1 g) with simple sample preparation. Given its sensitivity and efficiency, this method may be useful for monitoring TCC exposure in toxicological studies of animals. Triclocarban  is a chlorinated urea commonly used as a broad range antimicrobial in personal care products . Human eKow = 4.9 and Koc = 50,118 L/kg, respectively), and is environmentally persistent undergoing little degradation for months [i.e., treated wastewater sludge) are applied to agriculture fields as fertilizer, TCC is then transferred to the terrestrial environment raising safety concerns regarding the potential uptake of TCC into the food chain, allowing for a potential secondary human exposure route [After topical application, the bulk of TCC enters the wastewater treatment process where current treatment technologies can only transfer up to 79% of TCC to waste water sludge . With hir months . When bire route . Previoure route . These rre route .Methods of TCC detection have been reported in both solid and liquid biological matrices  . Oral TC13C6-TCC, quantitation reference) and 13C6-2,4,5-trichlorophenoxyacetic acid  were purchased from Cambridge Isotope  and prepared in methanol. Stock solutions of TCC (5 mg/mL) were prepared in acetone and TCC standards  were prepared from stock solution in methanol. TCC standards, 13C-TCC and TCPAA were stored at −20 °C until use.Acetone (99.5% purity), acetonitrile (99.9% purity), methanol (99.9% purity) and water (purity grade: Optima™) were purchased from Thermo Fisher Scientific . TCC (99% purity), carbon-13 labeled TCC  22 followed by 4 weeks washout with 2020X only. Feces were collected prior to treatment initiation at PND 22 and after 12 days of treatment. Fecal samples were also collected throughout the washout period at 2, 8, and 28 days after the withdrawal of TCC exposure. Samples were snap frozen and stored at −80 °C until analysis. The Animal Use and Care Committee at University of Tennessee, Knoxville, approved all research protocols used in this report. The studies were conducted in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.Feces were collected from female Sprague Dawley rats . Briefly, rats (n = 4 per group) were weight ranked and randomized to control or TCC treatment groups and fed Harlan chow diet (2020X) or 2020X supplemented with 0.2% w/w TCC 13C6-TCC was added to 0.1 g thawed feces and thoroughly mixed with a countertop vortex at maximum speed for 1 minute. Next, 5 mL of 80:20 acetonitrile/H2O was added to each sample and vortexed at maximum speed for 30 s. After vortexing, samples were sonicated for 30 min and centrifuged at 1500 rpm for 8 min at 21 °C followed by 0.45 µm filtration prior to blow down under nitrogen flow to 1 mL. Samples were reconstituted to 2 mL with a 1:1 mixture of methanol/H2O. A 300 µL aliquot of the mixture plus 6 µL of 2500 ng/mL 13C6 TCPAA was added to auto-sampler vials prior to analysis.To extract TCC from fecal samples, 50 µL of 500 ng/mL m/z: 312.72 and 160.00, respectively [2O with 0.02% acetic acid (mobile phase A) and methanol (mobile phase B). The analyte was separated using a gradient program starting with Time (minute) = 0, A = 40%, B = 60% at 0.3 mL/min; Time = 3, A = 2%, B = 98% at 0.3 mL/min; Time = 5.5, A = 2%, B = 98% at 0.3 mL/min; Time = 5.6, A = 2%, B = 98% at 0.35 mL/min; Time = 12, A = 2%, B = 98% at 0.35 mL/min; Time = 12.05, A = 40%, B = 60% at 0.35 mL/min; Time = 18.5, A = 40%, B = 60% at 0.35 mL/min and Time = 18.6, A = 40%, B = 60% at 0.3 mL/min. The MS conditions used in the method were set as follow: negative Electrospray Ionization (ESI); 200 °C for capillary temperature; 425 °C for vaporizer temperature; 20 (Arb) for sheath gas pressure; 2 (Arb) for Aux gas; and 1.5 mTorr for collision gas pressure. Collision energy was set 17 for TCC and 13C-TCC, and at 16 for 13C-TCPAA. Product ions were monitored at m/z 200.700 for 13C-TCPAA; m/z 160.000 for TCC; and m/z 159.700 for 13C-TCC. The signal:noise ratio was set at ˃3.Instrumental protocols followed were from EPA Method 1694  and as f© (version 2.1) software was utilized to acquire and analyze data. Concentration of TCC in the sample was determined as the peak area ratio of TCC/13C-TCC as compared to the calibration curve derived from TCC concentrations: 2.5, 7.5, 37.5, 125, and 500 ng/mL. Quan Browser in Thermo Xcalibur 2.2® was used to set up the calibration curve . A quadratic log-log calibration curve was used for quantitation. Calibrants were weighted by the inverse of the square of their quantity (1/X2).Thermo Xcalibur13C-TCPAA, and that of a 10 ng/mL concentration of TCC standard in fecal matrix are shown in 13C-TCC to characterize the performance of the assay in the presence of matrix. The LOD and the LOQ of the TCC in the fecal matrix were 69.0 and 92.9 ng/g feces, respectively. To determine intra-assay variability, concentration of 10 or 350 ng/mL TCC standard was spiked into control feces (n = 4 per concentration). Day-to-day inter-assay variability was calculated from 4 extractions over a period of 4 days with TCC spiked at both 10 and 350 ng/mL. Percent relative standard deviation (%RSD) of intra-assay variability was 22.4% at 10 ng/mL and 4.99% at 350 ng/mL respectively; the average recovery of TCC was 87.7% at 10 ng/mL and 120% at 350 ng/mL; the inter-assay variability (%RSD) was 30.6% (relative recovery of 73.5%) at 10 ng/mL and 14.1% (relative recovery of 130.6%) at 350 ng/mL over the period of 4 days. The accuracy and precision of the assay in a single extraction day were assessed by repeat analysis of 15 control replicates spiked with either 25 ng/mL or 100 ng/mL TCC  and utilized a liquid extraction method (Acetonitrile/H2O: 80/20). Our method was based on EPA Method 1694, which is optimized for Group 3 Compounds [


In solid-state anaerobic digestion (AD) bioprocesses, hydrolytic and acidogenic microbial metabolisms have not yet been clarified. Since these stages are particularly important for the establishment of the biological reaction, better knowledge could optimize the process performances by process parameters adjustment.VS-1 of fermentative products under wet (10 and 14% TS), dry (19 to 28% TS), and highly dry (28 to 33% TS) conditions, respectively. Furthermore, both wet and dry fermentations showed acetic and butyric acid metabolisms, whereas a mainly butyric acid metabolism occurred in highly dry fermentation.This study demonstrated the effect of total solids (TS) content on microbial fermentation of wheat straw with six different TS contents ranging from wet to dry conditions (10 to 33% TS). Three groups of metabolic behaviors were distinguished based on wheat straw conversion rates with 2,200, 1,600, and 1,400 mmol.kgSubstrate conversion was reduced with no changes of the metabolic pathways until a clear limit at 28% TS content, which corresponded to the threshold value of free water content of wheat straw. This study suggested that metabolic pathways present a limit of TS content for high-solid AD. At present, anaerobic digestion (AD) of agricultural waste such as manure and lignocellulosic residues represents an economical, environmental, and societal opportunity to produce massively renewable energy (methane and hydrogen) and agricultural amendment through a digestate of high agronomic quality ,2. The Aet al.[et al.[et al. [et al. [Clostridium in inhibited systems at high TS content. So far, the effect of TS content on dry microbial fermentation has rarely been evaluated. Only two studies compared the effects of a gradient of TS content  on biohThe first two steps of AD, hydrolysis and acidogenesis, are commonly considered as the limiting steps of solid waste degradation ,10,15. Dc was estimated at 2.4 ± 0.2 g.gTS-1, which corresponds to a critical TS content of 29.4 ± 1.6% g.g-1[Wheat straw had an initial TS content of 95.6 ± 0.7% and a volatile solids (VS) content of 88.9 ± 0.7%. Wheat straw fiber analysis indicated the following fractionation (based on TS content): 9.4 ± 0.6% of soluble fraction, 33.0 ± 1.1% of hemicellulose, 43.7 ± 1.3% of cellulose, and 6.4 ± 0.8% of lignin. According to drying test results, the critical water content of the straw w.6% g.g-1. ProductTS-1 for 10 and 14% TS, between 22 and 26 NmL.gTS-1 from 19 to 28% TS, and less than 20 NmL.gTS-1 for 28 and 30% TS. Based on kinetics and production of gaseous compounds, two distinct behaviors within the four replicates were observed at 28% TS: two replicates (named 28a% TS) had a similar trend to the experiments carried out at lower TS values, while two other replicates (named 28b% TS) behaved as at higher TS content. Other indicators are presented in Figure 2/gaseous products), and the biohydrogen yield. The time to achieve 90% of the hydrogen production was affected by the TS content: it was less than 4 days between 10% and 28a% TS (fast reaction rate), and more than 8 days between 28b% and 33% TS (slow reaction rate). Moreover, the hydrogen content was strongly impacted and significantly decreased with the increase of TS content, from about 55% of hydrogen at 10% TS to 15% at 33% TS. Hydrogen production was lowered by the increase of TS content and three groups were distinguished : at 10 and 14% TS, hydrogen production was higher than 15 NmL.gTS-1, between 8 and 9 NmL.gTS-1 at 19 to 28% TS, and lower than 5 NmL.gTS-1 at 28 and 33% TS. Based on the correlation of Monlau et al.[TS-1. The hydrogen potential of the wheat straw estimate was higher than the 17.8 NmL.gTS-1 of hydrogen obtained at 10% TS. Even though the hydrogen potential is higher than generally reported in the literature , the re-1 of soluble phase at 10, 14, 19, 24, 28a, 28b, and 33% TS, respectively. The decrease of water content leads to high apparent metabolite concentrations. These concentrations are theoretically highly inhibitory for methanogenic activity, even though they are classically observed with no inhibition in dry AD [2, CO2, VFA, ethanol, and lactate) was normalized by the amount of TS in the medium. The corresponding data are presented in Additional file P value of 5.7e-12, <0.001): 10 and 14% TS with approximately 2,288 ± 168 mmol.kgTS-1 of fermentative products, 19 to 28a% TS with approximately 1,610 ± 41 mmol.kgTS-1, and finally 28b and 33% TS with approximately 1,389 ± 38 mmol.kgTS-1.In addition to production of gaseous products, soluble fermentative end-products were simultaneously produced. Total concentration of soluble metabolites increased with the increase in TS content, with 3.6 ± 0.2, 5.4 ± 0.3, 5.6 ± 0.5, 6.4 ± 1.0, 8.0 ± 0.4, 9.8 ± 1.2, and 11.9 ± 1.4 g.Ln dry AD ,28, beca2 and CO2) was in the same order of magnitude, between 57 and 67% of the sum of metabolites. However, as revealed by the hydrogen/carbon dioxide ratio, three groups were distinguished : 10 and 14% TS with a hydrogen/carbon dioxide molar ratio of 1.06 ± 0.14, 19 to 28a% TS with 0.46 ± 0.05, and 28b to 33% TS with 0.23 ± 0.09. On the overall range of TS content, the hydrogen associated VFAs, that is acetic and butyric acids, represented between 20 and 32% of the sum of metabolites. However, their distribution revealed only two groups based on the butyric/acetic acid molar ratios : from 10 to 28a% TS with an average value of 0.69 ± 0.32, and 28b and 33% TS with a significantly higher ratio of 2.47 ± 0.59. Homoacetogenic activity was quantified by estimating a theoretical molar balance between the hydrogen produced or consumed for the production of acetic, propionic, and butyric acids from the glucose equivalent and the experimental amount of hydrogen . The difference indicated the hydrogen consumed, mainly by homoacetogenesis: 462, 312, 357, 174, 81, 303, and 359 mmol.kgTS-1 of missing hydrogen at 10, 14, 19, 24, 28a, 28b, and 33% TS, respectively. If for every condition the molar balance was in agreement with the accumulation of acetic acid, at 28b and 33% TS a lack of hydrogen was observed that could not be explained by any metabolite production. The other fermentative products  are associated with no hydrogen-producing pathways and corresponded to less than 7% of the total fermentative products for 10 and 14% TS, and more than 13% from 19 to 33% TS. The product distribution within this category was similar in most of the operating conditions for ethanol  and propionic acids  corresponding to 25 ± 7% and 56 ± 8% of the other metabolites, respectively. The main differences of the ‘no hydrogen’ metabolism were the measurement of 17% of lactate at 33% TS  and approximately 15% of caproic acid at 10 and 14% TS , and a decrease of valeric acid quantity at 10 and 14% TS .Table In this study, the increase in TS contents had two consecutive consequences on acidogenesis: a sharp reduction of 25% in wheat straw conversion between 14 and 19% TS, followed by a clear shift in metabolite distribution at 28% TS. A microbial community analysis was performed on the final media to verify the origin of the metabolite distribution. Additional file TS-1), a fast reaction, and a hydrogen-producing pathway-oriented metabolism (more than 50% of the gaseous products). Fermentative end-products corresponded mainly to hydrogen and carbon dioxide , acetic and butyric acids , and ethanol and propionic acids (for the last 7%). Additionally, butyric/acetic acid ratios of 0.4 and 0.8, observed respectively at 10 and 14% TS, are low when compared to the overall stoichiometric ratio of 1.5 for hydrogen production reported by Guo et al.[The group W, that is 10 and 14% TS batch tests, corresponds to water-saturated conditions usually described as wet digestion for AD systems. Wet digestion is characterized by a liquid medium, which allows homogeneity in the bioreactor, full access of the degrading microorganisms to the substrate, and dilution of inhibitory products (VFAs) . Under tuo et al.. These luo et al.. Overalluo et al.,20.TS-1 of fermentative product) was likely caused by the decrease of substrate accessibility due to the reduction of water content. Indeed, switching from wet to dry conditions makes it more difficult for microorganisms to access solids [In group D, that is batch tests operated between 19 and 28a% TS, metabolite distribution and reaction rate were both similar to group W. The distribution of soluble fermentative products corresponded mainly to acetic and butyric acids , with a butyric/acetic acid ratio ranging from 0.4 to 1.0. These ratios are consistent with literature data for dark fermentation under dry conditions . Here ags solids . This hys solids . The limTS-1 of butyric acid against 163 mmol.kgTS-1 of hydrogen). Although the existence of hydrogen-consuming pathways that could have redirected microbial activity (equation 4 and 5), the low quantity of acetic and propionic acids fails to explain the occurrence of high concentration of butyric acid concomitant to a low hydrogen yield. These observations may suggest a butyric pathway with no hydrogen production [Group HD, that is 28b and 33% TS batch tests, was characterized by very low hydrogen production, low hydrogen/gaseous product ratio, and slow fermentation rates requiring 8 days to reach the maximum hydrogen productivity. The substrate conversion was reduced by approximately 40% of group W and 15% of group D. Thus, by reducing the water content, its effect was increased in group HD, with a slight increase in ethanol and propionic acid production. This observation could partly explain the reduction of hydrogen content in gas phase (only 18%) by a higher production of carbon dioxide. If the amount of acetic acid is similar to group D, a very high concentration of butyric acid was measured . Since eoduction . Such a et al.[Clostridium) and specific Archaea for groups W, D, and HD. To explain this phenomenon, Staley et al.[TS.g-1). Therefore, at higher TS contents, no free water was available for wheat straw degradation. Interestingly, the limit between the dry and highly dry group [et al.[In addition, the value of 28% TS corresponded to a critical TS content. Two duplicates (28a%) behaved like experiments in group D 19 and 24% TS), while the two others (28b%) were classified in group HD. Interestingly, similar shifts were previously reported and discussed in studies dealing with organic solids AD . Even thet al. found they et al. suggesteey et al.. These aey et al.. It was ey et al.. Based o% TS, whip [et al.,32. Sincp [et al.. FurtherThe present study investigates the effect of TS content on wheat straw acidogenesis from wet to highly dry conditions 10 to 33% TS)  was harvested in July 2010 at an organic farm located in the Hérault region of France. A bale was homogenized manually, aliquoted in plastic bags, and stored at ambient temperature. The straw was then milled at 1 mm and sieved between 0.4 and 1 mm. The Van Soest fractionation was performed with the fiberbag system . The proportion of free and bound water was assessed with a drying test of wheat straw, according to the protocol proposed by García-Bernet et al.[c (water content expressed in g.gTS-1).Wheat straw  was applied to the inoculum to eliminate any methanogenic activity [Four different digestates were sampled in AD plants treating organic waste to obtain a high microbial diversity in the initial anaerobic inoculum. A mix of inocula was prepared to equalize the contribution of each inoculum source (25% in VS basis). Two digestates were collected from two upflow anaerobic sludge blanket (UASB) digesters treating effluents from a sugar factory or sludge from a wastewater treatment plant under mesophilic and thermophilic conditions, respectively. Two other digestates were sampled in two dry anaerobic digesters treating the organic fraction of municipal waste and operated under mesophilic and thermophilic conditions. The digestates were centrifuged at 3,000 activity ,26. It hTS.g-1. In each 600 mL flask, the medium was composed of: 21.4 ± 0.1 g of straw, 69.3 ± 1.4 mL of inoculum, 16.0 ± 0.3 g of 2-(N-morpholino) ethanesulfonic acid (MES) buffer, and 10 mL of a 3.2% NaOH solution to maintain a pH at 5.7 ± 0.1 and 4.1 ± 0.1 mL of a trace element solution with the following composition: 1.5 g.L-1 of FeCl2,H2O; 62 mg.L-1 of H3BO3,H2O; 117 mg.L-1 of MnSO4,H2O; 26 mg.L-1 of CoCl2,6H2O; 120 mg.L-1 of ZnCl2; 28 mg.L-1 of NiCl2,6H2O; 38 mg.L-1 of CuCl2,2H2O; 31.8 mg.L-1 of NaMoO4,2H2O; and 4.4 mL of HCL 37%. Distillated water addition was calculated by mass balance on both TS and VS contents  in order to obtain the required TS contents: 259.2 ± 1.9, 130.3 ± 1.0, 67.6 ± 0.7, 30.6 ± 0.3, 5.1 ± 0.1, and 0.0 ± 0.0 mL for 9.5, 14.2, 18.8, 24.0, 28.1, and 33.2% TS, respectively. The batch tests were then incubated at 35°C ± 1 for 11 days.Batch tests were operated in four replicates, with six different TS contents ranging from wet to dry conditions: 10, 14, 19, 24, 28, and 33 gAll batch reactors were connected to a multiplexed R3000 micro-gas chromatograph (μGC) with two analytical capillary columns  to monitor gas production on line. The first column was dedicated to carbon dioxide analysis and corresponded to a 5Ǻ molecular sieve (10 m length and 0.32 mm diameter) with argon as carrier gas at a pressure of 30 PSI. The second column dedicated to oxygen, hydrogen, nitrogen, and methane analysis was a PLOT Q (8 m length and 0.32 mm diameter) with helium as carrier gas (20 PSI). The injector and column temperatures were 90°C and 80°C, respectively. The detector was a micro-thermal conductivity detector (μTCD). Multiplexing the channels allowed the simultaneous connection of 28 batch tests with a measure of the total gas production every 4 hours, by pressure measurement. To maintain a constant pressure in headspace, the gas composition was evaluated by sampling only when pressure was higher than 1.1 bars.-1 was used as carrier gas. Main fermentation metabolites were analyzed after filtration at 0.2 μm by HPLC equipped with an HPX-87H column  at 35°C. The eluting solution corresponded to a 0.005 M H2SO4 water solution at a flow rate of 0.4 mL.min-1.Reactors were sampled at the beginning (after the inoculation phase) and the end of each experiment. Samples were homogenized with a magnetic mixer (10 and 14% TS series) or by hand-shaking (19 to 33% TS series). In order to extract the liquid fractions of the reactors a gravimetric dilution to 5% TS was performed. The liquid phase was then extracted after filtration with a microfiber filter GF/D . The pH was measured immediately after dilution. VFAs were measured with a gas chromatograph Varian 580  composed of an Elite-FFAP Crossbond Carbowax 15 m column connected to a flame ionization detector (FID) at 280°C. Nitrogen at 6 mL.minP value at 0.05. The notations of significance levels of P values were: * < 0.05; ** < 0.01; *** < 0.001; and not significant (ns), >0.05.The R software (version 2.15.1) coupled with Rcmdr package version 1.8-4 was used for statistical significance measurement, using analysis of variance (ANOVA) with a AD: Anaerobic digestion; ANOVA: Analysis of variance; CE-SSCP: Capillary electrophoresis single strand conformation polymorphism; D: Dry fermentation; FID: Flame ionization detector; HD: Highly dry fermentation; HPLC: High performance liquid chromatography; MES: 2-(N-morpholino)ethanesulfonic acid; PCA: Principal component analysis; TS: Total solids; UASB: Upflow anaerobic sludge blanket; VFA: Volatile fatty acid; VS: Volatile solids; W: Wet fermentation; μGC: micro-gas chromatograph; μTCD: micro-thermal conductivity detector.The authors declare that they have no competing interests.JCM designed and carried out the experiments, analyzed results, and wrote the manuscript. ET, RE, and CD assisted and validated the experimental design and reviewed the manuscript. NB, JPD, and JPS coordinated the project and reviewed the manuscript. All authors read and approved the final manuscript.Distribution of fermentative products for the six conditions tested. The 28% TS condition is a threshold value and is split into two groups. This table provides the original data of Figure Click here for filePrincipal component analysis (PCA) of bacterial communities was characterized using CE-SSCP profiles including discriminant peaks. Since the PCA shows an absence of correlation between profiles and TS content, butyric acid production can be attributed to a metabolic shift and not to the emergence of new population.Click here for file


In Volume 92, Issue 8, August 2014, page 565, the second sentence of the findings section of the abstract should read: “In 56% (62/111) of the samples, it exceeded the Bangladeshi threshold of 50 μg/l; the mean concentration being 54.5 μg/l (range: 0.1–193.1).”


Consecutive two-dimensional frameworks comprised of molecular or cluster building blocks in large area represent ideal candidates for membranes sieving molecules and nano-objects, but challenges still remain in methodology and practical preparation. Here we exploit a new strategy to build soft single-layer ionic organic–inorganic frameworks via electrostatic interaction without preferential binding direction in water. Upon consideration of steric effect and additional interaction, polyanionic clusters as connection nodes and cationic pseudorotaxanes acting as bridging monomers connect with each other to form a single-layer ionic self-assembled framework with 1.4 nm layer thickness. Such soft supramolecular polymer frameworks possess uniform and adjustable ortho-tetragonal nanoporous structure in pore size of 3.4–4.1 nm and exhibit greatly convenient solution processability. The stable membranes maintaining uniform porous structure demonstrate precisely size-selective separation of semiconductor quantum dots within 0.1 nm of accuracy and may hold promise for practical applications in selective transport, molecular separation and dialysis systems. Membranes composed of ionic frameworks offer applications in nano-size separation thanks to their highly regular pores. Here, the authors devise such a system composed of polyoxometalates and organic pseudorotaxanes, and demonstrate their ability to separate mixed sized CdTe quantum dots. Ultrathin porous membranes210111211121417181920212311VO404− (PWV4−) cluster nodes in water. Because negative charges of the POM are delocalized and moveable4142Polyoxometalates262728303132333435363738391H NMR and electrospray ionization mass spectra (ESI-MS) (1H NMR spectra in D2O (2O (The bolaform cationic molecules (Azo-Tr/TeEG·2Br) comprised of two cationic azobenzene (Azo) groups connecting with a tri-/tetra-ethylene glycol (Tr/TeEG) spacer were synthesized and their chemical structures were characterized by (ESI-MS) . The rea(ESI-MS) , and thea in D2O . Upon ad D2O  shown in 4− cluster in the final complex after separation from solution. Combined these results with elemental analysis , both intensities of positive and negative Cotton effects gradually decrease. As is known, the deviation of transition moment for guest molecule from normal axis of CD cavity leads to the weakness of induced chirality until to silence464− cluster and Azo-TrEG@CD2+ by simply monitoring induced Cotton signals of Azo groups. The intensity plots of Cotton effects at 360 and 440 nm versus the molar ratio of PWV4− to Azo-TrEG@CD2+ illustrate a definite 1:2 stoichiometry at the turning point when the charges of PWV4− are fully neutralized by Azo-TrEG@CD2+  measurements further demonstrate the micrometre-scale sheets of singly layered ionic supramolecular assembly . The wriTo demonstrate the role of α-CD in synergistic ionic self-assembly for 2D framework structure, we repeat the preparation of single layered self-assembly by using β-CD to replace α-CD. As predicted, we do not observe any expected layer assemblies, but instead, nubbly and distorted bulk aggregations are found . This di−2, 2 cm in diameter) was prepared from filtration of [Azo-TrEG@CD][PWV] solution  on a supporting polycarbonate filter under the vacuum pressure of −2,000 Pa, as shown in The uniform mesh-like structure and the flexibility of the single-layer frameworks in aqueous solution exhibit the processing feature and simplify the fabrication of nanoporous membrane to a facile suction filtration under a slightly reduced pressure. Typically, the Tr-membrane  and the mixture of α-, β- and γ-CDs, which are positive, negative and nonionic at different pH conditions while maintaining the size less than 2 nm, were chosen as filtered chemical objects. The rhodamine B and xylenol orange filtrates were detected by UV–vis spectra , and CD D=3.3 nm in diameter with green emission (λmax=533 nm) and the bigger one (TG-2) has the size D=4.4 nm in diameter with red emission (λmax=611 nm) after considering the thickness of modified surface layer. The mixture solution of TG-1 and TG-2 was filtered through Tr-membrane and the filtrate was monitored by emission spectrum. Interestingly, the luminescent photographs (λmax=523 nm) of the emission band of the filtrate, the full-width at half-maximum (FWHM) narrows from 41.4 to 39.1 nm. Apparently, this spectral change corresponds to particle size having decreased to ∼3.0 nm, while the larger size part of TG-1 and whole TG-2 are blocked by the supramolecular polymer membrane, as supported by red luminescence on filtered membrane . With this fast and facile filtration, we provide an alternative analysis method for size distribution of QDs in addition to high-resolution TEM.The pore of prepared single-layer framework is adjustable and can be modulated by simply increasing the length of flexible spacer: TeEG. The obtained [Azo-TeEG@CD][PWV] displays the whole characteristics of [Azo-TrEG@CD][PWV] on framework structure and filtration property except the increased pore size (2.4−4.1 nm) after considering the shrinkage and stretching of TeEG chain . The TG-Generally, the aforementioned separation is independent of the surface species stabilizing QDs because when 3-mercaptopropionic acid-modified QDs in size of 4.0 (MPA-1) and 4.8 nm (MPA-2) are used in the filtration, we also obtain identical separation effect . By evalThe separation efficiency of QDs is carried out by evaluating relative fluorescence intensity before and after the filtration. As shown in −2, ∼0.43 μm) spreading on a support bearing average pore of 220 nm in diameter is 1.49∼1.66 MPa with the test area of 3.8 × 10−6 cm2  for TG-1 and TG-2 mixture solution were consecutively carried out by using the same Tr-membrane. After the continuous separation, a slight decrease of separation efficiency is found but there is no significant change by examining the emission band and FWHM of the filtrate , implyin10−6 cm2 , reveali10−6 cm2 , a new T10−6 cm2 , demonst4− as capper and node in aqueous solution, the obtained IOIF supramolecular polymer effectively overcomes the interlayer interaction and structure stiffness. The prepared single-layer framework not only integrates inorganic clusters and rotaxane-type units into branched polymeric architectures but also possesses highly ordered nanoporous mesh and good solution processability. This type of soft framework structure constructed by ionic bonding without preferential direction and its distinctive properties such as stability and plasticity offer an unprecedented opportunity to fabricate ultrafiltration membrane towards precisely screening nanoparticles. Because of the facile methodology and uniform nanoporous structure, the membranes prepared with 2D IOIF assemblies hold steady promise for practical applications in selective transport, molecular separation and dialysis systems. Meanwhile, the synergetic ionic self-assembly strategy can be hopefully extended to diverse IOIFs for advanced supramolecular materials.Overall, we have created a facile and convenient strategy for the fabrication of free-standing single-layer 2D IOIFs. In contrast to the known driving forces, electrostatic interaction was employed as the main binding force for the supramolecular hybrid frameworks. Through the synergetic ionic self-assembly of cationic pseudorotaxane unit as bridging stick and anionic PWV2O5 and distilled prior to use. N,N-dimethylformamide (DMF) was dried with CaH2 for several days and distilled before using. Doubly distilled water (Milli-Pore 18.2 MΩ cm−1) was used in the experiment. The polycarbonate membranes with 0.2-μm pore size are the product of Whatman Filters . The polyanionic cluster PWV4− was prepared following the published procedureThe general chemicals, α-, β-, γ-CD , rhodamine B and xylenol orange, are the products of TCI Chemicals (China) Pvt. Ltd. Other chemicals and solvents were purchased from Beijing Chemical Reagent Industry and used as received. Acetonitrile was dried over P1H NMR spectra were recorded on a Bruker AVANCE 500 MHz spectrometer while the chemical shifts were corrected by the solvent value (δ=4.79 p.p.m. for D2O and δ=2.50 p.p.m. for DMSO-d6). UV–vis spectra were carried out on a spectrometer (Varian CARY 50 Probe). The fluorescent spectra were carried out using the spectrophotometer (Shimadzu RF-5301PC). Circular dichroism spectroscopy were performed on a Bio-Logic MOS-450 spectropolarimeter in water with step size of 1 nm and speed of 5 nm s−1 at 25 °C. AFM images were taken with a SPA-300HV  under ambient conditions. AFM was operated in the tapping mode with an optical readout using Si cantilevers. Scanning electron microscope images were acquired on a JEOL FESEM 6700F field emission scanning electron microscope . TEM and HRTEM were conducted on a JEOL JEM 2010 electron microscope under an accelerating voltage of 200 kV without staining and energy-dispersive X-ray spectrum was collected on a FEI Tecnai F20 microscope operating at an accelerating voltage of 200 kV. XRD data were recorded on a Rigaku SmartLab X-ray diffractometer using Cu Kα1 radiation at wavelength of 1.542 Å. MALDI-TOF mass spectra was recorded on an autoflex MALDI-TOF/TOF  mass spectrometer equipped with a nitrogen laser . The ESI-MS/MS spectra were carried out by POEMS inductively coupled plasma mass spectrometer . X-ray photoelectron spectra was carried out on an ESCALAB 250 spectrometer with a monochromic X-ray source  and the charging shift was corrected by the binding energy of C(1 s) at 284.6 eV. Inductively coupled plasma atomic emission spectrometry (ICP-AES) was carried out on the PerkinElmer Optima 3300DV . Organic element analysis was carried out on the vario MACRO cube CHNS .a, sulfonated compound of glycol b, coupling product c was prepared according to the literatures60c bearing TrEG or TeEG chain  was added to a 10 ml round bottom flask containing 5 ml of DMF and 2 ml of pyridine. The reaction mixture was heated under stirring in an oil bath at 100 °C for 48 h. Then the reaction solution was cooled to room temperature and transferred dropwise to 500 ml of ethyl acetate. The yielded precipitate was collected by filtration and washed with ethyl acetate  for three times to give the product . 1H NMR  for Azo-TrEG·2Br: δ 8.87  p.p.m. , 8.57 , 8.07 , 7.62 , 7.57 , 7.51 , 6.92 , 5.75 , 4.10 , 3.86  and 3.77 . 13C NMR : δ 161.7, 152.8, 146.3, 146.2, 145.0, 136.4, 130.1, 128.7, 124.9, 123.0, 115.3, 70.1, 68.9, 67.8 and 62.9. ESI-MS (m/z) [M]2+: calculated for C42H42N6O4: 347.4; found: 347.4. 1H NMR  for Azo-TeEG·2Br: δ 9.26  p.p.m. , 8.67 , 8.22 , 7.98−7.80 , 7.72 , 7.17 , 5.97 , 4.21 , 3.79  and 3.65−3.50 . 13C NMR  δ 162.1, 152.8, 146.63, 145.57, 145.4, 136.8, 130.4, 129.0, 125.3, 123.3, 115.6, 70.4, 70.3, 69.3, 68.2 and 63.3. ESI-MS (m/z) [M]2+: calculated for C44H46N6O5: 369.2; found: 369.6.The bolaform cationic organic compounds were synthesized according to the route in −1) was constructed by simply mixing the as-prepared Azo-TrEG@CD·2Br and PWV4− solution that is prepared by dissolving PWV4−  in 60 ml water according to certain stoichiometry.In a typical procedure, Azo-TrEG·2Br  and α-CD  were mixed and dissolved in 60 ml water under sonication for 0.5 h to generate pseudorotaxane unit Azo-TrEG@CD·2Br. The final framework assembly of [Azo-TrEG@CD][PWV] (0.17 mg ml−1) was filtered over a supporting filter  under the preset vacuum pressure. Water (10 ml) was subsequently used to wash the membrane. Under the same vacuum pressure, the IOIF membranes with various thicknesses were prepared by using 20 ml sample solution with concentrations: 0.02, 0.04, 0.06 and 0.08 mg ml−1, and corresponding thicknesses are measured to be 0.20∼0.35, 0.43, 1.43 and 2.19 μm, respectively.The IOIF membranes were prepared by a simple filtration of sample solutions containing single-layer IOIF self-assemblies through a commercial filter with even dispersed pores in certain size under vacuum pressure of −2,000 Pa. After washing with water, the prepared nanofiltration membrane has the same size as the effective filtration area of supporting substrate used in the installation. In a typical procedure, 20 ml as-prepared [Azo-TrEG@CD][PWV] solution  like a soap film and cast it on a copper grid. The procedure was repeated three times for one sample to ensure enough amounts of samples being attached on the copper grid. During the measurement, longer time exposure to the high-energy electron beam will destroy the framework structure, resulting in less ordered structure. The HRTEM images were tracked within a quite short time. In order to obtain a clear high contrast image, we used a smart camera technique for image collection, which was based on a continuous acquisition of images to get high image contrast. Because of the heating disturbance of the sample during the electron beam irradiation, the image superposition brought slight ghosting phenomenon, which causes the size of inorganic clusters seemed larger than their ideal dimension.We used a thin copper ring to spread a thin layer of [Azo-Tr/TeEG@CD][PWV] solution  and then grinding it into powder. The film sample was prepared by the filtration of [Azo-TrEG@CD][PWV] solution  over a supporting filter , drying in oven at 40 °C for 48 h.The powdered sample was prepared by the lyophilization of the [Azo-TrEG@CD][PWV] solution (0.17 mg mlDc) of CdTe QDs was calculated according to the following published equationDc=(9.8127 × 10−7)λ3−(1.7147 × 10−3)λ2+(1.0064)λ−194.84, where λ (nm) is the maximum wavelength corresponding to the first excitonic absorption peak of QDs. The full diameter of surface stabilized CdTe QDs (average diameter D) was calculated by summing the molecular length of ligands (l) and the calculated Dc value, where the molecular length of ligand stabilizer is estimated ∼0.46 nm for TG and ∼0.65 nm for MPA, simulated by ChemBio 3D (12.0 version). Thus, the diameters (D) of four QDs were calculated to be 3.3 nm for TG-1, 4.4 nm for TG-2, 4.0 nm for MPA-1 and 4.8 nm for MPA-2.The core diameter .Supplementary Figures 1-52, Supplementary Tables 1-5 and Supplementary References


To assess EQ-5D-5L (5L) validity in patients with acute stroke, in comparison with EQ-5D-3L (3L).Cross-sectional study of 408 patients during index hospitalization. We compared 5L and 3L in terms of feasibility, frequency of unique health states, ceiling effect and discriminatory power (informativity). We assessed construct validity in terms of known-groups validity and convergent validity of 5L dimensions with other stroke outcome measures.The overall proportion of patients with acute stroke reporting ‘no problems’ with 3L—6.1 % was further reduced to 5.6 % with 5L (relative reduction of 8.2 %). The highest improvement in relative discriminatory power, when moving from 3L to 5L, was noticed in pain/discomfort and anxiety/depression dimensions . Known-groups validity tests confirmed prior hypotheses: Health state utilities were lower in following subpopulations—females, patients with high modified Rankin Scale (mRS) score, low Barthel Index (BI) or VAS score, patients with subarachnoid hemorrhage or intracerebral hemorrhage, and when proxy respondent was used. Convergence of EQ-5D-5L dimensions with mRS, BI and EQ VAS was improved or at least the same as for 3L dimensions.Results support the validity of the EQ-5D-5L descriptive system as a generic health outcome measure in patients with acute stroke, demonstrating some psychometric advantages in comparison with EQ-5D-3L. Three level EQ-5D is, probably, the most widely used generic health status questionnaire in patients experiencing stroke , 2. An eRecently, the EuroQol group has introduced new, five level, version of the EQ-5D (EQ-5D-5L) . JanssenThe aim of our study was to assess the validity of the EQ-5D-5L, in comparison with EQ-5D-3L, in acute stroke patients.Adult patients with cerebral infarction, intracranial or subarachnoid hemorrhage  were included into a single center cross-sectional study. A diagnosis had to be confirmed by clinical and neuroimaging examinations. Patients had to be Polish language native speakers. Patients in coma were excluded. In case of aphasia or dementia, the survey was completed by a family member serving as a proxy respondent.The survey took place during index hospitalization (median 8 days since admission). The degree of disability due to stroke was assessed with the modified Rankin Scale (mRS) , physicaWe compared 5L and 3L in terms of feasibility (proportion of missing answers), frequency of unique health states, ceiling effect (proportion of ‘no problem’ answers) and discriminatory power (informativity) , 9. We aThe proportion and level of logical inconsistencies in pairs of 5L and 3L answers was analyzed as described by Janssen et al. . InconsiH′), which represents the absolute amount of captured informativity and the Shannon Evenness Index (J′), which reflects the rectangularity of a distribution regardless of the number of levels, as described elsewhere [H′ approaches 1.58 (3L) or 2.32 (5L) and J′ approaches 1.0, indicating maximum informativity captured by the instrument.To assess discriminatory power, we calculated the Shannon Index , stroke type according to ICD-10, stroke outcome according to mRS, BI and EQ VAS . We hypors < 0.20), poor (rs = 0.20–0.34), moderate (rs = 0.35–0.50) or strong (rs > 0.50) [Convergent validity was assessed by examining the strength of association between 5L and 3L dimensions with mRS score, BI score and EQ VAS and by comparing 5L and 3L dimensions between themselves using Spearman’s rank correlation coefficient . Strengt > 0.50) . We hypoThe study data were analyzed using StatsDirect ver. 2.8.0 statistical software.From July 2009 to May 2010, 408 patients , mean age 69.0 years were enrolled  and nearly perfect informativity of 5L UA  and SC dimensions , in patients with stroke. Nevertheless, the highest improvement in informativity, when moving from 3L to 5L, was noticed in PD and AD dimensions . The total number of unique health states was 213 for 5L  and 62 for 3L .A total of 2.9 % 5L and 3.7 % 3L questionnaires had at least one missing answer, indicating good feasibility of both instruments in patients with stroke. For 5L, missing values ranged from 0.25 % in MO to 1.5 % in UA. The overall proportion of inconsistent 5L responses (in comparison with 3L responses) was 3.5 %, ranging from 2.2 % for MO to 5.0 % for UA and with 86 % of inconsistencies being level 1, as defined by Janssen et al. . The proResults for known-groups construct validity are shown in Table Moderate to strong correlations were found between 5L and mRS, BI and EQ VAS, with a minimum of −0.37 between PD and BI, and a maximum of 0.79 between UA and mRS .Furthermore, our results support convergence validity of EQ-5D-5L with other stroke measures. As expected, we found moderate to strong correlations between 5L dimensions and mRS, BI or VAS. All coefficients were slightly higher or at least the same for 5L, in comparison with 3L.Our results are in general in line with these obtained by other authors in different populations , 9, 11. Our study has some limitations. Quality of life questionnaires were administered in the fixed order. This could affect the proportion of missing answers, which was lower for 5L (administered first). The desirable solution would be to present instruments in a random order. Second, there were no other questionnaires presented between 5L and 3L. The risk is that memory effects may affect the comparison of these two versions.Although, there is some evidence supporting the use of proxy respondents for 3L , 23, to To conclude, evidence supports the EQ-5D-5L descriptive system as a valid generic health outcome measure in patients experiencing acute stroke, with some psychometric advantages in comparison with the EQ-5D-3L.


A pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) is a signature of favorable prognosis in breast cancer. The aim of this study was to assess the accuracy of magnetic resonance imaging (MRI) in predicting the pCR after NAC. 265 women with stage II or III breast cancer who underwent surgery after NAC were retrospectively investigated for MRI findings before and after the NAC. Correlation of pCR with an “imaging complete response” (iCR), defined as no detectable tumor on all serial images with dynamic contrast-enhanced T1-weighted imaging, was evaluated with respect to each tumor subtype. Of 265 cases, 44 (16.6 %) and 24 (9.1 %) were diagnosed as iCR and pCR, respectively. Nineteen of the 44 iCR cases (43.2 %) were assessed as pCR, and 216 (97.7 %) of the 221 non-iCR cases were assessed as non-pCR. The accuracy (ACC), the pCR predictive value (PPV) and the non-pCR predictive value (NPV) were 88.7, 43.2, and 97.7 %, respectively. When assessed according to each tumor subtype, the ACC, PPV and NPV were 93.2, 21.4 and 100 % for luminal subtype, 70.8, 0 and 89.5 % for luminal/HER2 subtype, 75, 57.1 and 88.8 % for HER2-enriched subtype, and 90.9, 72.7 and 97 % for triple-negative subtype, respectively. MRI is a valuable modality for predicting pCR of breast cancer after NAC treatment. However, its accuracy varies greatly in different breast cancer subtypes. Whereas MRI closely predicts pCR in the triple-negative subtype, iCR in the luminal subtype is often an over-estimation. On the other hand, residual lesions identified by MRI are reliable markers of non-pCR for the luminal subtype. Neoadjuvant chemotherapy (NAC) is one of the recommended treatments for operable invasive breast cancer. Published clinical trials have shown that breast cancer patients with pathological complete responses (pCR) to NAC have significantly better prognosis than those without pCR  are widely used for the assessment of therapeutic effect.MRI is a useful device to assess the size and extent of lesions in breast cancer. The size of the invasive component of the tumor estimated by MRI closely correlates with that determined pathologically. MRI is also capable of detecting small residual cancer nests after NAC  evaluated by MRI.2, adriamycin 50 mg/m2, fluorouracil 500 mg/m2, q3w), AC , and CEF  therapy. The taxane regimens were 12 cycles of weekly paclitaxel at a dose of 80 mg/m2 or 4 cycles of tri-weekly docetaxel at a dose of 75 mg/m2. In Japan, postoperative administration of trastuzumab was approved for health insurance coverage in 2008. In the present study, none of the patients were administered trastuzumab preoperatively.Standard anticancer drugs including anthracycline and/or taxane were administered to all patients as NAC. Sixty five patients received an anthracycline-based combination regimen, 22 received only taxane and the remaining 178 were treated with the anthracycline-based combination regimen followed by taxane. The former involved 4–6 cycles of CAF  at a rate of 3 mL/s, followed by a 20 mL saline flush at the rate of 3 mL/s.MRI examinations were performed using a 1.5T MRI unit  and a commercially-available dedicated four-channel breast array coil. Patients underwent MRI in the prone position. Our imaging protocol included a localizing sequence followed by unilateral fast spin-echo T2-weighted coronal imaging  with fat suppression by chemical shift-selective imaging sequences. Other parameters were as follows: field of view, 260 mm; section thickness, 3 mm and interslice gap, 0 mm. This examination was followed by combined dynamic contrast-enhanced unilateral coronal breast imaging. An enhanced T1-weighted examination 3D gradient echo sequence with fat suppression by spectral inversion recovery was performed before and after contrast material injection. The image parameters were as follows: TR/TE/FA, 3.6 ms/1.0 ms/15°; FOV, 26 × 26 cm; matrix, 320 × 240; section thickness, 3.0 mm; interslice gap, 0 mm and acquisition time, 60 s. A dynamic study in the coronal plane was performed before and 60, 120, 180 and 240 s after starting intravenous injection of 0.2 mmol/kg of gadodiamide hydrate  Guidelines.Detailed evaluation of surgical specimens was carried out. Partial mastectomy specimens were cut into 5 mm thick serial sections and formalin-fixed paraffin blocks were made from the all sections. The all blocks cut into thin slices and were examined microscopically. In total mastectomy specimens, the location of the tumor bed was firstly identified from the clinical information and gross observation of the cut surfaces. Next, many tissue blocks broadly distributed around the tumor bed were sampled and microscopically examined.pCR was defined as no invasive cancer cells in any slices of the resected breast specimen. pCR was allowed for in situ carcinoma and for positive lymph nodes.Tumors were classified into four subtypes based on the two HRs, the estrogen receptor (ER) and the progesterone receptor (PgR), and HER2. The status of these biomarkers was estimated using biopsy samples obtained before chemotherapy. Immunohistochemical staining for ER and PgR was performed using antibody clone 1D5 for ER and for clone PgR636 for PgR . Positive reactions for ER and PgR were defined as nuclear staining in ≥10 % of cancer cells, and negative reactions were defined as staining in <10 %. HR-positivity was defined as positivity for ER and/or PgR. Immunohistochemical staining for HER2 was performed using the Hercep Test (Dako Japan Inc.). Expression of HER2 protein was classified into 4 strata, i.e., 0, 1+, 2+ and 3+. In the 2+ cases, HER2 genetic testing by fluorescence in situ hybridization (FISH) was performed using the PathVysion HER2-DNA Probe Kit . Both protein and genetic status were estimated based on the guidelines for HER2 testing in breast cancer, as recommended by the American Society of Clinical Oncology/College of American Pathologists . The Fisher’s exact test was used to compare iCR and pCR. P < 0.05 was taken to indicate statistical significance in all instances.Informed consent for the use of specimens was obtained preoperatively from all patients participating in this study, which was approved by the Institutional Review Board of the authors’ institution and was disclosed information.All patients studied were female with a mean age at the beginning of treatment of 49.9 years (range 25–78). Tumors were at Stage II (172 cases) or Stage III (93 cases) with a tumor size >2 cm  or/and positive for lymph node metastasis . The latter included seventeen cases of T1 with positive lymph node metastasis (6.4 %). Total mastectomy was performed on 165 patients (62.3 %) with the remaining 100 undergoing partial mastectomy (37.7 %). ER, PgR and HER2 were positive in 70.6, 46.4 and 21.1 % of cases, respectively. Data on at least one of the biomarkers were missing for 4 patients’ biopsies from other hospitals. Regarding subtype criteria combining HR and HER2, 161 cases were classified as luminal subtype (60.8 %), 24 were luminal/HER2 (9.1 %), 32 were HER2-enriched (12.1 %) and the remaining 44 were triple-negative (16.6 %).Of the total of 265 patients, 44 were diagnosed as iCR by MRI 16.6 %) and 24 as pCR (9.1 %). Of the 44 iCR patients, 19 were also diagnosed as pCR (43.2 %) and the remaining 25 as non-pCR (56.8 %). Of the 221 patients who were non-iCR, 216 were also diagnosed as non-pCR (97.7 %) but the remaining 5 were diagnosed as pCR (2.3 %). Thus, iCR was significantly correlated with pCR in the whole cohort , pCR predictive value (PPV) and non-pCR predictive value (NPV) were estimated as 79.2, 89.6, 88.7, 43.2 and 97.7 %, respectively (Table Proportions of patients with iCR in each subtype were as follows: 8.7 % 14/161) luminal, 20.8 % (5/24) luminal/HER2, 43.8 % (14/32) HER2-enriched and 25.0 % (11/44) triple-negative. In contrast, proportions of patients with pCR in each subtype were 1.9 % (3/161) for luminal, 8.3 % (2/24) luminal/HER2, 31.3 % (10/32) HER2-enriched and 20.5 % (9/44) triple-negative. Statistically significant correlations between iCR and pCR were present for luminal (P = 0.001), HER2-enriched (P = 0.008) and triple-negative (P < 0.001) subtypes, but not for the luminal/HER2 subtype (P = 1.000) . When assessed for subtype distribution, proportions of non-pCR in iCR cases were 78.6 % 11/14) for the luminal subtype, 100 % (5/5) for the luminal/HER2 subtype, 42.6 % (6/14) for the HER2-enriched subtype and 27.3 % (3/11) for the triple-negative subtype (Table 1/14 for Reciprocally, we found that MRI discordantly diagnosed five cases of pCR as non-iCR. Fibrous granulation tissue that may have caused the misdiagnosis was observed in all five cases. Figure In the present study, we investigated the accuracy of MRI for predicting pCR of breast cancer after NAC, especially focusing on its value in respect to each tumor subtype. When all cases were considered together, the iCR results were found to correlate significantly with pCR. These results are consistent with previously reported data (Straver et al. In the present study, cases with results discordant between iCR and pCR were carefully analyzed for pathological findings. MRI diagnosed 25 cases of non-pCR as iCR. There likely to be at least two different reasons for this misdiagnosis. For HER2-enriched and triple-negative subtypes, the small volume of the residual cancer cells, often present with a scattered distribution pattern, could be responsible for their evading detection by MRI. In the cases of luminal subtype, MRI may fail to detect tumors with low nuclear grade despite a relatively large tumor size. The proportion of non-pCR in the iCR group was disturbingly high in this subtype, despite the majority of the cases having relatively large tumors and showing slight therapeutic responses. These cases all had low nuclear grade tumors. Reciprocally, MRI diagnosed five cases of pCR as non-iCR. These five shared the common findings of fibrosis and granulation occupying the tumor bed of the surgical specimen. Because the fibrous granulation tissue contained numerous small vessels and inflammatory cells, it is most likely that these NAC-responsive structures mimicked the remnant tumor in the MRI findings (Woodhams et al. In conclusion, subgroup analysis with respect to each tumor subtype enhances the accuracy of MRI for predicting pCR after NAC treatment of breast cancer. It is an especially valuable technique for the prediction of pCR in triple-negative breast cancer. Now, we are planning a study which confirms this result with lager patients.


The objective of this study is to evaluate the effect of oral sodium bicarbonate on circulating FGF23 levels in patients with CKD.The regulation of fibroblast growth factor-23 (FGF23) secretion in patients with chronic kidney disease (CKD) is incompletely understood. An 2 and serum bicarbonate between 20–24 mEq/L were treated with placebo for 2 weeks, followed by increasing doses of oral sodium bicarbonate  in 2 week intervals for a total of 6 weeks. C-terminal FGF23 levels were measured at the initial visit, after 2 weeks of placebo and after 6 weeks of bicarbonate therapy. Wilcoxon matched-pairs signed-rank test was used to compare FGF23 before and after sodium bicarbonate.This was a single-blind pilot study. Twenty adults with estimated glomerular filtration rate between 15–45 mL/min/1.73 mp = 0.048) and this persisted after excluding participants who received activated vitamin D.After 6 weeks of oral sodium bicarbonate, the median FGF23 increased significantly from 150.9 RU/mL (IQR 107.7–267.43) to 191.4 RU/mL (IQR 132.6–316.9)  on April 23, 2009.The study was registered at ClinicalTrials.gov ( Fibroblast growth factor 23 (FGF23) is a hormone produced in osteocytes and osteoblasts –3. Its pin vitro study demonstrated that metabolic acidosis increased FGF23 concentration and RNA expression in mouse bone  90.2-289.4). Nine participants were receiving vitamin D at baseline . Among them, one participant (participant #3) had paricalcitol dose increased during the study, and one participant (participant #16) had very high FGF23 at baseline (824.9 RU/mL). None of the participants received phosphate binders during the study period. Fifteen participants were receiving diuretics. Two of them had diuretic dose increased prior to their last visit while others remained on the same dose throughout the study.Enrollment is shown in Fig. p < 0.001). No statistically significant changes were observed in 25-OH vitamin D, 1,25-OH vitamin D, serum calcium, serum phosphate, urine calcium, urine phosphate, urine sodium, fractional excretion of phosphate or sodium although the p-value was marginally significant for urine sodium (p = 0.07)   or after excluding participant #16, who had a very high FGF23 at baseline (p = 0.01). The median FGF23 increased after excluding all 9 participants on vitamin D or after excluding participant #3, who had an increased paricalcitol dose during the study, but it was not statistically significant (p = 0.08 for both) (Table FGF23 increased in 14 (70 %) participants. The median FGF23 increased significantly from 150.9 RU/mL (IQR 107.7–267.43) to 191.4 RU/mL (IQR 132.6–316.9) (p = 0.21). There was no significant association of FGF23 with 25-OH vitamin D, serum calcium, urine calcium or urine sodium. FGF23 was inversely associated with 1,25-OH vitamin D (p = 0.005), and positively associated with serum and urine phosphate  (Table There was no significant association between bicarbonate and FGF23 (log-transformed)  Table .Table 4BThis is the first human study evaluating the effect of oral sodium bicarbonate on the regulation of circulating FGF23. We found that oral sodium bicarbonate did not decrease FGF23 in patients with CKD and mild acidosis. Contrary to our hypothesis, there was a statistically significant increase in C-terminal FGF23 levels after 6 weeks of oral sodium bicarbonate  study, the median C-terminal FGF23 was 145 RU/mL (IQR 96–239) in patients with a mean eGFR of 42.8 ± 13.5 ml/min/1.73 m2 [The FGF23 levels were similar to the levels reported by others. The median C-terminal FGF23 was 157.4 RU/mL IQR 90.2–289.4) with a mean eGFR of 32.9 ± 8.9 ml/min/1.73 m0.2–289.4/1.73 m2 . In a co/1.73 m2 . Isakovain vitro data observed by Krieger et al. [in vitro data may differ from in vivo due to other unmeasured factors. In an in vivo study, Leibrock et al. [There are several explanations for why our findings differ from the r et al. , who fouk et al.  examinedk et al. , 20. FGFk et al. . In clink et al. , 22 examk et al. , 24, andk et al. . If alkaSecond, the increased FGF23 might not be induced by the alkalinizing effect of sodium bicarbonate as we did not find a statistically significant association between bicarbonate and FGF23 Table . The assp = 0.07) and the result was similar after excluding the 2 participants who had their diuretic dose increased  guidelines, which suggest treatment of acidosis based on serum bicarbonate alone .in vitro work [Our pilot study showed that FGF23 levels increased after short-term oral sodium bicarbonate in patients with CKD and mild metabolic acidosis. This might not be due to the alkalinizing effect of sodium bicarbonate, but at the very least, we observed no reduction in FGF23 that would be predicted based on prior tro work . The NKFtro work –41, and 1,25-OH vitamin D, 1,25-dihydroxyvitamin D; CKD, chronic kidney disease; CRIC, Chronic Renal Insufficiency Cohort; eGFR, estimated glomerular filtration rate; FGF23, fibroblast growth factor 23; IQR, interquartile range; PTH, parathyroid hormone


With the exception of 3-OHBaP at a low spiking level, the average recoveries were greater than 80%. The method has good accuracy (72.1–107.7%) and reproducibility (1.8–11.4%) and was successfully used to study the uptake of pyrene, benzo[a]pyrene, and benzo[a]anthracene from cigarette smoke. The results indicated that urinary 1-OHP concentration in the smoking group (66.58 ± 70.91 ng/g creatinine) was higher than that observed in the nonsmoking group (58.16 ± 49.48 ng/g creatinine). Urinary 3-OHBaA concentrations in nonsmokers and smokers with 8 mg and 10 mg tar cigarettes were 10.98 ± 4.39 ng/g creatinine, 11.01 ± 13.30 ng/g creatinine, and 9.17 ± 12.89 ng/g creatinine, respectively. Urinary 3-OHBaP concentrations in nonsmokers and smokers with 8 mg and 13 mg tar cigarettes were 1.30 ± 0.20 ng/g creatinine, 2.83 ± 1.78 ng/g creatinine, and 6.00 ± 4.44 ng/g creatinine, respectively. Urinary 1-OHP levels exhibited a significant correlation with BaP yield in cigarette smoke under the Canadian intense smoking condition ( Polycyclic aromatic hydrocarbons (PAHs) mainly from incomplete combustion of organic matter are an important class of environmental contaminants, which is found in food –3, feed High performance liquid chromatography (HPLC) with fluorescence detection has been widely used in the determination of urinary OH-PAHs, particularly 1-OHP –17. ThisGas chromatograph (GC) and gas chromatography-mass spectrometer (GC-MS) methods have also been used to detect OHPAHs –23. ReceOwing to the fact that LC tandem mass spectrometry (LC-MS/MS) can be used to quantify a wide range of substances in complex biological matrixes at low levels with high specificity and short chromatographic run times, LC time-of-flight MS with electrospray ionization (ESI)  and LC-MIn this study, we describe a LC-MS/MS method for simultaneous determination of urinary 1-OHP, 3-OHBaP, and 3-OHBaA. The proposed method has good precision and accuracy, very high sensitivity, an extraction procedure suitable for processing large batches of samples, and a relatively short chromatographic run time (12 min). This method was successfully applied to determine 1-OHP, 3-OHBaP, and 3-OHBaA levels in urine samples obtained from 81 smokers and 58 nonsmokers.9-1-OHP internal standard  was purchased from Toronto Research Chemicals Inc. . β-Glucuronidase-arylsulfatase  was purchased from Merck . HPLC-grade acetonitrile was obtained from TEDIA Company Inc. .1-OHP was obtained from Dr. Ehrenstorfer GmbH . 3-OHBaP was purchased from the Institute for Reference Materials and Measurements . 3-OHBaA was purchased from MRIGlobal  and the Dμg/mL of 1-OHP and 3-OHBaP was dissolved/diluted in acetonitrile to obtain separate stock solutions of each compound. 3-OHBaA was diluted in the same way to a concentration of approximately 1 μg/mL. The D9-1-OHP  solution was diluted in acetonitrile to concentrations of 50 μg/mL as a stock solution and 100 ng/mL as a working solution. Aliquots of the three standard stock solutions were mixed, and 100 μL of internal standard solution (100 ng/mL) was added. This solution was further diluted with acetonitrile to obtain a series of standard solutions ranging within 0.50–25.00 ng/mL for 1-OHP, 0.10–5.00 ng/mL for 3-OHBaP, and 0.25–12.50 ng/mL for 3-OHBaA. All stock and standard solutions were stored at −80°C.Approximately 2 C, where C is the concentration of each calibration standard. Correlation coefficients  were required to be greater than or equal to 0.99. The limits of quantification (LOQs) were calculated using the background noise level estimated from the peak-to-peak baseline near the analyte peaks. The precision and accuracy of the method were assessed by intra- and interday validation experiments. The intra- and interday accuracy and precision were obtained by determining the concentrations of 1-OHP, 3-OHBaP, and 3-OHBaA in five replicates of pooled blank urine samples for three separate batches. Precision was expressed as the relative standard deviation (RSD). Accuracy was expressed in terms of bias as the percent deviation of the mean determined concentration against the spiked concentration. The recoveries of 1-OHP, 3-OHBaP, and 3-OHBaA were determined by comparing the analyte responses obtained from pooled blank urine samples with those obtained from prepared urine samples that were spiked with equivalent analyte concentrations. Freeze-thaw stability was assessed by exposing samples with moderate concentrations to three freeze-thaw cycles. In each cycle, the samples were removed from the freezer, thawed (unassisted) to room temperature, kept at room temperature for 2 h, and refrozen at −80°C for 8 h.The method was validated for specificity, matrix effects, precision, accuracy, linearity, sensitivity, recovery, and stability according to US Food and Drug Administration (FDA) guidelines for the validation of bioanalytical methods . The speμm particle size, California, USA) and equilibrated with 40% solvent A (water) and 60% solvent B (acetonitrile) at 380 μL/min. The gradient conditions were 0–2.5 min, 40% A; 2.5–3.5 min, 10% A; 3.5–8 min, 10% A; 8–8.1 min, 90% A; 8.1–11 min, 90% A; 11–11.1 min, 40% A; 11.1–12 min, 40% A.Samples were analyzed by an Agilent 1200 rapid-resolution liquid chromatography instrument  on a Phenomenex Synergi Hydro-RP column  was used in APCI positive ionization mode. Mass detection conditions were as follows: ion spray voltage, 5500 V; ion source temperature, 400°C; nebulizer gas, nitrogen, 60 psi; curtain gas, nitrogen, 25 psi; collision gas (CID), nitrogen, setting 8. The MRM transitions of81 Chinese smokers and 58 Chinese nonsmokers from the city of Zhengzhou were enrolled in the study. For each subject, data regarding smoking habits, age, and job description were collected using a questionnaire administered by trained interviewers. The clinical portion of this study was conducted by the Institute of Clinical Pharmacology of Zhengzhou University in 2010. The study was approved by the Institutional Review Board for Zhengzhou University, China. All participants in the study gave written consent. The ages and occupations of the nonsmokers matched those of the smokers . The stuμL of internal standard solution (100 ng/mL) were added. After the addition of 30 μL β-glucuronidase/arylsulfatase, the conical flask was covered with aluminum foil and placed in a shaker bath overnight at 37°C to completely hydrolyze the conjugated PAH metabolites.Urine (10 mL) from each subject was transferred to a conical flask (25 mL). The pH of the solution was adjusted to 5.0 with 0.2 M HCl, and 2.5 mL of 0.5 M acetate buffer (pH 5.0) and 100 μL of methanol. The solution was filtered through a 0.22 μm filter and then stored at −20°C until LC-MS/MS analysis.The optimization of the experimental parameters associated with the retention and elution of PAH metabolites (from the solid-phase cartridge) resulted in the following procedures. Aqueous metabolite solutions were processed through a Supelco-ENVI C-18 cartridge  that was previously conditioned with 5 mL methanol and 5 mL water. All solutions were injected onto the cartridge with the aid of a 10 mL syringe (attached to the cartridge). The cartridge was sequentially rinsed with 10 mL water and 10 mL of 30% methanol in water. After the cartridge was dried completely, the trapped metabolites were eluted with 4 mL of methanol. The elution was evaporated to dryness under nitrogen gas and redissolved in 150 μL urine samples were pipetted into separate polypropylene tubes, and 25 μL of the internal standard solution (10 μg/mL) and 725 μL of water were added. The mixed samples were vortex-mixed for 2 min. After centrifugation for 15 min, the supernatants were filtered through a 0.22 μm syringe filter. 5 μL of the filtrates was then introduced into the LC-MS/MS system.Urinary cotinine levels were analyzed according to a previously published LC-MS/MS method . BrieflyμL of formic acid was added to a 1 mL aliquot of human urine sample, stirred, and centrifuged at 10000 rpm for 10 min. The mixture was filtered through a 0.22 μm polyethersulfone membrane. 5 μL of urine aliquot was transferred to an amber volumetric flask and brought to a total volume of 10 mL with water after being spiked with 100 μL of creatinine-d3 internal standard solution (1 μg/mL). 5 μL aliquot was then injected on-column for LC-MS/MS analysis.Urinary creatinine levels were analyzed according to a previously published LC-MS/MS method . BrieflyBaP yields under different machine smoking regimes using the appointed brands of cigarettes with different tar yields  were analyzed according to the reported method . CigaretμL internal standard solution was added. The calibration standard solutions were also processed with SPE using the same procedure applied to the real urine samples. The concentrations of the calibration standard solutions (in urine) were 0.008, 0.015, 0.030, 0.076, 0.152, and 0.379 ng/mL for 1-OHP; 0.002, 0.003, 0.006, 0.015, 0.030, and 0.076 ng/mL for 3-OHBaP; and 0.004, 0.008, 0.015, 0.038, 0.076, and 0.19 ng/mL for 3-OHBaA. All urine samples were stored at −80°C until analysis. Each analyte exhibited good linearity with a coefficient of determination (r) exceeding 0.99. Data analysis resulted in the following representative regression equations: Y = 0.454X + 2.06 for 1-OHP, Y = 0.215X + 0.236 for 3-OHBaP, and Y = 1.23X − 0.013 for 3-OHBaA, where Y indicates the analyte/IS ratio and X indicates the urine concentration. The slopes of these regression equations were reproducible for calibration curves determined on three separate occasions (over 3 days). Under optimized conditions, the LOQs  were 0.003 ng/mL for 1-OHP, 0.002 ng/mL for 3-OHBaP, and 0.004 ng/mL for 3-OHBaA.Calibration standard solutions were prepared by diluting standard solutions to the appropriate concentrations with pooled blank human urine while a 100 n = 6) are also shown in To determine the effects of endogenous interferences, the specificity was assessed by comparing the chromatograms of analyte-free human urine samples with those of the spiked urine samples. Under the described chromatographic conditions, the retention times of 1-OHP, 3-OHBaP, 3-OHBaA, and IS were 3.44, 5.94, 4.31, and 3.29 min, respectively. Representative chromatograms from a nonsmoker and smoker are shown in Figures The proposed method was successfully used to analyze 58 urine samples from 58 nonsmokers and 243 urine samples from 81 smokers ; the results are shown in P < 0.01) and between smokers with 10 mg tar cigarettes and 13 mg tar cigarettes (P < 0.05). However, the differences in urinary 3-OHBaA and 3-OHBaP between different groups of smokers were not significant (P > 0.05). The correlation between 1-OHP in 24 h urine and the nicotine metabolite cotinine, which is a recognized biomarker for tobacco exposure, was also studied .Because spot urine sampling may not provide a valid overview of the entire toxicant exposure profile  and is e studied . The corPyrene, benzo[a]pyrene, and benz[a]anthracene are ubiquitously present in the environment and cigarette smoke and are associated with a variety of adverse health effects. In mainstream cigarette smoke, the levels of benzo[a]pyrene and benz[a]anthracene ranged from 8.5 to 11.6 ng/cig and 20 to 70 ng/cig , respectUrinary 1-OHP concentrations in nonsmokers (58.16 ng/g creatinine) and smokers (66.85 ng/g creatinine) in this study were lower than those reported in France by Lafontaine et al. (84.88 ng/g creatinine and 277.6 ng/g creatinine) , which my = −0.3322x + 157.18 ; under the Canadian intense machine smoking regime , BaP yield was strongly related to urinary 1-OHP . The method was successfully used to assess urinary 1-OHP, 3-OHBaP, and 3-OHBaA in nonsmokers and smokers with different tar yield cigarettes. After creatinine correction, urinary 1-OHP and 3-OHBaP increased with tar and BaP contents in cigarette smoke. Urinary 1-OHP levels were significantly correlated with BaP yield in cigarette smoke under the Canadian intense smoking condition.