Mycobacterium rufum JS14T , a type strain of the species Mycobacterium rufum sp. . belonging to the family Mycobacteriaceae, was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil in Hilo  because it harbors the capability of degrading PAH. Here, we describe the first genome sequence of strain JS14T, with brief phenotypic characteristics. The genome is composed of 6,176,413 bp with 69.25 % G + C content and contains 5810 protein-coding genes with 54 RNA genes. The genome information on M. rufum JS14T will provide a better understanding of the complexity of bacterial catabolic pathways for degradation of specific chemicals. Polycyclic aromatic hydrocarbons, defined as organic molecules consisting of two or more fused aromatic rings in linear, angular, or cluster arrangement, mostly result from coke production, petroleum refining, fossil fuel combustion, and waste incineration . AlthougSphingomonas spp., Pseudomonas spp., Rhodococcus spp., Burkholderia spp., and Mycobacterium spp., have been investigated regarding whether they can metabolize PAHs. In particular, several Mycobacterium species have been reported to effectively degrade high-molecular-weight PAHs ) )  supplemed energy .Fig. 2A M. rufum JS14T are C18:1ω9c (36.72 %), C16:0 (26.24 %), C16:1ω7c + C16:1ω6c (9.40 %), C17:1ω7c (8.44 %), C14:0 (5.27 %), C18:0 (3.14 %), and C17:0 (1.94 %), respectively [Mycobacterium species [M. rufum JS14T revealed a major C24:0 peak and a trace of a C22:0 peak. The general characteristics of the strain are summarized in Table The main cellular fatty acids of ectively . The pro species , 19–21. M. rufum JS14T was selected for sequencing because of its effective ability to degrade PAH, as a model organism for a recalcitrant organic-pollutant-degrading bacterium. The genome sequencing was performed in September, 2014, and the Whole Genome Shotgun project was deposited in the DDBJ/EMBL/GenBank databases under the accession number JROA00000000. The version described in this study is the first version, labeled JROA00000000.1. The sequencing project information and its association with the Minimum Information about a Genome Sequence version 2.0 compliance [Strain mpliance  are descM. rufum JS14T from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (strain accession number DSM 45406T) was used for preparation of genomic DNA. The strain was cultured aerobically in a 250-mL Erlenmeyer flask containing 50 mL of tryptic soy broth , on a rotary shaker at 200 rpm and 30 °C. Genomic DNA was isolated from 50 mL of culture using the QIAamp® DNA Mini Kit  following the standard protocol recommended by the manufacturer. The quantity and purity of the extracted genomic DNA were assessed with a Picodrop Microliter UV/Vis Spectrophotometer  and Qubit® 2.0 Fluorometer (Fisher Scientific Inc.), respectively. Finally, a DNA concentration of 780.0 ng/μL and OD 260/OD 280 of 1.87 was determined.M. rufum JS14T was sequenced using the single-molecule real-time DNA sequencing platform on the Pacific Biosciences RS II sequencer with P5 polymerase - C3 sequencing chemistry  [The genome of ark, CA) . A 20-kbde novo using the RS hierarchical genome assembly process, version 3.3 in SMRT analysis software, version 2.2.0 (Pacific Biosciences) [All post-filtered reads were assembled ciences)  and resuThe protein-coding sequences were predicted by Prokaryotic Genome Annotation Pipeline, version 2.8, on the NCBI website (rev. 447580) . AdditioM. rufum JS14T was found to be 6,176,413 bp with the average G + C content of 69.25 %. The genome was predicted to contain a total of 5864 genes, which include 5810 protein-coding genes with 54 RNA genes . Of these, 4498 genes were assigned to putative functions, and 3669 genes (approximately 62.57 %) were assigned to the COG functional categories. The genome statistics are presented in Table The genome size of M. rufum JS14T was found to contain corresponding genes encoding proteins for the aromatic-compound degradation.Regarding the specific degradation capability toward the four-aromatic-ring-fused compound, fluoranthene –12, the nidA (PAH dioxygenase large subunit), nidB , phtAa , phtAb , phtB , phdE , and phdK (2-formylbenzoate dehydrogenase) were not identified  or toward low-molecular-weight PAHs  [M. rufum JS14T may provide important clues to the identity of the whole metabolic pathway for fluoranthene degradation. Just as a recent study on the functional pan-genome analysis of the genus Mycobacterium capable of degrading PAHs [One of native isolates from the petroleum-contaminated Hawaiian soil in Hilo ,  JS19b1T , is know JS19b1T , 11, 29.nthrene) . The gening PAHs , our datM. rufum JS14T was isolated from PAH-contaminated soil of a former oil gasification company site in Hilo  and was designated as a novel species that was named Mycobacterium rufum  [ strain) . In this


HIV-1 group M subtype of each sample was determined by phylogenetic analyses with the standard reference sequences. Among these infected individuals, 81.6 % (31/38) self-reported to be infected via sexual contacts, including 50.0 % (19/38) via heterosexual contact and 31.6 % (12/38) via homosexual contact. Among 34 samples with available pol or vif-env sequence, 19 (55.9 %) CRF01_AE, 7 (20.6 %) CRF07_BC, 3 (8.8 %) CRF08_BC, and 5 (14.7 %) inter-subtype recombinants were identified. No pure B, B′ and C subtypes were found in this cohort. The five recombinants contain one B/C, three CRF01/B and one CRF01/B/C recombinants. These results suggest that CRF01_AE was the most predominant HIV-1 group M subtype and CRF01_AE-involved recombinants were the major recombinant forms. Comparison showed that there was no obvious difference in HIV-1 group M subtype distribution between Jiangsu (including Suzhou and Suqian) and the surrounding provinces . CRF01_AE and CRF07_BC were the top two predominant HIV-1 genotypes in Jiangsu, and less and/or no pure subtype B and C was currently circulating here. We predicted that more CRF01/CRF07 recombinants, but fewer B/C recombinants will be generated in Jiangsu in future.Jiangsu is one province with severe HIV-1 epidemic in China. However, the molecular epidemiological characterizations of HIV-1 in many cities of Jiangsu remain unclear. A molecular epidemiological investigation was performed based on 38 HIV-positive samples collected from Suzhou and Suqian during 2011–2013. Five HIV-1 genomic fragments,  HIV/AIDS continues to be one of major public health issues in China. By the end of 2011, there were approximately 780,000  persons living with HIV/AIDS (PLHIV) in China  of HIV-1 to determine the subtype characterization of HIV-1 circulating in two cities (Suzhou and Suqian) of Jiangsu . Their locations in HXB2 are 683–1255, 2147–3462, 5084–5805, 5653–6454, and 6817–7381 nt, for p17, pol, vif-vpr, vpr-env, C2V3, respectively. Reverse transcription PCR reactions were performed using One-step RNA PCR kit . The PCR products were subjected to second PCR (nested PCR) using ExTaq . The primer pairs used in this study and the amplification fragments information were consistent with a previous study  and then subjected to the amplification of five HIV-1 genomic fragments (www.hiv.lanl.gov). The obtained sequences were aligned together with reference sequences using the Clustal W program implemented in MEGA 5.2  from Suzhou and 14 (36.8 %) from Suqian, were covered in this study. The social-demographic information, including gender, marital status, educational level, age, ethnic, occupation, risk behaviors, is listed in Table There are several light differences in social-demographic characterizations between Suzhou and Suqian although the little differences between two cities might be a result of a bias due to small sample size. Most HIV-infected individuals (62.5 %) in Suzhou had completed their high school education, higher than those (28.6 %) in Suqian. Heterosexual contact (58.3 %) was the likely predominant risk factor for HIV infection in Suzhou, while homosexual contact (46.2 %) was the major factor for HIV infection in Suqian. In addition, three individuals (21.4 %) in Suquan self-reported to have multiple sex partners, while no one was in Suzhou.p17, 32 (84.2 %) pol, 35 (92.1 %) vif-vpr, 32 (84.2 %) vpr-env and 34 (89.5 %) C2V3 fragments were successfully amplified and sequenced (Table vif-vpr (5084–5805 nt in HXB2) fragment overlapped with the vpr-env (5653–6454 nt in HXB2) fragment, we merged both two fragments into a vif-env sequence if both sequences available for the same sample. Finally, 27 merged vif-env sequences were obtained  ed Table , the vifp17, pol, vif-env and C2V3 fragments. Among 34 p17 sequences, 1 (2.9 %), 13 (38.2 %) and 20 (58.8 %) were identified to be HIV-1 group M subtype B, C and CRF01_AE, respectively  that clusters outside the subtype B clade, 11 (32.4 %) and 22 (64.7 %) were clearly identified as HIV-1 group M subtype C and CRF01_AE, respectively  was unable to cluster within the clades of known subtypes  that lack both pol and vif-env sequences were excluded in the statistics. Among the remaining 34 samples, 7 (20.6 %) CRF07_BC, 3 (8.8 %) CRF08_BC, 19 (55.9 %) CRF01_AE, and 5 (14.7 %) inter-subtype recombinants were found in two cities Suzhou and Suqian  CRF01_AE, 1 (9.1 %) CRF07_BC, 2 (18.2 %) CRF08_BC, and 2 (18.2 %) URFs were identified Fig. .In China, CRF01_AE was the most predominant strains circulating in the sexual transmission group , and CRF07_BC and CRF08_BC were major circulating among injection drug users (IDUs)  might be greater than reported data. In contrast, the numbers of subtypes B and C might be lower than reported data. Most recombinants in these regions were CRF01_AE-associated recombinants, different from the observation in southwestern China, where B/C recombinants were the most common recombinant forms  was similar to those in the surrounding provinces  (Wu et al. In this study, by characterizing the subtype distribution of HIV-1 group M in Suzhou and Suqian, we found that CRF01_AE was the most predominant HIV-1 group M subtypes in Jiangsu, and less and/or no pure subtype B and C was currently circulating here. We predicted that more CRF01/CRF07 recombinants, but fewer B/C recombinants will be generated in Jiangsu in future. Additional, we also suggest that the actual number of HIV-1 inter-subtype recombinants is larger and the genetic diversity of HIV-1 group M in Jiangsu is more complex than described here. Therefore, a molecular epidemiological investigation based on all high-risk groups in whole Jiangsu province is needed for providing detailed information for the prevention and control of HIV-1 in Jiangsu and even whole east China.


Chlorella containing different contents of lipids were scanned by NIRS and their fatty acid profiles were determined by GC-MS. NIRS models were developed based on the chemometric correlation of the near-infrared spectra with fatty acid profiles in algal biomass. The optimized NIRS models showed excellent performances for predicting the contents of total fatty acids, C16:0, C18:0, C18:1 and C18:3, with the coefficient of determination (R2) being 0.998, 0.997, 0.989, 0.991 and 0.997, respectively. Taken together, the NIRS method established here bypasses the procedures of cell disruption, oil extraction and transesterification, is rapid, reliable, and of great potential for high-throughput applications, and will facilitate the screening of microalgal mutants and optimization of their growth conditions for biodiesel production.The key properties of microalgal biodiesel are largely determined by the composition of its fatty acid methyl esters (FAMEs). The gas chromatography (GC) based techniques for fatty acid analysis involve energy-intensive and time-consuming procedures and thus are less suitable for high-throughput screening applications. In the present study, a novel quantification method for microalgal fatty acids was established based on the near-infrared spectroscopy (NIRS) technique. The lyophilized cells of oleaginous  To date, fossil-derived fuels have still served as the main energy sources ,2. The eChlorella spp. are thought to be promising candidates of biodiesel feedstocks in that they are able to grow robustly for high cell density, produce high level of triacylglycerol, and serve as an ideal source for making biodiesel . It. ItC. vuty acids . In contC. protothecoides and C. zofingiensis. This may indicate that the model built based on samples from a single strain is not suitable for other algal strains. In this context, we built new models by adding extra NIR spectra from 30 samples of C. zofingiensis and 69 samples of C. protothecoides. Briefly, 119 samples were randomly assigned to the calibration set, and the remaining 40 samples were assigned to the validation set. The means, maximum values, minimum values and standard deviation of TFA, C16:0, C18:0, C18:1, C18:2 and C18:3 contents of 159 samples determined by GC-MS were shown in CV-NIRS-WR II model, however, showed poor performance when predicting fatty acid composition in R2, RMSECV (mg/g cell), SEP (mg/g cell), r2, and RPD being 14.68, 0.988, 18.81, 24.16, 0.964, and 4.98, respectively. Although CVPZ-NIRS-WR II models had lower RPD values and higher RMSEC, RMSECV and SEP than CV-NIRS-WR II models for predicting C16:0, C18:0, C18:1 and C18:3 contents  content had good performance with R2 and RPD being 0.992 (0.985 and 0.979) and 7.45 (4.56 and 2.80), respectively. As for the model from C. zofingiensis, they were, respectively, 0.998 (0.997 and 0.989) and 9.58 (9.81 and 5.61). Although the models based on 3 Chlorella species are not as good as those based on individual species, they are feasible for mutant screening use.Based on the spectra of WR II (wavelength range of 1030–1500 and 1600–1880 nm) and fatty acid composition measured by GC-MS of all 159 samples, a series of new NIRS models, namely, CVPZ-NIRS-WR II, were created. Calibration and validation performances were calculated and shown in contents , they deNear-infrared spectroscopy (NIRS) consists of complex overtones and combinations of molecular vibrations . In contet al. [R2 being 0.97 and 0.98, and RPD being 5.37 and 7.35, respectively; in contrast, the prediction performance for C16:0  and C18:0  was far less acceptable. Fatty acids varying in chain length and unsaturation level possess different near-infrared spectra [R2 and RPD being 1.62–5.28 mg/g cell, 0.991–0.998 and 7.31–14.68, respectively , Chlorella protothecoides (CSIROCS-41), and Chlorella zofingiensis (ATCC 30412) were grown heterotrophically in the medium with a series of nitrate concentration. The inocula were prepared by culturing the microalgae in 500-mL Erlenmeyer flask with 200 mL of Kuhl medium at 25 °C for 4 days with orbital shaking at 150 rpm in the dark. The seeds of C. vulgaris, C. protothecoides and C. zofingiensis were inoculated to 200 mL of fresh modified Basal medium, modified Basal medium and modified Kuhl medium, respectively, at a 5% (v/v) inoculums size for batch culture in 500-mL Erlenmeyer flask. The modified Basal medium contained (per liter) different concentration of KNO3 from 0.75 to 10 g, 40 g glucose, 1.25 g KH2PO4, 1 g MgSO4·7H2O, 0.5 g EDTA·Na2, 0.1142 g H3BO3, 0.111 g CaCl2·2H2O, 0.0498 g FeSO4·7H2O, 0.0882 g ZnSO4·7H2O, 0.0142 g MnCl2·4H2O, 0.0157 g CuSO4·5H2O, 0.0049 g Co(NO3)2·6H2O, and 0.0071 g MoO3. The modified Kuhl medium was consisted of (per liter) different concentrations of KNO3 from 0.75 to 10 g, 40 g glucose, 0.62 g NaH2PO4·H2O, 0.089 g Na2HPO4·2H2O, 0.247 g MgSO4·7H2O, 14.7 mg CaCl2·2H2O, 6.95 mg FeSO4·7H2O, 0.061 mg H3BO3, 0.169 mg MnSO4·H2O, 0.287 mg ZnSO4·7H2O, 0.0025 mg CuSO4·5H2O, and 0.01235 mg (NH4)6MO7O24·4H2O. The pH values of modified Basal medium and modified Kuhl medium were adjusted to 6.1 and 6.5, respectively, prior to autoclaving. After inoculating, flasks were incubated at 25 °C in an orbital shaker at 150 rpm in the dark for 10 days. All samples were harvested and lyophilized for the collection of NIR spectra and fatty acid analysis. In total, 30 samples of C. zofingiensis, 69 samples of C. protothecoides and 60 samples of C. vulgaris, which contained various lipids contents, were collected and lyophilized.Three v/v) and 0.8 mg heptadecanoic acid in 0.8 mL hexane as the internal standard) overnight at 50 °C for transesterification to form fatty acid methyl esters (FAMEs). FAMEs were then extracted three times with hexane in a reciprocating shaker. The FAMEs were analyzed by using a GC-MS-QP 2010 SE (Electron Ionization type) gas chromatograph-mass spectrometer  and a Stabilwax-DA capillary column (30 m × 0.25 mm × 0.25 μm) . Helium was used as the carrier gas. The injection temperature, ion temperature and interface temperature were set at 250, 200 and 260 °C, respectively. The initial column temperature was set at 150 °C. The column temperature subsequently rose to 200 °C at 10 °C/min and then to 220 °C at 6 °C/min, followed by a hold at 220 °C for 10 min. FAMEs were identified by NIST 11 mass spectral library . The quantities of individual FAMEs were calculated by the peak areas according to the total ion chromatogram (TIC) using heptadecanoic acid as the internal standard.Twenty milligrams of lyophilized algal cells were incubated in a solvent mixture . Temperature and relative humidity conditions during scanning ranged from 22 to 26 °C and from 35% to 45%, respectively. About 200 mg biomass of each sample was packed into a 1.5-mL Eppendorf tube for collecting NIRS. Diffusely reflected radiation was detected with optical fiber probe from 1000 to 2499 nm at a 1 nm resolution. The NIRS of each individual sample were obtained by averaging 5 parallel spectra.Spectra data of all samples were converted and imported into the chemometrics software of the Unscrambler version 9.7 . First, spectra were pretreated with the approach of Savitzky-Golay smoothing filter to preserve the features of distribution. Second, the first order derivatives were computed by the convolution (Savitzky-Golay) method to reduce peak overlap and eliminate baseline shift . Then thR2. The regression models were tested with the validation sets and three parameters, namely SEP, r2 and RPD, were calculated to assess the predictability. Notably, in the same concentration range, the accuracy of prediction result increases with the RPD value. RPD values of <2 indicate that the prediction result by the model is unacceptable; RPD values of 2–5 indicate the model is suitable for screening; RPD values of >5 indicate the model is suitable for quality control and even process control, and RPD values of >8 indicate the model is suitable for all possible applications [R2 or r2 is, the more accurate the NIRS model is. As for RMSECV, RMSEC, and SEP, the smaller the better.In common, the calibration performance of each regression model was evaluated by RMSECV, RMSEC, and ications . BesidesChlorella species. In a word, compared to the traditional GC-mediated analyses, the reliable NIRS technique described here bypasses the involvement of cell disruption, oil extraction and transesterification and is thus easier to conduct and more environmentally friendly, and has great potential for screening purposes, in particular the high-throughput screening of oleaginous microalgal fatty acids for biodiesel uses.The key properties of biodiesel are largely determined by the fatty acid methyl ester profile. Therefore, when evaluating the feasibility of biodiesel feedstocks, the fatty acid composition should be considered as an important indicator. The present study developed a novel near-infrared spectroscopy (NIRS) technique for rapid and reliable analysis of fatty acids in microalgae, which can be done within a few seconds and requires only a small amount of samples. The optimized NIRS method demonstrated to have a good performance for the quantification of fatty acids across


Moreover, there could be an unexplored pathophysiological time window for the detection of CA125 in EOC, during which it is expressed on tumor cells prior to being shed into the bloodstream. A method for the in vivo evaluation of CA125 expression on ovarian neoplasms earlier along disease progression and/or recurrence can potentially contribute to better disease management. To this end, the present work utilizes an anti-CA125 monoclonal antibody (MAb) and a single-chain variable fragment (scFv) labeled with the positron-emitting radionuclide 64Cu for preclinical molecular imaging of CA125 expression in vivo.Epithelial ovarian cancer (EOC) is characterized by the overexpression of cancer antigen 125 (CA125), a mucinous glycoprotein that serves as a tumor biomarker. Early diagnosis of EOC is plagued by its asymptomatic nature of progression and the limitations of currently used immunoassay techniques that detect CA125 as a shed antigen in serum samples. Presently, there is no technique available for the Anti-CA125 MAb and scFv were prepared and functionally characterized for target binding prior to being tested as radiotracers in a preclinical setting.64Cu-labeled anti-CA125 MAb and scFv were obtained in specific activities of 296 and 122 MBq/mg, respectively. Both radioimmunoconjugate vectors demonstrated highly selective binding to NIH:OVCAR-3 cells and virtually no binding to SKOV3 cells. In vivo radiopharmacological evaluation using xenograft mouse models injected with 64Cu-labeled anti-CA125 MAb provided a standardized uptake value (SUV) of 5.76 (29.70 %ID/g) in OVCAR3 tumors 24 h post-injection (p.i.) versus 1.80 (5.91 %ID/g) in SKOV3 tumors. 64Cu-labeled anti-CA125 scFv provided an SUV of 0.64 (3.21 %ID/g) in OVCAR3 tumors 24 h p.i. versus 0.25 (1.49 %ID/g) in SKOV3 tumors. Results from small-animal PET imaging were confirmed by ex vivo autoradiography and immunohistochemistry.Immunoblotting, immunofluorescence, and flow cytometry revealed specific binding of CA125-targeting vectors to NIH:OVCAR-3 cells and no binding to antigen-negative SKOV3 cells. 64Cu did not compromise their immunoreactivity. Both radioimmunoconjugates presented specific tumor uptake and expected biological clearance profiles. This renders them as potential immuno-PET probes for targeted in vivo molecular imaging of CA125 in EOC.Radiolabeling of anti-CA125 MAb and scFv with The online version of this article (doi:10.1186/s13550-014-0060-4) contains supplementary material, which is available to authorized users. Despite its relative rarity in the general population and the availability of standard treatment through surgical intervention and platinum chemotherapy, epithelial ovarian cancer (EOC) continues to be the most insidious and lethal gynecologic malignancy in the western world. The Surveillance Epidemiology and End Results (SEER) program has estimated 21,980 new cases of ovarian cancer to occur in 2014, with 14,270 deaths resulting from this disease in the United States alone  FDG-PET has shown the highest sensitivity and accuracy in detecting recurrent ovarian cancer, while simultaneous investigations with CT, MRI, and ultrasound which primarily detect morphological features and anatomical changes that can be distorted on account of primary cyto-reductive surgery in patients yielded negative or equivocal results   was conjugated to anti-CA125 MAb and scFv to serve as a bifunctional chelator for 64Cu radiolabeling. Briefly, a 6 M excess of p-SCN-Bn-NOTA in DMSO was added to anti-CA125 MAb/scFv in 0.1 M sodium bicarbonate buffer pH 9.0 and allowed to react for 1 h at 37 C. NOTA-functionalized anti-CA125 MAb/scFv was purified from excess unconjugated bifunctional chelator while simultaneously achieving buffer exchange into 0.25 M sodium acetate pH 5.5 by using an Econo-Pac 10DG desalting column . Protein quantification of the column-eluted fractions was performed using A280 measurements on nanodrop 2000 (Thermo Scientific) and bicinchoninic assay  using a Synergy H1 multimode microplate reader for A562 absorbance read-out. The number of bifunctional chelates conjugated per MAb and scFv was determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS analysis and according to the method described by Cooper et al.  were calculated for each ROI. Additionally, radioactivity uptake was also analyzed as percentage of injected dose per gram tissue (%ID/g). Data are expressed as means ± SEM from n investigated animals.Small-animal PET experiments were performed on a MicroPET® R4 or INVEON PET scanner . Mice were anesthetized by inhalation of isoflurane in 40% oxygen/60% nitrogen, 1 L/min, while maintaining body temperature at 37°C throughout the experiment; 6 to 10 MBq of high specific activity radioimmunoconjugate (approximately 30 to 40 μg) in 150 to 200 μL of 0.25 M sodium acetate (pH 5.5) was administered intravenously via a tail vein catheter. For blocking experiments, 1 mg of unmodified anti-CA125 MAb was administered intraperitoneally 24 h prior to injection of the 64Cu-labeled anti-CA125 MAb via the tail vein. At 24 h p.i., the animal was euthanized by cervical dislocation. The tumor was submerged into OCT medium and flash frozen using a bath of dry ice-cooled methanol. Seven-micrometer slices were cut on a Leica CM 1850 cryostat .An NIH:OVCAR-3 tumor-bearing mouse was injected with 4.55 MBq of Tissue sections were placed into a BAS-Cassette  and exposed to a phosphor imaging plate  for 15 h at room temperature. The images were developed on a BAS-5000 reader  and analyzed with Adaptive Image Deconvolution Algorithm (AIDA) Image Analyzer v.450 software.Upon thawing, tissue sections were fixed with formalin for 30 min. The fixed sections were blocked overnight at 4°C using 0.12 mg/mL unconjugated goat anti-mouse Fab fragment  in 0.5% fish skin gelatin  pH 7.4, supplemented with 0.1% Triton X-100. The sections were rinsed 3× with Tris-buffered saline having 0.5% Tween 20 (TBST) for 5 min each. MAb-B43.13 was used as primary antibody at a dilution of 1:6,000 in Dako antibody diluent  and allowed to incubate overnight at 4 C. The sections were rinsed 3× with TBST and incubated with Alexa Fluor® 488 goat anti-mouse antibody  used as secondary antibody at 1:400 dilution in Dako antibody diluent for 2 h at room temperature. After three washes with TBST, the sections were rinsed with water and counterstained using Hoechst H33342 (2 μg/mL) for 5 min. The sections were rinsed and mounted under a coverslip using FluorSave . The slides were analyzed with a Zeiss Plan Apochromat 10X/0.45 na lens on a confocal laser scanning microscope (Zeiss LSM 710) using Z-stack for image acquisition. The images were registered using Zen 2011 software (Zeiss) and further processed with Adobe Photoshop CS6.To analyze the samples for immunohistochemistry, the same procedures for treatment, fixation, blocking, and probing of tissue with primary antibody were followed. DakoEnVision™ + HRP-conjugated anti-mouse antibody was used as a secondary antibody, and no counterstain was employed. The sections were observed with a Zeiss Fluar 2.5X/0.12 na lens on an Axioskop 2 Plus microscope. Images were processed and analyzed on AxioVision 4.7 (Zeiss) and further processed with Adobe Photoshop CS6.t test and were considered significant for p <0.05.All data are expressed as means ± SEM. Graphs were constructed using GraphPad Prism 4.0 (GraphPad Software). Where applicable, statistical differences were tested by unpaired Student's -9 M for MAb-B43.13 and 1.11 × 10-8 M for the scFv, with comparable dissociation constants in the range of 10-4s-1  with specific activity of 296 ± 37 MBq/mg for anti-CA125 MAb and 56% ±14% (n = 9) with specific activity of 122 ± 44 MBq/mg for anti-CA125 scFv  at 24 h p.i. rising to 7.06 ± 0.67 (41.66 ± 4.38 %ID/g) at 48 h p.i. (n = 4). NIH:OVCAR-3 xenograft mice pre-dosed with unlabeled MAb revealed a SUVmean of 2.90 ± 0.45 (13.10 ± 2.07 %ID/g) at 24 h p.i.  and 3.96 ± 0.76 (14.47 ± 3.01 %ID/g) at 48 h p.i. . Non-specific tumor uptake using 64Cu-labeled isotype IgG1 was found to have a SUVmean of 1.77 ± 0.44 (9.77 ± 0.44 %ID/g) at 24 h p.i.  with no further change at 48 h p.i. A SUVmean of 1.80 ± 0.69 (5.91 ± 3.31 %ID/g) 24 h p.i.  was observed in SKOV3 tumors.In NIH:OVCAR-3 tumors, 64Cu-labeled anti-CA125 scFv in xenograft mice revealed a SUVmean of 0.64 ± 0.04 (3.21 ± 0.29 %ID/g) 24 h p.i. (n = 3) in NIH:OVCAR-3 tumors and 0.25 ± 0.11 (1.49 ± 0.68 %ID/g)  in SKOV3 tumors.In contrast, PET studies using in vivo clearance of the radioimmunoconjugates was evaluated from the heart (representing blood pool content), liver, and kidneys  and 2.59 ± 0.20 (12.54 ± 1.67 %ID/g) (n = 4), respectively. SUVmean in the kidneys of these animals was 1.64 ± 0.09 (10.35 ± 1.06 %ID/g).After 24 h, SUVmean in the heart and liver of xenograft animals imaged with 64Cu-labeled anti-CA125 scFv led to a SUVmean (n = 3) of 0.31 ± 0.06 (1.55 ± 0.40 %ID/g) in the heart, 0.98 ± 0.11 (4.89 ± 0.89 %ID/g) in the liver, and 13.2 ± 1.2 (66.46 ± 9.20 %ID/g) in the kidneys after 24 h. This information demonstrated the different in vivo clearance profile between the full-length antibody and the fragment.In contrast, injection of The NIH:OVCAR-3 tumor tested positive for CA125 expression as seen from immunofluorescence and immunohistochemistry of tissue sections  overexpressing CA125  FDG in OVCAR3 tumors to be relatively low  [64Cu-NOTA-MAb B43.13 observed in CA125-negative SKOV3 tumors.The present study with CA125-targeting MAb-B43.13 and its derivative scFv in preclinical ovarian cancer models found a higher targeted tumor uptake of ct (EPR) , whose n64Cu-NOTA-MAb B43.13 could be attributed to a) its longer residency time in circulation, b) consistent non-specific muscle uptake of the radiotracer in all experimental animals . However, NOTA has previously demonstrated high in vivo kinetic stability as a chelator for 64Cu2+ ions [in vitro challenge experiments for 64Cu-NOTA-MAb B43.13 in the presence of an excess of purified SOD showed negligible trans-chelation  pharmacological evaluation of the immunoconjugates. He was responsible for writing the manuscript. MeW was responsible for animal experiments, including analysis of the PET data. MoW was supporting all radiopharmacological experiments. DG supported Supporting information. A file containing supplementary table and figures. (DOCX 3 MB)Additional file 1:Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Below are the links to the authors’ original submitted files for images.


Extracellular pyrophosphate is a potent endogenous inhibitor of vascular calcification, which is degraded by alkaline phosphatase (ALP) and generated by hydrolysis of ATP via ectonucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1). ALP activity  represents the maximal activity , but not the real activity . For the first time, the present study investigated extracellular pyrophosphate metabolism during hemodialysis sessions (including its synthesis via eNPP1 and its degradation via ALP) in physiological conditions.vs. 89.3 ± 3.6 IU/L) and 48% under physiological conditions (3.5 ± 0.2 IU/L vs. 5.2 ± 0.2 IU/L). Pyrophosphate synthesis by ATP hydrolysis remained unaltered post-hemodialysis. Post-hemodialysis plasma pH (7.45 ± 0.02) significantly increased compared with the pre-dialysis pH (7.26 ± 0.02). The slight variation in pH (~0.2 units) induced a significant increase in ALP activity (9%). Addition of phosphate in post-hemodialysis plasma significantly decreased ALP activity, although this effect was not observed with the addition of urea. Reduction in phosphate levels and increment in pH were significantly associated with an increase in physiological ALP activity post-hemodialysis. A decrease in plasma pyrophosphate levels (3.3 ± 0.3 μmol/L vs. 1.9 ± 0.1 μmol/L) and pyrophosphate/ATP ratio (1.9 ± 0.2 vs. 1.4 ± 0.1) post-hemodialysis was also observed.45 patients in hemodialysis were studied. Physiological ALP activity represents only 4–6% of clinical activity. ALP activity increased post-hemodialysis by 2% under ideal conditions (87.4 ± 3.3 IU/L Extraction of uremic toxins, primarily phosphate and hydrogen ions, dramatically increases the ALP activity under physiological conditions. This hitherto unknown consequence of hemodialysis suggests a reinterpretation of the clinical value of this parameter. Vascular calcification (VC), or ectopic calcification in vessels, is highly prevalent in patients with chronic hemodialysis, and is associated with cardiovascular events and all-cause mortality,2. Elevaex vivo medial calcification in aortic ringsATP at a final concentration of 1 μmol/L and 10 μCi/mL, respectively. Production of PPi, Pi, and ATP was determined by chromatography on PEI-cellulose plates, and they were developed with 650 mmol/L KH2PO4 pH 3 as previously described[To analyze the products released during ATP hydrolysis, 50 μL of human plasma was incubated with 50 μL of Hank’s salt solution containing ATP and [γdescribed,18. AftePPi was measured  with an Results are represented as mean ± standard error of the mean (SEM). Experiments shown in Figs H. Using this method, the maximum activity of ALP, which strictly correlates with the amount of ALP enzyme present in serum or plasma, can be detected; however, ALP activity in physiological conditions, e.g., in normal plasmatic conditions, has not yet been studied. Therefore, we examined ALP activity in both maximal and physiological conditions. Maximal ALP activity was observed to slightly increase after the hemodialysis session , although all ALP activity values were found to be within the normal range (45–129 IU/L). Physiological ALP activity significantly increased (P < 0.001) in post-hemodialysis plasma (5.2 ± 0.2 IU/L) compared with pre-hemodialysis plasma (3.5 ± 0.2 IU/L) in all samples. Pre- and post-hemodialysis physiological ALP activities were found to be 4.1% and 5.9% of the maximal ALP activity, respectively (relative ALP activity). ALP activity in physiological conditions was observed to increase by approximately 40% after a hemodialysis session. There was a significant correlation between pre- and post-hemodialysis ALP activities in maximal (P < 0.001), physiological (P < 0.001), and relative (P = 0.011) ALP activities; however, the R2 values differed and poor in the case of physiological and relative ALP activities  post-hemodialysis . This correlation was observed to be inverted in post-hemodialysis samples  due to the dramatic reduction in phosphate levels. Furthermore, both calcium and ionized calcium levels were significantly (P < 0.001) increased in post-dialysis plasma in 86.6% of paired samples analyzed . The Ca/PPi ratio was also significantly (P < 0.001) increased, by approximately 2-fold  post-hemodialysis (2.3 ± 0.3 μmol/L vs. 1.8 ± 0.3 μmol/L) but to a lesser extent than PPi (P = 0.02) post-hemodialysis (1.9 ± 0.2 vs. 1.4 ± 0.1). Furthermore, there was a significant correlation (P = 0.003), suggesting that the reduction in the PPi/ATP ratio is a consequence of the increase in ALP activity.Plasma ATP levels were significantly decreased  . AdditioH. Post-hemodialysis plasma pH (7.45 ± 0.02) was significantly increased (P < 0.001) compared with the pre-dialysis pH (7.26 ± 0.02) in all samples analyzed (H (approximately 0.2 units) was observed to induce a significant increase in ALP activity (approximately 9%) at a pH of 7.45 compared with a pH of 7.25 .As shown analyzed . The sli of 7.25 . Finally 7.25 (H  and the +) are the most common substances that accumulate in blood as a consequence of renal dysfunction, resulting in hyperphosphatemia and acidosis in chronic hemodialysis patients[More than 5000 substances accumulate as a consequence of renal dysfunction, and produce complex and multiple biochemical and physiological abnormalities (uremic syndrome). Phosphate (Pi) and hydrogen ions  is associated with an increased risk of VC,4. SeverPlasma PPi concentration was reported to be reduced by 32% after standard hemodialysis, primarily as a consequence of dialytic clearance. The autThese new findings may support a new therapeutic treatment against ALP during dialysis to reduce PPi hydrolysis. Recent studies using rat and mice models showed that daily injections of exogenous PPi prevent VC,24–26. G+ levels during hemodialysis.In conclusion, our study revealed three important concepts. First, measurements of ALP in clinical practice represent the maximal activity in ideal conditions but not the actual activity under normal or physiological conditions. Physiological ALP activities represent 4–6% of the maximal activity. Second, physiological ALP activity increases after dialysis clearance, reducing PPi availability. Finally, the increase in ALP activity may be primarily explained by a decrease in phosphate and H


IKr) plays a critical role in cardiac repolarization. Although IKr is known to be regulated by both α1- and β1-adrenergic receptors (ARs), the cross-talk and feedback mechanisms that dictate its response to α1- and β1-AR activation are not known. In the present study, IKr was recorded using the whole-cell patch-clamp technique. IKr amplitude was measured before and after the sequential application of selective adrenergic agonists targeting α1- and β1-ARs. Stimulation of either receptor alone (α1-ARs using 1 μM phenylephrine (PE) or β1-ARs using 10 μM xamoterol (Xamo)) reduced IKr by 0.22 ± 0.03 and 0.28 ± 0.01, respectively. The voltage-dependent activation curve of IKr shifted in the negative direction. The half-maximal activation voltage (V0.5) was altered by −6.35 ± 1.53 and −1.95 ± 2.22 mV, respectively, with no major change in the slope factor (k). When myocytes were pretreated with Xamo, PE-induced reduction in IKr was markedly blunted and the corresponding change in V0.5 was significantly altered. Similarly, when cells were pretreated with PE, Xamo-induced reduction of IKr was significantly attenuated. The present results demonstrate that functional cross-talk between α1- and β1-AR signaling regulates IKr. Such non-linear regulation may form a protective mechanism under excessive adrenergic stimulation.The rapidly activating delayed rectifier potassium current ( Specifically, from a holding potential of −40 mV, currents were activated by a variable test pulse from −40 to +40 mV , followed by a return pulse to −40 mV (duration 775 ms) to evoke outward tail currents. This protocol was applied to measure each peak tail current at repolarization after different test voltages. Activation curves were fit to a single-power Boltzmann equation: IKr recordings, the pipette solution contained (in mmol/L) KCl 140, CaCl2 1, MgCl2 2, HEPES 10, EGTA 11, Na2-ATP 5, creatine phosphate  5, pH 7.2 adjusted with 8 M KOH. The bath solution contained (in mmol/L) NaCl 140, KCl 3.5, CaCl2 1.5, MgSO4 1.4, HEPES 10, pH adjusted 7.4 with 10 M NaOH. Nifedipine (10 μM) and Chromanol 293B (10 μM) were added to the bath solution to block the L-type calcium and delayed rectifier potassium (IKs) currents, respectively. Na2-ATP, EGTA, creatine phosphate, l-glutamic acid, HEPES, taurine, bovine serum albumin (BSA), nifedipine, chromanol 293B, PE, and dofelitide were purchased from Sigma , collagenase II from Worthington , and Xamol-hemifumarate from Santa Cruz . All other reagents were obtained from Amresco .For For stock solutions, nifedipine and chromanol 293B were dissolved in dimethyl sulfoxide (DMSO) (10 mM concentration); Xamo in distilled water (10 mM concentration); PE and dofetilide in distilled water (1 mM). All stock solutions were stored at −20 °C except nifedipine which was stored at 4 °C. Before experiments, aliquots of the stock solutions were diluted with the extracellular solution to the desired test concentrations of 1 μM (PE and dofetilide) and 10 μM . The final concentration of DMSO was less than 0.5% in extracellular solution, which was determined to have no confounding effects on the currents of interest.t test. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA), with a post-hoc comparison using a Newman–Keuls test. A p value less than 0.05 was considered statistically significant.Currents were acquired with Pulse + Pulsefit V8.53  and analyzed by SPSS 18.0 software . Data were expressed as mean ± S.E.M. Statistical significance was evaluated using the unpaired Student’s hERG channel activation is strongly modulated by acute stimulation of α1-ARs and β1-ARs. Remarkably, stimulation of either receptor class markedly attenuates the inhibitory effects of the other class on IKr Further investigations are required to elucidate the molecular mechanisms that underlie the functional cross-talk or to identify putative intermediate proteins in the signal transduction pathways involved in modulation of IKr current via adrenergic activation.


Bacillus amyloliquefaciens B10-127 exhibited an excellent ability for industrial-scale microbial fermentation of 2,3-butanediol  from biodiesel-derived glycerol. However, the accumulation of by-products  and the 2,3-BD yield remains prohibitively low for commercial production.B. amyloliquefaciens. Firstly, extra copies of NADH/NAD+ regeneration system were introduced into B. amyloliquefaciens by co-overproduction of glycerol dehydrogenase and acetoin reductase, which resulting in improvement of 2,3-BD production and suppression of by-products accumulation. Subsequently, the transcriptional regulator ALsR under the control of a moderate promoter PbdhA was introduced into B. amyloliquefaciens, which increased carbon flux to 2,3-BD branch. Finally, a three-stage dissolved oxygen control strategy were proposed based on analysis of the characteristic of 2,3-BD fermentation, and a two-stage pH control strategy were proposed based on different pH preferences of ACR for reduction and oxidation. Following these strategies, a high titer (102.3 g/L), yield (0.44 g/g), and productivity (1.16 g/L/h) of 2,3-BD were achieved.Several strategies were developed to manipulate the carbon flux to 2,3-BD branch in a designed B. amyloliquefaciens should be an excellent candidate for producing 2,3-BD on an industrial scale.To our knowledge, this is the highest reported 2,3-BD production using biodiesel-derived glycerol as substrate, and this designed  InteresKlebsiella strains can catalyze pure glycerol into 2,3-BD, but these reactions generate large quantities of 1,3-PD [Klebsiella pneumoniae is a pathogenic microorganism, it does not conform to the safety regulations of industrial-scale fermentation [Enterobacter aerogenes FMCC-10; however, the efficiency of the production was still much too low for an economic process.To date, many studies on 2,3-BD production have focused on sugar fermentation. Despite the high-effective productivities that have been achieved via the conversion of glucose , the relf 1,3-PD , 9. Impoentation  obtainedB. amyloliquefaciens readily produces 2,3-BD from biodiesel-derived glycerol in the presence of beet molasses as a co-substrate [Previously, we reported that ubstrate . In fed-+-dependent glycerol dehydrogenase (GDH) oxidizes glycerol to dihydroxyacetone, which is subsequently oxidized to pyruvate. Acetolactate synthase (ALS) catalyzes the in vivo coupling of two pyruvate molecules to form acetolactate, which is then decarboxylated to acetoin by acetolactate decarboxylase (ALDC). Finally, acetoin is reduced to 2,3-BD by an NADH-dependent acetoin reductase (ACR) [B. amyloliquefaciens, such as succinate, lactate, and acetate, which negatively regulate the 2,3-BD yield and increase the costs of downstream separation and purification. Furthermore, large-scale microbial 2,3-BD production requires efficient and economical fermentation processes. Thus, in this work, we focused on improving strains to produce 2,3-BD with high yield, using low price substrates (raw glycerol and molasses) to lower the cost of feedstock, and optimizing the operation mode to make the process more efficient  . Bacterit Scheme .Scheme 1+ to NADH. So, over-production of glycerol dehydrogenase could increase not only the dehydrogenation of glycerol but also the level of available NADH.Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms, and for value-added utilization of glycerol in the industry that catalyzes the dehydrogenation of glycerol to dihydroxyacetone. This reaction is coupled to the reduction of oxidized NADK. pneumoniae ATCC 25955, there are two GDHs (DhaD and GldA) [B. amyloliquefaciens, and generated recombinant strains DH and GL, respectively. As shown in Table In the genome sequence of nd GldA) . So, the+-dependent DhaD oxidizes glycerol to dihydroxyacetone, with concomitant reduction of NAD+ to NADH. In contrast, the NADH-dependent ACR reduces acetoin to 2,3-BD, with concomitant oxidation of NADH to NAD+. So, co-overproduction of DhaD and ACR may enhance 2,3-BD production [dhaD gene harbored in pMA5-acr was overexpressed in the strain GA. The plasmid genetic rate remained about 95 %, indicating that the pMA5-acr-HapII-dhaD was stably expressed in the strain GA. The specific activities of GDH (0.67 ± 0.06 U/mg) and ACR (0.58 ± 0.05 U/mg) in the strain GA were 4.58-fold and 3.66-fold higher than in the strain B10-127, respectively.The NADoduction . InspiredhaD and acr on cell growth and 2,3-BD production were also investigated. As shown in Fig. B. amyloliquefaciens increased the highest 2,3-BD titer by 13.6 %, while decreasing the acetoin concentration by 64.6 % , possibly because introducing extra copies of DhaD/ACR enzymes into B. amyloliquefaciens accelerated the NADH/NAD+ regeneration rate without influencing the NAD+/NADH levels. Two reasons may account for this phenomenon. First, over-production of NAD+-dependent DhaD increased the rate of glycerol consumption and NADH level. Second, in the glycerol metabolism, the 2,3-BD branch is primarily responsible for oxidizing NADH, and when NADH-dependent ACR is overproduced, the 2,3-BD branch gains a competitive advantage over the end products of pyruvate-deriving pathways (such as lactic acid and succinic acid), through the enhanced availability of NADH. So, when extra copies of DhaD/ACR enzymes were introduced into B. amyloliquefaciens, it was found that overexpressing the NADH/NAD+ regeneration system effectively improved 2,3-BD production and inhibited by-products accumulation.The effects co-overexpression of alsSD operon in B. subtilis [alsSD [alsR prevents the transcription of alsSD [There are three key enzymes involved in 2,3-BD branch, i.e. α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC), and acetoin reductase (ACR). To make the 2,3-BD branch gains a competitive advantage over the end products of pyruvate-deriving pathways , in our pre-test, we attempted to overexpress ALS and ALDC to enhance acetoin production. And we succeeded in increasing the activity of these enzymes by more than 50-fold, however, 2,3-BD production was not significantly enhanced and cell growth was markedly inhibited. ALS and ALDC are encoded by the subtilis . It has s [alsSD  and thatof alsSD .alsR and pMA5-PbdhA-alsR constructed using the strong HpaII and moderate PbdhA promoters to express ALsR, respectively [alsR under the control of two different promoters (HpaII and PbdhA) were cloned into B. amyloliquefaciens B10-127, respectively. 2,3-BD production was improved by regulating ALsR expression using either of the two promoters. However, although ALsR expression was higher under the control of the stronger promoter (HpaII) than the moderate promoter (PbdhA), moderate enhancement of ALsR expression was more benefic to improve 2,3-BD production. Compared with the parent strain, 2,3-BD concentration increased by 9.2 % and 15.4 % under the control of and PbdhA, respectively. Furthermore, the rates of cell growth and glucose consumption under the control of HpaII were lower than under the control of PbdhA. Zhang et al. [The recombinant plasmids pMA5-HpaII-ectively . In the g et al.  also repbdhA promoter to express ALsR in B. amyloliquefaciens GA. The plasmid genetic rate of the resulting GAR recombinant remained stable at about 95 %, indicating that the pMA5-dhaD-HapII-acr-PbdhA-alsR was stably expressed in the GAR strain. The specific activities of ALS (0.82 ± 0.07 U/mg) and ALDC (0.25 ± 0.03 U/mg) in the GAR strain were respectively 1.02-fold and 1.76-fold higher than in the GA strain.So, we selected the moderate PAs shown in Fig. B. amyloliquefaciens, 2,3-BD plays a major role in oxidizing NADH. To secure NADH and C, it must compete with other end products of pyruvate-deriving pathways. Thus, this study characterized the metabolic flexibilities of B. amyloliquefaciens in response to over-expression of the dhaD, acr and alsR genes. For this purpose, the concentrations of major metabolites of both strains  were determined . As shown in Table B. amyloliquefaciens.In the aerobic glycerol metabolism of Oxygen supply is one of the most important variables in the 2,3-BD fermentation  found thB. amyloliquefaciens GAR under different oxygen supply methods by changing agitation speeds. Based on the analysis of two kinetic parameters including specific cell growth rate (μx) specific glycerol consumption rate (μs) and specific 2,3-BD formation rate (μp)  . The repKlebsiella sp., at neutral pH, it synthesizes acetic acid and ethanol, but below pH 6, 2,3-BD and ethanol are produced. However, the optimum pH for 2,3-BD production strongly depends on the microorganism and substrate used. Voloch et al. [K. oxytoca. For E. aerogenes, Converti et al. [K. pneumoniae a pH above 6 causes a sharp decrease in the activity of α-acetolactate synthase . Previously, the results also clearly showed that pH-dependent 2,3-BD production from glucose of B. amyloliquefaciens with the maximum production was at initial pH 6.5, and in the initial stage of fermentation, it was good for 2,3-BD synthesis without external pH control [B. amyloliquefaciens has been classified as GRAS  by the US Food and Drug Administration [B. amyloliquefaciens is an economically valuable goal.Another major influence on 2,3-BD production is the pH  reportedh et al.  found thi et al.  and Perei et al.  all expei et al.  found th control . Industrstration . TherefoDuring the production of 2,3-BD from glycerol, acetoin is the precursor of 2,3-BD. So, it is very important that maintain the suitable conditions to steadily biosynthesize 2,3-BD from acetoin. Previously, it was found that the ACR, which catalyzes the interconversion between acetoin and 2,3-butanediol , 33. We Basing on the above pH preference, a two-stage control strategy was proposed to improve 2,3-BD formation Fig. b. At the+ regeneration system effectively improved 2,3-BD production and inhibited by-products accumulation. Medorate expression of ALsR made the 2,3-BD branch gain a competitive advantage over the end products of pyruvate-deriving pathways. The carbon flux from acetoin to 2,3-BD was manipulated by using two-stage controlled pH and dissolved oxygen strategies. Finally, a high titer (102.3 g/L), yield (0.44 g/g), and productivity (1.16 g/L/h) of 2,3-BD were achieved. To our knowledge, this is the highest reported 2,3-BD production using biodiesel-derived glycerol as a substrate, and this designed B. amyloliquefaciens should be an excellent candidate for producing 2,3-BD on an industrial scale.Overexpressing the NADH/NADB. amyloliquefaciens B10-127, which has been deposited in the China Center for Type Culture Collection (CCTCC) under collection number CCTCC M 2012349. K. pneumoniae ATCC 25955 was obtained from American Type Culture Collection. The recombinant derivatives of Escherichia coli/B. subtilis shuttle plasmid vector pMA5-HapII were hosted in E.coli JM109. This shuttle vector introduced the expression cassette for hyper-expression of dhaD, gldA, acr and alsR into B. amyloliquefaciens B10-127.Strains, plasmids and primers used in this study are listed in Table B. amyloliquefaciens and E. coli were cultured in Luria-Bertain (LB) medium. When necessary, ampicillin or kanamycin was added into the culture medium. For 2,3-BD production, B. amyloliquefaciens was inoculated into 10 mL LB medium added with 40 g/L glycerol and cultivated overnight with agitation  at 37 °C. After 12 h, 2.5 mL of the seed culture (OD600 = 5.0–6.0) was inoculated into fermentation medium (crude glycerol (80 g/L), beet molasses (15 g/L) (Addition of beet molasses could enhance glycerol assimilation [2HPO4 (3 g/L), MgSO4·7H2O (0.3 g/L), FeSO4·7H2O (0.05 g/L), pH6.5). Waste glycerol comprised of 88 % (w/w) glycerol, 6–9 % (w/w) water, 4–6 % (w/w) ash, 4 % (w/w) chlorides, and 0.2 % (w/w) methanol. The composition of beet molasses was 56.6 % (w/w) sucrose, 0.08 % (w/w) glucose, 23.8 % (w/w) water, 10 % (w/w) sulfated ash, 7.3 % (w/w) colloidal substances, and 2.1 % (w/w) nitrogen.milation  ), corn Batch fermentation was carried out at 37 °C in 250-mL flasks or a 5-L bioreactor  containing 2.5-L initial medium ) at 37 °C and airflow rate 0.66 vvm. The fed-batch (inoculated with 4 % v/v seed culture) was cultivated in a 5-L stirring bioreactor with a working volume of 2.5 l (15 g/l molasses). Glycerol at 80 % (w/v), or a solution of 80 % glycerol, 15 % molasses, and 5 % H2O, was fed into the bioreactor to maintain the glycerol concentration between 20 and 50 g/l from 5 to 88 h. Supplementation was ceased after 88 h to minimize the glycerol residue in the final broth .dhaD and gldA (encoding glycerol dehydrogenases) from K. pneumoniae ATCC 25955 were amplified by PCR technique using primers P1/P2 and P4/P5, respectively. The purified PCR products were separately double-digested by BamH I and Mlu I, and then ligated to the corresponding sites of the pMA5 plasmid. The recombinant plasmid pMA5-dhaD and pMA5-gldA were generated.The gene dhaD gene, containing the HapII promoter from the pMA5-dhaD plasmid, was then PCR-amplified using primers P2 and P3. The amplified HapII-dhaD gene was inserted into the Mlu I site of the previously constructed plasmid pMA5-acr [acr-HapII-dhaD plasmid. The PbdhA-alsR gene cut from the previously constructed plasmid pMA5-PbdhA-alsR [EcoR V and Hind III sites of the pMA5-acr-HapII-dhaD plasmid to create the pMA5-acr-HapII-dhaD-PbdhA-alsR plasmid. These constructed plasmids were isolated from E. coli JM109 and subsequently transformed into B. amyloliquefaciens according to published method [B. amyloliquefaciens was also tested as described in our previous study [The pMA5-acr  to creatdhA-alsR  was insed method . Furtherus study .2PO4, 2.2 mM Na2HPO4, and 8.5 mM NaCl; pH 7.4) and then suspended in this buffer. Cells were disrupted using a sonicator  for 20 min with chilling. The lysed cells were centrifuged at 12,000 rpm for 25 min at 4 °C, and the supernatant was used for enzyme assays. The ALS and ALDC assays were performed according to published procedures [The cell pellets collected by centrifugation were suspended and washed with 0.1 M potassium phosphate buffer (pH7.0) at least for three times. For determining DhaD or GldA activity , the celocedures .600 in a UV–visible spectroscopy system . The dry cell weight (DCW) was calculated from the optical density using a calibration curve for the strain. The composition of the fermentation broth  was determined by high-performance liquid chromatography (HPLC) [+ and NADH concentrations were measured by procedures described previously [The cell mass concentration was determined from the ODy (HPLC) . The inteviously . All assX, h−1), specific substrate consumption rate  and specific 2,3-BD formation rate  were estimated from experimental or fitted data of cell growth , residual substrate concentration , and 2,3-BD production  by Eqs. (The specific cell growth rate μX, h−, specifi


The Actionable questionnaire is an 8-item tool to screen patients with multiple sclerosis (MS) for neurogenic bladder problems, identifying those patients who might benefit from urological referral and bladder-specific treatment. The original scoring yields a total score of 0 to 24 with cut-off point 6. A simplified scoring, yielding a total score of 0 to 8 with cut-off point 3, has been developed in urogynaecological patients, but has not been investigated in MS.One-hundred-and-forty-one MS patients completed the Actionable on two occasions. We compared the test performance of the simplified scoring with cut-off point 3 with that of cut-off point 2, using the original scoring with cut-off point 6 as a gold standard. The following measures were calculated: True Positives (TP), True Negatives (TN), False Positives (FP), False Negatives (FN), Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Accuracy. The associations between positive test result and urological treatment, and bladder-specific drug treatment were calculated.P = 0.0331), which was not the case with cut-off 3. Only a positive test according to the original scoring was associated with both urological treatment (P = 0.0119) and bladder-specific medication (P = 0.0328).For cut-off point 3 the outcomes  were: TP 43.26 %, 40.88 %; TN 29.79 %, 32.85 %; FP 0.00 %, 0.00 %; FN 26.95 %, 26.28 %; Sensitivity 0.62, 0.61; Specificity 1.00, 1.00; PPV 1.00, 1.00; NPV 0.53, 0.55; Accuracy 0.73, 0.74; and for cut-off point 2: TP 59.57 %, 59.85 %; TN 26.95 %, 31.39 %; FP 2.84 %, 1.46 %; FN 10.63 %, 7.30 %; Sensitivity 0.85, 0.89; Specificity 0.90, 0.96; PPV 0.95, 0.98; NPV 0.72, 0.81; Accuracy 0.87, 0.91.  Cut-off 3 completely prevented FP outcomes, but wrongly classified 26 % of the patients as negative (FN). Cut-off 2 reduced the FN to 7–10 %, with low FP values (2.84–1.46 %). With cut-off 2, the percentage of patients screened positive was higher in the Progressive group (75.00 %) than in the Relapsing Remitting group (56.25 %) (Our findings suggest that in MS patients the simplified Actionable scoring is more accurate with cut-off point 2 than with cut-off point 3, especially by substantially reducing FN outcomes; and that in MS the original Actionable scoring seems preferable. The Actionable questionnaire is a short, 8-item, psychometrically validated instrument that was developed in response to the growing need to screen patients with multiple sclerosis (MS) for neurogenic bladder problems . The queAs reported by Bates et al.  the itemrd or 4th right hand box incurred a point, and consequently the Actionable score ranged from 0 (no checked 3rd or 4th right hand box) to 8 (8 checked 3rd or 4th hand boxes). It is conceivable that this simplified scoring system appeals to health care professionals using the Actionable questionnaire as a routine standard of care in their daily practice, as well as to patients who use the questionnaire for self-screening in the context of self-management. Interestingly, in the simplified scoring the cut-off point indicating a need for urological evaluation of bladder function was 3 ) age was 47.7 (10.40) years , the mean (SD) disease duration 10.1 (8.0) years , and the mean (SD) EDSS score 4.3 (1.8) . Eighty patients were relapsing remitting, 48 progressive and in 13 patients the disease course was unknown. Forty-five (31.91 %) patients were on bladder medication, whereas 96 (68.09 %) were not. Fifty-six (39.72 %) patients were seen by an urologist for overactive bladder symptoms, whereas 85 (60.28 %) were not. The Day 8 assessment was completed by 137 patients.The mean, SD, minimum and maximum values of the Actionable scores at Day 1 (Test 1) and at Day 8 Test 2) according to the original scoring system 0 to 24) and to the simplified scoring system (0 to 8) are presented in Table  accordin and to tFor each of the three modes of screening  the percentages of positive and negative patients that were treated by an urologist for MS-related bladder problems, and the percentages of patients that were taking medication for MS-related bladder problems are presented in Table P = 0.0121 and P = 0.0331, respectively). This was not the case with the use of the simplified scoring and cut-off 3 .When comparing the RRMS group with the progressive group, it was found that with the original scoring  and with the simplified scoring and cut-off 2, the percentage of patients that was screened positive was higher in the progressive group  than in the RRMS group   to the cut-off point 6 of the original scoring would result in a cut-off of 1.83–1.78 , which is indeed closer to 2 than 3.If the simplified scoring (0 to 8) of the Actionable questionnaire is used, in MS patients the cut-off point 2 is likely to be more accurate than the cut-off point 3, especially by preventing a considerable number of patients from being withheld referral to an urologist . Further, our data suggest that preferentially the Actionable item scoring should be as described originally by Bates et al., from 0 to 3, which results in a total score ranging from 0 to 24 . Future


Kidney Disease Improving Global Outcomes (KDIGO) 2013 updated the classification and risk stratification of chronic kidney disease (CKD) to include both the level of renal function and urinary albumin excretion (UAE). The update subclassifies the previous category of moderate renal impairment. There is currently limited information on the prevalence of CKD based on this new classification in United States (US) adults with type 2 diabetes mellitus (T2DM). The objective of this study was to provide such estimates, for T2DM both overall and in those ≥ 65 years of age. We used the continuous National Health and Nutrition Examination Survey (NHANES) 1999–2012 to identify participants with T2DM. Estimated glomerular filtration rate (eGFR) and UAE were calculated using laboratory results and data collected from the surveys, and categorized based on the KDIGO classification. Projections for the US T2DM population were based on NHANES sampling weights.2) was 22.0% overall and 43.1% in those age ≥ 65 years. Prevalence of more severe renal impairment (eGFR < 45 ml/min/1.73 m2) was 9.0% overall and 18.6% in those age ≥ 65 years. The prevalence of elevated UAE (> 30 mg/g) was 32.2% overall and 39.1% in those age ≥ 65 years. The most common comorbidities were hypertension, retinopathy, coronary heart disease, myocardial infarction, and congestive heart failure.A total of 2915 adults diagnosed with T2DM were identified from NHANES, with 1466 being age ≥ 65 years. Prevalence of CKD based on either eGFR or UAE was 43.5% in the T2DM population overall, and 61.0% in those age ≥ 65 years. The prevalence of mildly decreased renal function or worse (eGFR < 60/ml/min/1.73 mThis study confirms the high prevalence of CKD in T2DM, impacting 43.5% of this population. Additionally, this study is among the first to report US prevalence estimates of CKD based on the new KDIGO CKD staging system. Type 2 diabetes mellitus (T2DM) is the leading cause of chronic kidney disease (CKD) in the United States (US), with previous estimates suggesting close to 40% of patients with T2DM having evidence of CKD. ConcordBased on the significant impact of CKD, Kidney Disease Outcomes Quality Initiative (KDOQI) and Kidney Disease: Improving Global Outcomes (KDIGO) recommend a focus on early identification of CKD. This early identification may allow for treatment directed at CKD to slow or prevent progression, and treatment of associated complications and comorbidities such as cardiovascular disease,5.KDOQI originally proposed a uniform CKD staging and classification system based on estimated glomerular filtration rate (eGFR). The oriet al.[2 (Stage 3 CKD), these risks substantially increased when eGFR declined below 45 ml/min/1.73 m2[et al. advocated for subclassification of Stage 3 to reflect these findings. A recent meta-analysis provided further evidence to support the division of Stage 3 CKD into 3a and 3b based on the increased risk for progression to ESRD, and cardiovascular and all-cause mortality at the GFR threshold of < 45 ml/min/m2[Soon after the original KDOQI CKD staging system was published, Go et al. reportedn/1.73 m2. Based oml/min/m2.cause of CKD, gFR category, and albuminuria category (CGA). The cause category is based on the presence or absence of systemic disease. The GFR categories acknowledge the need to subdivide Stage 3 CKD, classified as G3 under the new classification system, into G3a (eGFR 45–59) and G3b (eGFR 30–44). The albuminuria categories are based on the presence and severity of albuminuria. This new classification was developed to allow risk stratification based on progression of CKD and cardiovascular and all-cause mortality.Based on this new evidence, developed since KDOQI, KDIGO recently updated the classification system for CKD. This neThe prevalence of diabetes in the older adult population (age > 65) is greater than 20%. This older adult group also has a higher prevalence of comorbid conditions  compared to younger populations. Based oTo date, there are no US estimates of the prevalence of CKD in T2DM based on this new classification system, particularly for stages G3a and G3b. The objective of this study was to provide current national estimates of the prevalence of CKD in the overall T2DM population, and in the subpopulation aged ≥ 65 years using this new KDIGO classification.National Health and Nutrition Examination Survey (NHANES) 1999–2012 data were used for this study. NHANES is a cross-sectional survey designed to monitor the health and nutritional status of the civilian non-institutionalized US population. NHANES NHANES participants with T2DM were identified using previously published criteria to differentiate T2DM from type 1 diabetes mellitus (T1DM),12. The Comorbidities were determined based on participant self-reporting of diagnosis by a doctor or other health-care professional. The identification of hypertension was additionally based on measured mean systolic blood pressure/diastolic blood pressure > 140/90 mmHg, or self-reported use of antihypertensive agents, including diuretics, beta-blockers, calcium-channel blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, or other antihypertensive agents .To ensure comparability with standard lab assays and consistency when testing methods or instruments change across different survey cycles, NHANES conducts quality assurance and monitoring, and provides specific calibration recommendations in analytical notes for affected lab tests and survey cycles. Serum c2 , G2 60–89 ml/min/1.73 m2 (mildly decreased), G3a 45–59 ml/min/1.73 m2 (mildly to moderately decreased), G3b 30–44 ml/min/1.73 m2 (moderately to severely decreased), G4 15–29 ml/min/1.73 m2 (severely decreased), and G5 < 15 ml/min/1.73 m2 (kidney failure). Albuminuria categories were based on the UACR: A1 < 30 mg/g , A2 30–300 mg/g (moderately increased), and A3 > 300 mg/g (severely increased). eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation[CKD was categorized based on the new KDIGO classification recommendations. GFR catequation. BecauseTo provide an estimate of the US prevalence of CKD in the overall and age ≥ 65 years T2DM population, the observations from the NHANES were weighted to account for the complex design of NHANES and age-A total of 2915 adults with T2DM were identified from NHANES 1999–2012. We combined the NHANES 1999–2012 data because none of the linear trend tests for eGFR and UACR were statistically significant, and numerical values also appeared comparable across these survey cycles (data not shown). The sample demographics and projected national estimates are presented in Table 2, and the mean (SE) urinary albumin excretion (UAE) was 109.1 (1.4) mg/g. The overall prevalence of CKD based on either eGFR (CKD-EPI) or albuminuria criteria was 43.5%, with 95% confidence interval (CI) 41.6-45.4%. The prevalence of mildly decreased to more severe renal impairment based on eGFR below the threshold of 60 ml/min/1.73 m2 was 22% (95% CI 20.4-23.5%). Based on the new subdivision of stage 3 CKD into 3a/3b, the prevalence of Stage 3a CKD (eGFR > 45–59 ml/min/1.73 m2) was 12.9% (95% CI 11.5-14.3%) (Table 2) was 9% (95% CI 7.8-10.3%). The prevalence of elevated UAE based on UACR ≥ 30 mg/g was 32.2% (95% CI 30.0-34.3%), 24.4% (95% CI 22.3-26.4%) with moderately increased (UACR ≥ 30–300 mg/g) and 7.8% (95% CI 6.5-9.1%) with severely increased (UACR > 300 mg/g) UAE.The national estimate of mean (SE) eGFR (CKD-EPI) for the T2DM population was 80.5 (0.5) ml/min/1.73 m2. The overall prevalence of CKD using either eGFR (MDRD) or albuminuria criteria was 45.1%, 95% CI 43.0-47.1%. The prevalence of mildly decreased to more severe renal impairment based on eGFR below the threshold of 60 ml/min/1.73 m2 was 24.0%, 95% CI 22.1-26.0%; 14.9% (95% CI 13.2-16.6%) had eGFR (MDRD) 45–59 ml/min/1.73 m2  eGFR was 79.1 (0.6) ml/min/1.73 mA total of 1466 NHANES participants were aged ≥ 65 years, representing 50.3% of the analysis sample. The projected mean (SE) age for this population was 73.4 (0.1) years, and the mean (SE) diabetes duration was 12.9 (0.3) years. Non-Hispanic whites comprised 72.6% of the study population age ≥ 65 years, non-Hispanic blacks 12.4%, and Mexican Americans 5.0%. The most common self-reported comorbidities were hypertension (80.9%), retinopathy (22.2%), coronary heart disease (19.9%), myocardial infarction (18.2%), and congestive heart failure (16.9%).2 was 43.1% (95% CI 39.5-46.6%). Stage G3a (eGFR 45–59 ml/min/1.73 m2) represented 24.5% (95% CI 21.6-27.4%) (Table 2 was 18.6% (95% CI 15.9-21.2%). The prevalence of increased UAE based on UACR ≥ 30 mg/g was 39.1% (95% CI 36.3-41.8%). Moderately increased UAE (ACR 30–300 mg/g) was present in 29.3% (95% CI 26.4-32.2%), and severely increased UAE (ACR > 300 mg/g) was present in 9.8% (95% CI 7.5-12.0%).For the age ≥ 65 years subgroup, the prevalence of CKD using either eGFR (CKD-EPI) or albuminuria criteria was 61.0%, 95% CI 57.9- 64.1%). The prevalence of mildly decreased to more severe renal impairment based on eGFR (CKD-EPI) below the threshold of 60 ml/min/1.73 m.1%. The 2. The overall prevalence of CKD using either eGFR (MDRD) or albuminuria criteria was 61.8% (95% CI 58.5-65.0%). The prevalence of mildly decreased to more severe renal impairment based on eGFR (MDRD) below the threshold of 60 ml/min/1.73 m2 was 44.2% (95% CI 40.3-48.1%); 26.1% had eGFR 45–59 ml/min/1.73 m2 (95% CI 22.9-29.3%)  eGFR was 64.8 (0.8) ml/min/1.73 met al.[et al. report was based on a smaller sample size (the more recent NHANES data were not available at the time of their study) with a lower mean age, and did not attempt to exclude T1DM or those diagnosed with T2DM age < 30 years. A previous report by Coresh et al.[Based on a nationally representative sample, this report is the first to provide estimates of the prevalence of CKD in the US population with T2DM based on the new KDIGO classification. These new estimates provide insights on the prevalence of the GFR categories G3a and G3b and the albuminuria categories. A recent report by Plantinga et al. using NHsh et al. observedAnother recent report on the prevalence of CKD in diabetes based on NHANES 2005–2010 observed a lower prevalence of CKD[Consistent with previous reports, the overall prevalence of CKD based on eGFR was lower using CKD-EPI compared to MDRD,22. BaseThe prevalence of CKD in the age ≥ 65 years population was higher than in the overall T2DM population. This is consistent with previous reports of increasing prevalence of CKD and inciet al.[The recognition of the presence of CKD and classification based on the severity of CKD was first proposed by KDOQI in 2002. The basis of the staging was to provide stage-specific treatment recommendations,5 and alet al.. Consideet al.,26. Mediet al.. Other met al.. This cuThe information related to the T2DM population aged ≥ 65 years may be relevant to those who provide care and make decisions specifically for this population, such as Medicare Part D pharmacy benefit plans. The relevance of this information in this older adult group is highlighted in the ADA 2014 standards of care, where tThis study has several limitations. We focused specifically on T2DM, and therefore made an attempt to exclude NHANES participants with T1DM based on previously reported methodologies. The empirical algorithm based on age at diabetes onset and early initiation of insulin therapy may exclude younger-onset T2DM patients and include older-onset T1DM patients. The determination of the presence of kidney disease based on eGFR and albuminuria was made based on a single random sample of the laboratory values, so the chronicity of the observations could not be determined. The cause of CKD could not be determined based on the data collected in NHANES, so we could not categorize CKD according to the cause category of the new KDIGO classification. Due to the cross-sectional nature of NHANES, outcomes such as CKD progression and mortality could not be assessed.2, impacts < 10% of T2DM patients. This study also provides estimates of the distribution of albuminuria by severity within the categories of eGFR, providing insights into the distribution of risk categories for progression to ESRD and mortality. Additionally, this study provides estimates of the prevalence of comorbidities such as hypertension, retinopathy, and cardiac disease in people with T2DM, based on a nationally representative sample. This information may be useful to clinicians, policy makers, and entities focused on population health management.This study confirms the high prevalence of CKD in T2DM, with 43.5% of individuals having evidence of CKD, and provides insights into prevalence of all CKD categories based on the new KDIGO staging system. The prevalence of more severe impairment of renal function, defined as eGFR < 45 ml/min/1.73 mCause of CKD, GFR category, and Albuminuria category; CI: Confidence interval; CKD: Chronic kidney disease; CKD-EPI: Chronic Kidney Disease Epidemiology Collaboration; eGFR: Estimated glomerular filtration rate; ESRD: End-stage renal disease; IDMS: Isotope dilution mass spectrometry; KDIGO: Kidney Disease: Improving Global Outcomes; KDOQI: Kidney Disease Outcomes Quality Initiative; MDRD: Modification of Diet in Renal Disease; NHANES: National Health and Nutrition Examination Survey; NHIS: National Health Interview Survey; T1DM: Type 1 diabetes mellitus; T2DM: Type 2 diabetes mellitus; UAE: Urinary albumin excretion; UACR: Urinary albumin to creatinine ratio; US: United States.ACE: Angiotensin-converting enzyme; ADA: American Diabetes Association; CGA: RAB and MFTR are employees of Janssen Scientific Affairs, LLC, Raritan, NJ. YW and VZ are employees of Janssen Research & Development, LLC, Titusville, NJ. All authors are stockholders of Johnson and Johnson.RAB conceived the study concept, participated in the design of the study, participated in the interpretation of the data, and led the development of the manuscript. YW conceived the study concept, led the design of the study, helped with the statistical analysis, and helped in the development of the manuscript. VZ participated in the design of the study, performed the statistical analysis, and helped in the development of the manuscript. MFTR participated in the design of the study and the interpretation of the data, and helped in the development of the manuscript. All authors have read and approved the final manuscript and have participated in alignment with the ICMJE standards.


In brief, fosfomycin, levofloxacin, and rifampin alone or in combination with linezolid were tested in vitro against planktonic and biofilm-embedded MRSA infection in three MRSA stains. The synergistic effects between linezolid and the other three antibiotics were assessed by fractional inhibitory concentration index (FICI) and time-kill curves, where the combination of linezolid plus fosfomycin showed the best synergistic effect in all strains. For further evaluation in vivo, we applied the combination of linezolid and fosfomycin in a catheter-related biofilm rat model and found that viable bacteria counts in biofilm were significantly reduced after treatment (P < 0.05). In summary, we have shown here that the combination of linezolid and fosfomycin treatment had improved therapeutic effects on biofilm-embedded MRSA infection both in vitro and in vivo, which provided important basis for new clinical therapy development.As long-standing clinical problems, catheter-related infections and other chronic biofilm infections are more difficult to treat due to the high antibiotic resistance of biofilm. Therefore, new treatments are needed for more effective bacteria clearance. In this study, we evaluated the antibacterial activities of several common antibiotics alone and their combinations against biofilm-embedded methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen that causes many catheter-related infections and other chronic biofilm infections [Methicillin-resistantfections . Vancomyfections . With hifections . Escherichia coli. In another study, Oliva et al. [ Enterococcus faecalis biofilm.To solve this problem, a potential way is to reassess the older generations of antibiotics and investigate the novel combinations of existing agents . For exaa et al.  showed t Enterobacteriaceae and >90% multidrug resistant P. aeruginosa [ in vitro [ in vivo studies need to be conducted to evaluate the potential of linezolid combination regimens in clinical treatments against biofilm-associated MRSA infections. In our study, the effects of fosfomycin, rifampin, and levofloxacin alone or in combination with linezolid on viable bacterial counts in biofilms were evaluated both in vitro and in vivo, which provided thorough information about the antibiofilm effects of linezolid combination regimens.Linezolid is the first synthetic oxazolidinone antibiotic that blocks protein synthesis by preventing the formation of the initiation complex. It has been approved as an alternative drug for the treatment of staphylococcal skin and soft-tissue infections or pneumonia . Chronicruginosa . For MRSruginosa . Linezolin vitro , 9, 13.  in vitro [ in vitro and in vivo studies. The strains were clinically isolated from PLA General Hospital and were identified by the automated VITEK-2 system  with a rapid latex agglutination test. Staphylococcus aureus ATCC 25923 was used as the quality control strain.In the previous study, we found 98.04% synergy of the combination of linezolid and fosfomycin against 102 planktonic MRSA strains by fractional inhibitory concentration index (FICI)in vitro . Therefo in vitro study. Fosfomycin sodium  and linezolid  were used in the biofilm-infection rat model.Fosfomycin, rifampin, and levofloxacin  were used for the5 cfu/mL MRSA prepared in broth. Three MRSA strains  were determined in FIC assay. The plates were incubated at 37°C for 24 hours and visually inspected for turbidity to determine the growth. Experiments were performed in triplicate. The interactions between two tested antimicrobials were evaluated by FICI, calculated as follows: FICI =  + . The FICI was interpreted as follows: FICI ≤ 0.5, synergy; 0.5 < FICI ≤ 4.0, indifference; FICI > 4.0, antagonism [Synergy of antibiotics was assessed using the checkerboard broth microdilution method assays as described previously . In brie5 cfu/mL in 10 mL of Muller-Hinton Broth (MHB)  for inoculation. The concentrations of fosfomycin were adjusted to 1/16x (4 mg/L), 1/8x (8 mg/L), 1/4x MIC (16 mg/L), and 1/2x MIC (32 mg/L). Each concentration was tested alone or in combination with 1/2x MIC (1 mg/L) linezolid for time-kill curve assays. Bacterial counts were done at 0, 2, 4, 8, 12, and 24 h by spreading 10-fold serial dilutions onto Muller-Hinton agar plates . The experiments described above were repeated three times. Synergy was defined as more than 2 lg 10 cfu/mL decrease between the combination and its most active constituent after 24 h (at least one of the drugs must be present at a concentration that does not affect the growth curve of the tested organism), and the number of viable organisms in the presence of the combination must be ≥2 lg 10 cfu/mL below the starting inoculum [Time-kill experiments were performed on MRSA 154311 according to the Clinical and Laboratory Standards Institute (CLSI) methodology . Briefly8 cfu/mL). 2 mL of MHB II and 100 μL of bacterial suspension were added to each well containing 3 catheter disks . The plates were then incubated at 37°C for 5 days, and MHB II was renewed daily. The catheter disk with biofilm model used in this study was based on several published articles that focused on antibiofilm effects of some antimicrobial agents [μL linezolid at 1/2x MIC, 1/4x MIC, and 1/8x MIC, together with fosfomycin or levofloxacin at 1/2x MIC, 1/4x MIC, and 1/8x MIC, and rifampin at a fixed concentration of 1 mg/L, according to previous studies [ in vivo study in biofilm-infection rat model. Each treatment had six catheters. Bacteria counts were repeated three times.Synergy test was performed on all MRSA strains. Biofilm was cultivated on disks in 24-well plates. Bacterial suspension was adjusted to 0.5 McFarland . Each catheter was placed into a 5 mL tube containing 3 mL MHB for culture. 100 μL MRSA suspension at 0.5 McFarland which includes 1.5 × 107 bacteria cells was inoculated into each tube (5 × 105 cfu/mL) and cultured for 5 days at 37°C. The catheters were the same as those used in in vitro experiments. Twenty-eight Wistar male rats, weighing 200–250 g, were anesthetized with intraperitoneal injection of 4% chloral hydrate at 0.01 mL/g. Then the catheters with biofilms were rinsed with sterile physiological saline solution and implanted at dorsal midline, as previously described [ in vitro synergy test. Real-time monitoring of WBCs changes in rats reflected infection conditions. Twenty microliters of blood was drawn from the tail vein of each rat before infection and at days 1, 3, and 5 during therapy. WBCs were examined using an animal blood analytical instrument . The data are presented as mean ± standard deviation (SD) (n = 7).MRSA 154311 biofilm was grown on catheters . SPSS 12.0 was used for statistical analyses. Differences with P < 0.05). In addition, 1/2x MIC (1 mg/L) of linezolid combined with 1/4x MIC (16 mg/L) or 1/2x MIC (32 mg/L) of fosfomycin exhibited synergistic effect. These results indicated that the linezolid-fosfomycin combination had improved efficiency against planktonic MRSA.The MICs and FICI of the tested antimicrobial agents were summarized in  in vitro results, we chose the combination of linezolid plus fosfomycin and further investigated its antibacterial effect against strain 154311 MRSA biofilm in vivo.P < 0.05), and LIN + FOS group, with the colony counts reduced by 4.5 lg cfu/mL, showed significant lower colony numbers than any other groups (P < 0.05).At 7 days after infection, the colony counts in biofilms of control, LIN, FOS, and LIN + FOS groups were 7.46 ± 0.35, 5.97 ± 0.97, 5.26 ± 0.29, and 2.98 ± 0.40 lg cfu/mL, respectively. Both linezolid and fosfomycin alone showed decreased colony counts compared to control group  were evaluated before infection and at 1, 3, and 5 days after infection. WBC results were as follows: control group: 8.15 ± 2.21 (100%), 6.88 ± 2.08 (100%), 10 ± 2.1 (100%), and 16.42 ± 4.89 (33.33%); LIN: 10.09 ± 3.04 (100%), 9.44 ± 4.64 (100%), 11.2 ± 2.58 (100%), and 14.54 ± 2.61 (71.43%); FOS: 8.30 ± 1.74 (100%), 8.40 ± 2.35 (100%), 9.81 ± 1.27 (100%), and 16.2 ± 2.11 (57.14%); LIN + FOS: 9.01 ± 3.11 (100%), 9.96 ± 2.57 (100%), 9.96 ± 2.05 (100%), and 13.47 ± 1.96 (85.71%). WBCs of the four groups were all continuously increasing from day 0 to day 5. At day 5, the WBCs of LIN + FOS group was 13.47 ± 1.96, lower than the other groups. But no significant difference among groups was observed.To examine the effects of the drugs on inflammation of the biofilm-infected rats, the values and normal rate of WBCs (defined as the number of rats whose WBCs counts ranged from 2.9 to 15.3 × 10 in vitro, compared with other combinations. A synergistic effect of linezolid and fosfomycin has been observed in vitro in previous studies. Pachón-Ibáñez et al. evaluated the efficacy of the combination of fosfomycin and linezolid against Staphylococcus aureus strain using time-kill curve. Compared with the initial bacterial counts, a nearly 3 lg cfu/mL reduction at 24 h time point was observed from the treatment of 1x MIC linezolid plus 1x MIC fosfomycin [In this study, the combination of linezolid and fosfomycin displayed the most effective antibacterial effect against MRSAsfomycin . Similar in vitro. Several mechanisms have been proposed to explain why only a few antibiotics are capable of fighting against biofilms. Fosfomycin has been reported to be involved in some combination therapies that showed increased therapeutic effects. The low molecular weight of fosfomycin could partially explain its facilitated effect in antibacterial activity against biofilm-related organisms [Our study also demonstrates that linezolid plus fosfomycin is an effective combination against MRSA biofilmrganisms . Fosfomyrganisms . In addi in vivo studies, Mihailescu et al. and Baldoni et al. [Fori et al. , 23 usedi et al. .White blood cells are a part of the immune system that helps body fight infection. The endotoxins produced by bacteria may elicit immune responses , which rAs reported previously, high concentrations of antibiotics might be necessary to treat biofilm-related infections. However, high doses of linezolid or fosfomycin can cause some adverse effects, such as thrombocytopenia and peripheral neuropathy . The tre in vitro and in vivo. These results provided important basis for developing new regimens to treat patients with biofilm-associated MRSA infections, especially for catheter-related infections.In conclusion, we found linezolid plus fosfomycin combination showed antibiofilm effect against MRSA strains both


Its effects on angiogenesis in cervical cancer- (CaSki-) implanted nude mice and its mechanisms on hypoxia-inducible factor-1α and vascular endothelial growth factor expression were investigated. Female BALB/c nude mice were divided into control (CON) and CaSki-implanted groups (CaSki group). One month after the injection with cervical cancer cells, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. The microvascular density (MVD) was evaluated using the CD31 expression. VEGF, VEGFR-2, and HIF-1α expression were also detected by immunohistochemistry. The MVD in CaSki + vehicle group was significantly increased compared to the CON + vehicle group. Interestingly, when treated with THC at all doses, the CaSki group showed a significant smaller number of the MVD. The CaSki + vehicle group also showed significantly increased VEGF, VEGFR-2, and HIF-1α expressions, but they were downregulated when mice were treated with THC at all doses. THC demonstrated an inhibitory effect against tumor angiogenesis in CaSki-implanted nude mice model. This effect is likely to be mediated by the downregulation of HIF-1-α, VEGF expression, and its receptor. THC could be developed into a promising agent for cancer therapy in the future.Tetrahydrocurcumin (THC), one of the important  Cervical tumors frequently have a high vascular density which is responsible for spontaneous bleeding, a common symptom of cervical cancer. There is an urgent need for the development of novel agents to combat this type of cancer. Antiangiogenic agents have been identified as promising treatments that may enhance therapeutic outcomes when used in combination with chemotherapy. Currently, there are a number of biological agents in clinical development that inhibit tumor angiogenesis by targeting vascular endothelial growth factor (VEGF) and its signaling.α (HIF-1α) [VEGF, also known as vascular permeability factor (VPF), is a major mediator of tumor angiogenesis and is tightly controlled by oxygen tension . Hypoxia(HIF-1α) . VEGF pr(HIF-1α) . VEGF ca(HIF-1α) . With th(HIF-1α)  which in(HIF-1α) . VEGF wa(HIF-1α) , 8. Our (HIF-1α) .VEGF binding sites were identified on vascular endothelial cells and called VEGFR-1 (Flt-1)  and VEGF Curcuma longa. Pharmacological outcomes of CUR have been widely reported. Our earlier study showed that CUR had anticancer and antiangiogenic effects in cervical cancer xenografts in nude mice [β-diketo moieties similar to CUR [α and VEGF/VEGFR-2 pathway.Curcumin (diferuloylmethane) (CUR) is a phenolic compound from the plantude mice . Howeverr to CUR . We alsor to CUR , and thi2 in a humidified atmosphere.Cervical cancer cells (CaSki) were purchased from the American Type Culture Collection. The cell lines were cultured in an MEM medium supplemented with 10% fetal bovine serum. All cultures were maintained in an incubator at 37°C with 5% COn = 6), (2) controls supplemented with THC (500 mg/kg) , (3) CaSki-implanted mice supplemented with corn oil , (4) CaSki-implanted mice supplemented with THC (100 mg/kg) , (5) CaSki-implanted mice supplemented with THC (300 mg/kg) , and (6) CaSki-implanted mice supplemented with THC (500 mg/kg) .BALB/c-nude female mice weighing about 20–25 g were used. The animal experiments were conducted according to the guidelines on experimental animals of The National Research Council of Thailand (1999). The mice were divided into 6 groups: (1) controls supplemented with corn oil . When the tumor volume was 100–120 mm3, the mice were randomized. Following this, the mice were supplemented daily with vehicle or THC at the doses of 100, 300, or 500 mg/kg body weight for one month.For the CaSki groups, a suspension of 10 × 102 mL MEM  was subc i.p). Fluorescence tracers 0.1 mL of 0.5% fluorescein isothiocyanate- (FITC-) labeled dextran  were injected into the jugular vein. The tumor microvasculature was visualized under a confocal microscope.On study day 30, the mice were anaesthetized with sodium pentobarbital  for 30 min at room temperature. The tissue slide samples were incubated with primary monoclonal antibody CD31  (1 : 500) at 4°C overnight. The slide was then gently rinsed with PBS and developed by the Envision system/HRP  for 30 min and substrate-chromogen for 10 min at room temperature. The nuclei were counterstained with Mayer's hematoxylin.After the microvascular study, the mice were sacrificed and the tumors were fixed in 10% formalin. Immunohistochemistry was performed using 5 2).To quantify angiogenesis, microvessel density (MVD) was assessed by immunostaining with the anti-CD31 antibody as previously described . The sec∞   at 4°C overnight. The slide was then gently rinsed with PBS and developed by the Envision system/HRP  for 30 min and substrate-chromogen for 10 min at room temperature. The nuclei were counterstained with Mayer's hematoxylin.The tissue slide samples were incubated with primary monoclonal antibody VEGF  (1 : 75), antibody VEGFR-2 (1 : 250) , and antibody HIF-1∞ were analyzed. Staining intensity (I) was scored as 0 (none), 1+ (weak), 2+ (moderate), and 3+ (strong). The percentage of positive stains (P) was scored as 0, 0% immunopositive cells; 1, ≤25% positive cells; 2, 26–50% positive cells; 3, ≥51–75% positive cells; 4, ≥76%. The sum of both (I) and (P) scores was evaluated for each case and a final score was assigned 0 (negative), 1–3 (weak expression), 4-5 (moderate expression), and 6-7 (strong expression) [The sample image from each slide was subtracted from the corresponding background image. The image threshold was standardized to delineate the labeled structures and then applied to all images of an individual experiment. The staining intensity and the percentage of positive stained VEGF, VEGFR-2, and HIF-1ression) .t-test was applied for comparison of the means of two groups (Control and CaSki + vehicle groups), and analysis of variance was used for the means of multiple groups. The correlation between CD31 expressions and VEGF, VEGFR-2, and HIF-1∞ was assessed with Pearson correlation test. For all of the value differences, P value less than 0.05 was considered significant.Data were expressed as means with standard error. SPSS.13 software was used for statistical analysis. Student's unpaired The confocal fluorescent images of the microvasculature for controls (a and b), CaSki + vehicle group (c), and CaSki + THC groups (d–f) are demonstrated in A detection of the CD31 antigen expression was used to determine the quantity of the angiogenesis process. CD31 primarily indicated the presence of endothelial cells in histological tissue sections. P < 0.001). The MVD was significantly decreased by all doses of treatments with THC  .We measured the microvascular density (MVD), as determined by CD31 staining, to assess the quantity of the angiogenesis process. In Figures P < 0.001).Figures P < 0.001). VEGF in CaSki + THC100 was 31.17 ± 0.95%, in CaSki + THC300 was 33.33 ± 1.45%, and in CaSki + THC500 was 29.67 ± 1.33%. In the same fashion, the percentage of positive staining of VEGFR-2 was also attenuated by the treatments with THC ).In all CaSki + THC groups, the percentage of positive staining of VEGF was significantly lower than in the CaSki + vehicle group . Similarly, staining intensity scores for VEGFR-2 expression were significantly reduced in all treated groups which were 1.50 ± 0.22, 1.50 ± 0.22, and 1.33 ± 0.21 in CaSki + THC300, CaSki + THC500, and CaSki + THC300 groups, respectively (P < 0.001).Staining intensity scores for VEGF expression were significantly reduced in CaSki + THC100 (mean score = 1.33 ± 0.21), CaSki + THC300 group (mean score = 1.33 ± 0.21), and CaSki + THC500 group (mean score  = 1.17 ± 0.17)  whereas the control group had weak VEGF expression . The total scores for VEGFR-2 in the CaSki + vehicle group also had strong VEGFR-2 expression  whereas the control group had weak VEGFR-2 expression .In the treated groups, the total scores for VEGF expression revealed weak expressions in CaSki + THC100 , CaSki + THC300 , and CaSki + THC500 groups . The total scores for VEGFR-2 expression also showed weak expression in all treated groups with the mean total scores of CaSki + THC100, CaSki + THC300, and CaSki + THC500 at 3.50 ± 0.22, 3.50 ± 0.22, and 3.00 ± 0.00, respectively.α expression was found in the CaSki + vehicle group (α expression. The quantitative data showed the percentage of positive staining of HIF-1α expression significantly increased in the CaSki + vehicle group (95.67 ± 0.42%) compared to the control group (16.67 ± 0.95%) (P < 0.001) .Overall, an increase in both nuclear and cytoplasmic HIF-1le group . Again, < 0.001) . The perα expression in CaSki + vehicle group was high (mean score = 2.97 ± 0.03) but was low (mean score = 1.12 ± 0.05) in the control group (α expression were significantly reduced in CaSki + THC100 (mean score = 1.30 ± 0.19), CaSki + THC300 (mean score = 1.39 ± 0.19), and CaSki + THC500 (mean score = 1.25 ± 0.17) groups (P < 0.001).The intensity score of HIF-1ol group . Howeverα expression showed that the CaSki + vehicle group had strong HIF-1α expression  whereas the control group had weak HIF-1α expression . The HIF-1α expression extent and intensity scores in the treated group revealed that weak expressions were found in CaSki + THC100 , CaSki + THC300 , and CaSki + THC500 groups , respectively.The total score for HIF-1r = 0.96 and r = 0.92, resp., P < 0.001) as well as the respective intensity scores . Furthermore, the percentage of positive cells and intensity scores for VEGF expression were positively correlated with both VEGFR-2 ) and HIF-1α expression ).The percentage of stained positive cells for both VEGF and VEGFR-2 expression was positively correlated with MVD in CaSki-implanted mice  implanted nude mice and study the mechanisms of THC on hypoxia-inducible factor-1r = 0.96). These results were similar to our previous finding that VEGF played an important role in tumor biological behavior and neovascularization in cervical cancer-implanted mice models [VEGF plays a pivotal role in the control of angiogenesis as well as tumor growth and metastasis , 20. Oveα under hypoxia conditions [2 availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. During tumor progression, the tumor mass develops hypoxic zones where HIF-1α can induce the accumulation of VEGF proteins [α significantly increases in CaSki-implanted mice. Moreover, we also found that HIF-1α expression strongly correlated with VEGF expression (r = 0.99). Lee and his coworkers demonstrated that a hypoxic zebrafish tumor model offers hypoxia-induced angiogenesis in promoting tumor cell dissemination and metastasis [The expression of VEGF is controlled by HIF-1nditions . HIF-1 iproteins . In the tastasis . Howeverγ-PKC-MAPK pathway for signaling [r = 0.98). Therefore, the VEGF-VEGFR-2 system is an important target for antiangiogenic therapy in cancer.Once VEGF binds to its high affinity receptors , it promotes a signaling cascade that ultimately produces increased endothelial cell survival, proliferation, vascular permeability, migration, and invasion , 20. Altignaling . The preAntiangiogenic therapy of cancers offers the possibility of a low toxicity treatment without acquisition of drug resistance due to the genetic stability, homogeneity, and low mutational rate of endothelial cells . The curα expression (Our previous study found that CUR was effective at doses of 1,000 mg/kg to inhibit tumor growth and angiogenesis in CaSki-implanted mice models . Interespression . This su in vivo, and it may play a crucial role in CUR-induced biological effects. In contrast to CUR, THC is stable in phosphate buffer and in saline at various pH values [α, and VEGF as well as VEGFR-2 protein expression. These results clearly suggest that THC inhibits tumor angiogenesis by downregulation of HIF-1-α, VEGF/VEGFR-2 pathway.THC is one of the major metabolites of CURH values . Interes in vivo, markedly inhibited tumor angiogenesis in CaSki-implanted female nude mice models by downregulation of HIF-1-α, VEGF/VEGFR-2 pathway. THC should be further investigated for its potential as a candidate for the development of an additional and/or alternative agent for the treatment of human cervical cancer.Our study shed new light on our understanding of the mechanisms of THC in cervical cancer intervention. We demonstrated that THC, one of the active anticancer forms of CUR