Bifidobacterium right after treatment (p = 0.027) till postnatal week three (p = 0.028). Long treatment caused Bifidobacterium abundance to remain decreased till postnatal week six (p = 0.009). Antibiotic treatment was effective against members of the Enterobacteriaceae family, but allowed Enterococcus to thrive and remain dominant for up to two weeks after antibiotic treatment discontinuation. Community richness and diversity were not affected by antibiotic treatment, but were positively associated with postnatal age (p < 0.023) and with abundance of Bifidobacterium (p = 0.003). Intravenous antibiotic administration during the first postnatal week greatly affects the infant’s gastrointestinal microbiota. However, quick antibiotic treatment cessation allows for its recovery. Disturbances in microbiota development caused by short and, more extensively, by long antibiotic treatment could affect healthy development of the infant via interference with maturation of the immune system and gastrointestinal tract.Antibiotic treatment is common practice in the neonatal ward for the prevention and treatment of sepsis, which is one of the leading causes of mortality and morbidity in preterm infants. Although the effect of antibiotic treatment on microbiota development is well recognised, little attention has been paid to treatment duration. We studied the effect of short and long intravenous antibiotic administration on intestinal microbiota development in preterm infants. Faecal samples from 15 preterm infants (35 ± 1 weeks gestation and 2871 ± 260 g birth weight) exposed to no, short (≤ 3 days) or long (≥ 5 days) treatment with amoxicillin/ceftazidime were collected during the first six postnatal weeks. Microbiota composition was determined through 16S rRNA gene sequencing and by quantitative polymerase chain reaction (qPCR). Short and long antibiotic treat ment significantly lowered the abundance of The online version of this article (10.1007/s10096-018-3193-y) contains supplementary material, which is available to authorized users. Over the last several years, the intestinal microbiota has been well recognised as a major contributor to human health and disease . DevelopEscherichia coli are associated with onset of neonatal sepsis [Antibiotic treatment is common practice in the neonatal ward for the prevention and treatment of sepsis, which is one of the leading causes of mortality and morbidity in preterm infants. Antibiotics, such as amoxicillin, ceftazidime, erythromycin and vancomycin, are frequently used as they target a broad spectrum of pathogens. Intrauterine infections are a common cause of preterm birth; thus, many preterm infants are born with suspicion of infection and are, therefore, treated with antibiotics from birth onwards. In preterm infants, group B streptococci and l sepsis . Since sThe applied antibiotic strategies in neonatology led to decreased mortality and morbidity rates. However, there is a risk of impeding gut microbiota development and increasing antibiotic resistance . It has This study was part of an observational, non-intervention study involving (pre)term infants admitted to the hospital level III NICU or the level II neonatal ward of Isala in Zwolle, the Netherlands. The ethics board from METC Isala Zwolle concluded that this study does not fall under the scope of the Medical Research Involving Human Subjects Act (WMO). Informed consent was obtained from both parents of all individual participants included in the study. For faecal microbiota profiling, 15 late preterm infants  were longitudinally sampled during the first six postnatal weeks, resulting in a total of 95 samples. Sampling days for each infant can be found in Table DNA was extracted from faeces by the repeated bead beating plus phenol/chloroform method, as described previously . DNA wasBifidobacterium-optimised 357F and 926Rb primers, as described previously [http://www.ebi.ac.uk/ena) under study accession PRJEB19937.Amplification of the V3–V5 regions of the 16S rRNA gene was performed using the eviously . For eaceviously . SequencPyrosequencing data were analysed using the QIIME software package (v1.8)  applyingU-test and Kruskal–Wallis test were applied for dependent, two groups of independent and more than two groups of independent samples, respectively. To study (dis)similarities in microbiota composition and relate changes in microbiota composition to clinical data, principal component analysis (PCA) and redundancy analysis (RDA) were performed using the Canoco multivariate statistics software v5. For RDA, factors were considered significant when the Bonferroni-corrected p-value was below 0.05. Co-occurrence patterns were determined by Spearman correlation using the taxa that remained after the OTU table was filtered for OTUs present in less than 25% of the samples. Visualisation was done using the Gephi-0.9.1 platform (https://gephi.org) and Adobe Illustrator CS6.To identify bacterial taxa that were significantly different in abundance between control, ST and LT infants, the non-parametric Kruskal–Wallis test with Monte Carlo permutation  was applied. The Kruskal–Wallis test was done using absolute read counts for each taxonomic group and after the OTU table was filtered for OTUs present in less than 25% of the samples. To compare richness and diversity between samples, the Wilcoxon signed-rank test, the Mann–Whitney Bifidobacterium [Enterococcus [Bifidobacterium and Enterococcus was initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 20 s and elongation at 72 °C for 50 s, followed by a melt-curve from 60 °C to 95 °C with 0.5 °C steps. The program for amplification of Enterobacteriaceae was initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55.8 °C for 20 s and elongation at 72 °C for 20 s, followed by a melt-curve from 60 °C to 95 °C with 0.5 °C steps. Standard curves contained 101–109 16S rRNA copies/μL and were performed in triplicate.Real-time PCR amplification and detection were performed on a CFX384™ real-time PCR detection system (Bio-Rad). The reaction mixture was composed of 5 μL iQ™ SYBR® Green Supermix, 0.2 μL forward and reverse primers 10 nmol), 1.6 μL nuclease-free water and 3 μL of DNA template (2 ng/μL). Primers used targeted total 16S , Bifidob nmol, 1.rococcus  and Enterococcus . The proBifidobacterium, Enterococcus and Enterobacteriaceae, respectively. To identify bacterial taxa that were significantly different in abundance between control, ST and LT infants, the non-parametric Kruskal–Wallis test with Monte Carlo permutation  was applied.Data were analysed using the CFX Manager™ software (Bio-Rad). Relative abundances of the taxa were determined by dividing the taxa-specific 16S rRNA gene copy number by the total 16S rRNA gene copy number. Quantitative polymerase chain reaction (qPCR) and pyrosequencing data had a Spearman correlation of 0.758, 0.729 and 0.822 for Bifidobacterium throughout the first six postnatal weeks, with an average relative abundance of 45% in meconium, increasing towards 73% at postnatal week six , dominance of Actinomyces at postnatal weeks three and four in another infant (infant B), members of Proteobacteria were only identified in three infants and, in three infants, Enterococcus was a dominant member in the meconium sample  and at postnatal weeks one , two  and three  compared to control infants. In LT infants, Bifidobacterium abundance also remained lower during postnatal weeks four (p = 0.086) and six (p = 0.009), whereas this could not be observed in ST infants. qPCR analysis confirmed the significantly lower quantity of Bifidobacterium right after a short (p = 0.033) or long (p = 0.035) antibiotic treatment compared to control infants. Enterococcus became a dominant member of the community in multiple ST and LT infants during the first postnatal week, which was not observed in any of the control infants   analysis and RDA. Temporal microbiota development was different between control, ST and LT infants p = 0.00  community richness and diversity were not affected by antibiotic treatment, but were associated with postnatal age and with the dominance of specific bacterial taxa, leading to differences in microbial networks.Intravenous antibiotic administration for the prevention and treatment of infection and sepsis occurs frequently in preterm infants during the neonatal period. Therefore, studying the side effects of antibiotic treatment, including the effect on microbiota development, is of great relevance. The idea that short antibiotic use negatively affects clinical success and induces antibiotic resistance is gradually being replaced by the aim to avoid antibiotic overuse . In thisBifidobacterium throughout the first six postnatal weeks. Bifidobacterium species are considered beneficial early-life colonisers, and are found in high abundance in term, vaginally delivered, breast-fed infants [Bifidobacterium abundance and allowed Enterococcus to thrive. It must be noted that Bifidobacterium abundance was already lower in meconium samples of ST and LT infants compared to control infants, most likely a result of the relatively late  defecation of meconium samples by most preterm infants. Enterococcus remained dominant for up to two weeks after antibiotic treatment discontinuation. This might possess a health risk for the infants, as some Enterococcus species emerged from gut commensals to nosocomial pathogens via the acquisition of multi-drug resistance and other virulence determinants [Bifidobacterium levels within the first six postnatal weeks. Although the differences in average Bifidobacterium abundance between ST and LT infants was less apparent using qPCR instead of sequencing, it did show that Bifidobacterium levels recovered in 4/5 ST and in only 2/5 LT infants. In addition, both methods indicate that long antibiotic treatment results in increased abundance of members of the Enterobacteriaceae family at postnatal week six.In the current study, 16S rRNA gene sequencing and qPCR analysis showed that preterm infants’ faecal microbiota was dominated by  infants . Short aBifidobacterium was negatively associated with abundance of other bacterial genera. Its dominance, however, allowed for higher community richness and diversity compared to dominance by other bacterial genera such as Enterococcus. We speculate that Bifidobacterium species control, but not outcompete, other bacterial species and that the microbial networks associated with Bifidobacterium species can, therefore, play an important role in early-life tolerance induction and immune system maturation. Microbiota profiles associated with antibiotic treatment could negatively influence immune system maturation via disturbance of the normal colonisation pattern. Indeed, previous studies showed that early-life antibiotic exposure increased susceptibility to immune-related diseases such as asthma and allergy, and associated this with perturbations in microbial composition [Antibiotic treatment did not affect community richness and diversity. However, richness and diversity were affected by postnatal age and by the dominance of specific bacterial taxa. Dominance of position , 33.In addition to antibiotic treatment, our findings show that postnatal age, gestational age, pre-eclampsia and maternal antibiotics influenced microbiota composition. The latter two highlight the importance of maternal health status on infant microbiota development. Previous studies showed that microbes can be vertically transmitted, and that maternal health status, such as bodyweight and antibiotic use, affect infant microbiota development , 13, 21.Overall, our findings show that intravenous antibiotic administration during the first postnatal week greatly affects the gastrointestinal microbiota community structure in preterm infants. However, quick cessation of antibiotic treatment allows for recovery of the microbiota. Disturbances in microbiota development caused by short and, more extensively, by long antibiotic treatment could affect healthy development of the infant via interference with maturation of the immune system and gastrointestinal tract. Clinicians should be aware of the disturbances that antibiotic treatment can cause and be strict in discontinuing antibiotic treatment as soon as possible to allow for a fast recovery of the microbiota community structure.ESM 1(DOCX 880 kb)Below is the link to the electronic supplementary material.


Herein, we report the first evidence that c-Met is palmitoylated and that palmitoylation facilitates its trafficking and stability. Inhibition of palmitoylation reduced the expression of c-Met in multiple cancer cell lines post-transcriptionally. Using surface biotinylation, confocal microscopy, and metabolic labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized c-Met and causes accumulation at the Golgi. Acyl-biotin exchange and click chemistry-based palmitate labeling indicated the c-Met β-chain is palmitoylated, and site-directed mutagenesis revealed two likely cysteine palmitoylation sites. Moreover, by monitoring palmitoylation kinetics during the biosynthesis and trafficking of c-Met, we revealed that stable palmitoylation occurs in the endoplasmic reticulum prior to cleavage of the 170 kDa c-Met precursor to the mature 140 kDa form. Our data suggest palmitoylation is required for egress from the Golgi for transport to the plasma membrane. These findings introduce palmitoylation as a critical modification of c-Met, providing a novel therapeutic target for c-Met-driven cancers. The c-Met receptor tyrosine kinase (RTK) is involved in embryonic development, tissue remodeling, and, when dysregulated, cancer progression . The c-Mc-Met is frequently overexpressed or mutated in cancers and this coincides with disease progression . Receptoin vivo [Our laboratory and others have found a connection between fatty acid synthase (FASN) activity and c-Met . Inhibitin vivo –30. Palmin vivo , 32. A nin vivo . The lacin vivo , 38. Giv14C-acetate incorporation into lipids  , 39, 40 o lipids . To deteo lipids . In addio lipids , but c-Mo lipids . Neithero lipids .In order to visualize the effect of 2BP on c-Met localization, DU145 cells were treated with 2BP for 3 or 6 hours, fluorescently labeled with antibody to c-Met, and analyzed by confocal microscopy. With increased duration of 2BP treatment, perinuclear accumulation of c-Met was apparent along with diminished plasma membrane expression, most notably at 6 hours Figure .We next sought to determine whether c-Met biosynthesis or degradation was affected by 2BP treatment. Quantitative reverse-transcriptase PCR was performed on DU145 cells treated with or without 2BP for 6 hours along with parallel western blot protein analysis Figure . There wcis-Golgi (GM130), and early endosomes (EEA1) , the ) Figure . With pr) Figure . No colo) Figure . It is pTo determine the degradation mechanism responsible for reduced c-Met levels, DU145 cells were treated with 2BP for 2, 4, or 6 hours in the presence or absence of the lysosome acidification inhibitor concanamycin A (Con) or the proteasome inhibitor MG132 (MG). Western blot analysis revealed that Con actually had an additive effect with 2BP treatment, and MG only partially prevented 2BP-induced c-Met loss Figure . To addrThese experiments suggest that inhibition of palmitoylation, at least in part, causes shedding of plasma membrane associated c-Met. To test this possibility, we collected conditioned media from DU145 cells treated with either HGF or EGF as positive controls or C75 to inhibit palmitoylation to detect possible increases in the previously published 90 kDa shed c-Met ectodomain fragment [2OH) [2OH) and that c-Met was biotinylated only when acyl-modified cysteines were reduced and exposed to biotin binding (+NH2OH)  . These s) Figure .2OH thereby removing any cysteine-linked Odya. Western blotting revealed c-Met pull-down was dependent on the click-reaction and was prevented by reducing agent, again supporting S-palmitoylation  or alkyne-linked 17-Octadecynoic acid (Odya) which are covalently cross-linkable with biotin –43. Aften Figure .The H1993 cell line with amplified c-Met, compared to the relatively low levels in DU145 cells, was used for several experiments hampered by limited c-Met levels. To test the reverse approach of detection, c-Met was immunoprecipitated from Odya-incubated H1993 cell lysates. Isolated protein was reduced with tris(2-carboxyethyl)phosphine (TCEP) to break disulfide bonds while maintaining the thioester-linked Odya and then click processed. Probing samples with streptavidin-HRP following SDS-PAGE again revealed Odya-incorporated c-Met Figure . To deteTo determine the site of acyl-group linkage we engineered fifteen individual HA-tagged cysteine (C/A) mutants representing all cysteines throughout the β-chain not previously shown to be involved in disulfide bonds, except C561 as a control . C/A mutTo determine which cysteine residue/s were required for trafficking of c-Met to the plasma membrane, we performed surface biotinylation after transfection with parental c-Met or C/A mutants. Surface expressed biotinylated protein was pulled-down and probed for HA. Western blot analysis and quantitation of replicate densitometry (140 kDa band) revealed most mutants trafficked to the plasma membrane comparable to parental c-Met except C882A and C894A, which aren't efficiently processed to the mature form, and control C561A which was expressed at the surface 40% less Figure . ResultsBased on the compelling data that c-Met is palmitoylated, we investigated the dynamics of palmitoylation during the lifespan of c-Met. Early studies on the biosynthesis of c-Met determined that the protein was synthesized as a 170 kDa precursor that is subsequently cleaved in the Golgi to α- and β-chains that are linked via disulfide bonds. Under reducing conditions, the mature β-chain migrates at 140 kDa by SDS-PAGE .cis-Golgi, or monensin  to prevent transport out of the Golgi network to the plasma membrane. When incubated with AHA for time points within 2 hours in combination with BF, the 170 kDa c-Met precursor accumulated having not entered the Golgi for precursor cleavage. Furthermore, CX treatment effectively prevented new synthesis of c-Met, and MN did not disrupt synthesis or cleavage of c-Met inside the Golgi  to inhibit protein synthesis, brefeldin A  to inhibit protein transport from the ER to the i Figure . Moreovei Figure . Prolongi Figure .With this information, we sought to determine the location and kinetics of c-Met palmitoylation. H1993 cells were labeled with Odya for time points within 2 hours in the presence or absence of CX, BF, or MN and western blot analysis was performed to observe palmitoylated c-Met. In the presence of CX treatment there is no detectable level of c-Met palmitoylation above the pull-down background (No Odya) Figure . TreatmeThis report is the first evidence that the RTK c-Met is palmitoylated, and that inhibition of palmitoylation reduces c-Met levels. Our data highlight the sensitivity of c-Met to inhibition of palmitoylation as compared to other lipid modifications. c-Met was also unique relative to other transmembrane proteins including integrin β4 and Ron, although EGFR levels were consistently reduced at longer time points. A recent report suggests FASN-dependent palmitoylation can elevate the activity and levels of a sub-population of EGFR . There icis-Golgi marker, suggesting c-Met is blocked along the biosynthetic route and possibly rerouted for degradation upon build-up. In accordance with these results, 2BP does not completely deplete c-Met protein but leads to a greatly reduced steady-state. Marked reduction of c-Met at the plasma membrane could be due to internalization or ectodomain shedding. Surface biotinylation assays indicated c-Met internalization was greatly reduced in response to FASN inhibition compared to basal or HGF-stimulated rates. In addition, we detected elevated levels of the 90 kDa c-Met ectodomain in media following treatment with a FASN inhibitor, suggesting inhibition of palmitoylation causes a loss of c-Met surface expression, at least in part, through ectodomain shedding. Blocking the proteasomal or lysosomal degradation pathways individually was unable to greatly prevent 2BP-induced c-Met loss. Multiple reports detail the paradoxical effects these degradation pathways have on c-Met trafficking and stability [Real-Time PCR and metabolic labeling of nascent protein indicated that c-Met was reduced post-translationally. c-Met expression diminished from the cell surface and accumulated in a perinuclear compartment colocalized with a tability –48. We fWe next determined that c-Met itself could be S-palmitoylated using an acyl-biotin exchange technique which has been used extensively to identify proteins with thioester-linked modifications. To support these data we used biorthogonal palmitate to biotin-label palmitoylated proteins following click chemistry. This relatively new technique allows for a more rapid and tractable study of palmitoylated proteins, compared to radiolabeling, with validated specificity –51. c-MeTo elucidate the dynamics of c-Met palmitoylation, we used inhibitors to block steps of c-Met biosynthesis. CX treatment completely blocked pulse labeling of c-Met and BF treatment greatly reduced detection of palmitoylated mature c-Met. We find it convincing that c-Met is palmitoylated prior to leaving the ER in its precleaved 170 kDa precursor structure. In this model, palmitate would be attached to precursor protein in the ER, likely catalyzed by a resident acyltransferase. Palmitate would remain attached to one or more cysteine residue/s within the β-chain through cleavage in the Golgi and as it is trafficked to the plasma membrane. Given that neither MN treatment nor HGF stimulation greatly affected the amount of c-Met palmitoylated over time, we predict that neither association with the plasma membrane nor internalization and recycling, control or are controlled by, dynamic palmitoylation. Rather, this is a stable attachment that remains throughout the duration of the molecule's life. Given these findings, it seems probable that the induction of c-Met shedding in response to inhibition of palmitoylation is an indirect effect – the perturbation of proteins that control the shedding process.Blocking palmitoylation did not prevent exit from the ER or cleavage in the Golgi, but it did impede exit of c-Met out of the Golgi through the secretory pathway. It is possible that the requirement of palmitoylation for c-Met to exit the Golgi is related to trans-Golgi lipid raft structures –54. In tMultiple methods were employed to identify the cysteine residue/s required for c-Met palmitoylation. Technical issues impeded complete consistent sequence coverage from mass spectrometry, and the labile nature of the lipid attachment made detection unreliable. Site-directed mutagenesis of all β-chain cysteine residues not involved in intramolecular disulfide bonds, except C561, revealed two sites that reduced Odya labeling of c-Met. C894 and C624 are evolutionarily conserved residues N-terminal to the transmembrane domain (aa933-955) residing extracellular at the plasma membrane. Importantly, C894A exhibits a clear defect in proteolytic processing as indicated by altered PAGE migration; however, given palmitoylation occurs before cleavage, the results still suggest this cysteine, along with C624, are probable palmitoylation sites.Type-1 membrane proteins are most commonly palmitoylated on cytosolic-facing cysteine residues; however, our evidence supports lumenal palmitoylation of c-Met in the ER resulting in an extracellular palmitoylated residue. Of the 21 DHHC family palmitoyl acyltransferases, DHHC12, 13, 14, and 16 are known to reside at the ER and were detected by PCR in the cell lines used to demonstrate c-Met palmitoylation (data not shown), 56. AltA parallel surface biotinylation experiment was performed to determine whether a direct relationship could be observed between impaired palmitoylation and trafficking of c-Met to the cell surface. As expected, C894A and C882A were not expressed at the plasma membrane because of the processing impairment. C561A was also expressed at the plasma membrane less efficiently; however, C624, which is palmitoylation impaired, seems to reach the plasma membrane equivalently.Although C624A and C894A greatly reduced palmitoylation, considering no single cysteine mutation completely ablated palmitoylation, surface expression or c-Met levels , we find it likely that multiple cysteine residues may be palmitoylated and required for proper trafficking of c-Met. Multi-site palmitoylation has been observed with integrin β4 and numerous other palmitoylated proteins . Future These findings reveal a novel post-translational modification that is necessary for the trafficking and stability of a clinically important receptor tyrosine kinase. There is abundant evidence implicating c-Met as a molecular target for cancer therapy, and its biology is important to embryonic development, liver regeneration, and fibrosis. Greater understanding of the factors regulating c-Met biosynthesis, trafficking, and activity is essential for future design of optimized therapeutic intervention. Exploiting the need for lipidation may serve as such a therapeutic modality.2. Monensin and brefeldin A were obtained from Biolegend , Y27637 from Stem Cell Technology , C75, 2-hydroxymyristic acid, and 17-Octadecynoic Acid from Caymen Chemicals , cycloheximide, recombinant EGF, geranylgeranyltransferase inhibitor, farnesyltransferase inhibitor, vinblastine, latrunculin, dansylcadaverine, ethylisopropyl amiloride, methyl-β-cyclodextrin, nystatin, and 2-bromopalmitate were obtained from Sigma-Aldrich . Click-iT® reaction buffers, azido-homoalanine, azide-palmitate, and methionine-free RPMI, were obtained from Invitrogen . Recombinant HGF was obtained from EMD Millipore . Streptavidin Sepharose High Performance beads were obtained from GE Healthcare .All cell lines used were obtained from ATCC  and have since been validated (Promega). DU145 (prostate carcinoma), H1993 (lung carcinoma), AU565, and HCC1806 (breast carcinoma) cells were maintained in RPMI 1640 media , MDA-MB-231 (breast carcinoma) cells were maintained in DMEM (Cellgro), and HEK293 cells in EMEM (Cellgro); each containing 10% fetal bovine serum (FBS) . Cells were kept at 37°C with 5.0% COCells were seeded to 70% confluency in 24-well plates before treatments. Inhibitors were spiked into the media and incubated for the indicated times prior to cell lysis. Lysates were taken in boiling Laemmli buffer  with 0.05% β-mercaptoethanol (BME) and boiled for 5 minutes. Samples were analyzed by SDS-PAGE and blotted with the indicated primary antibodies: c-Met CVD13 (1:1000)(Invitrogen), integrin β4 H-101 (1:1000), Ron-β C-20 (1:750), and EGFR 1005 (1:750), and β-tubulin . High sensitivity streptavidin-HRP was obtained from Pierce Thermo Scientific . Blots were subsequently probed with horseradish peroxidase-conjugated secondary antibodies , and detection was acquired with ECL (Amersham Biosciences).2 SYBR Green Flour FAST Mastermix  was used in qPCR reactions. Cycling conditions were 95°C 10 minutes, followed by 40 cycles of 95°C 10 seconds and 55°C 30 seconds. Melt curves were ran each time and reverse transcriptase negative and template negative controls were used. Reactions were carried out using a Bio-Rad CFX96 Real-Time PCR Detection System with Bio-Rad CFX Manager 3.0 software. Primers were designed using Integrated DNA Technologies  PrimerQuest software and sequences used include (5′ to 3′): Met forward: ACCGAAAGATAAACCTCTCATA, Met reverse: TGCTAGTGCCTCTTTACAC, GAPDH forward: GTCGGAGTCAACGGATTT, GAPDH reverse: AGTTGAGGTCAATGAAGGG.DU145 cells were seeded to 70% confluency in 10 cm dishes prior to treatment in serum-free media with or without 100 μM 2BP for 5 hours. Following cell collection and centrifugation, cells were resuspended in Trizol (Invitrogen) and RNA was isolated according to manufacturer instructions. The SuperScript First-Strand kit  was used for cDNA synthesis. RT14C-acetate  for 2 hours, washed with phosphate buffered saline (PBS), and lysed in hypotonic buffer (1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris-HCl, pH 7.5). Lipids were extracted with chloroform:methanol (2:1 v/v) and 14C-acetate incorporation was quantified by scintillation counting.To label newly synthesized fatty acid, cells were pulsed with 1 μCi Cells were grown to 50% confluency on glass coverslips in a 6-well plate. Following treatments cells were fixed in 4% PFA, washed twice with PBS, and blocked for 30 minutes in 10% donkey serum PBS with 0.1% saponin (DSP). Primary antibodies c-Met (1:70) (R&D Systems), GM130 (1:100), EEA1 (1:100), or calnexin (1:100) (BD Biosciences) were added simultaneously in DSP for 2 hours at room temperature. After washing three times with PBS, secondary antibodies (Jackson Immunoresearch) were added in DSP for 2 hours at room temperature. Coverslips were washed again three times with PBS and then mounted on coverslips with DAPI slowfade (Invitrogen). Representative images were taken on a Leica TCS SP5 Confocal Microscope at 60x magnification with oil immersion. Representative images are shown as enhanced using ImageJ software.Cells were grown to 80% confluency in 10 cm dishes. All dishes were pretreated with growth media containing fatty acid-free 10% FBS (Gemini) with vehicle or treatment for indicated times. Media is replaced with fresh fatty acid-free growth media containing alkyne-labeled  palmitic acid or azide-labeled  that was sonicated for 5 minutes to improve solubility with or without treatments. Cells were incubated for 4 hours to allow incorporation of palmitate orthologs. Cell lysates were taken in NP-40 lysis buffer  containing protease inhibitor cocktail (Roche) at 4°C. Lysates were processed by 30 minutes of end-over-end rotation at 4°C followed by removal of debris by centrifugation at 13,000xg for 5 minutes. Protein concentrations were determined and 200 μg of each sample were methanol-chloroform precipitated. Precipitated protein was resuspended in 50 μl of 1% SDS reaction buffer  and the click chemistry reaction was performed according to the manufacturer's protocol using the Click-iT® reaction buffer kit and corresponding biotin-based detection reagent (Invitrogen). Following linkage of the palmitate ortholog to biotin, protein was again precipitated and resuspended in 100 μl of 1% SDS. Once protein was solubilized, SDS was diluted out with 1 ml of NP-40 lysis buffer. Samples were precleared for 1 hour at 4°C with A/G agarose beads (Santa Cruz) and centrifuged at 13,000xg for 5 minutes. After preclear, 100 μl of prewashed streptavidin-conjugated sepharose beads  were added to each supernatant. Samples were rotated end-over-end overnight at 4°C. IP samples were washed 5 times (50 volumes) in NP-40 lysis buffer, before protein was eluted with 30 μl of Laemmli buffer with BME. Samples were analyzed by western blot using antibody to c-Met (Invitrogen).Cell lines were grown to 70% confluency in 10 cm dishes. Cells were incubated in methionine-free growth media (Invitrogen) containing 10% FBS for 1 hour prior to treatments. Any pretreatment inhibitor was spiked into the well for the last 30 minutes of the methionine-starve period. Media was then replaced with fresh methionine-free growth media containing 10% FBS with or without 30 μM azido-homoalanine (AHA) (Invitrogen) to label nascent proteins with or without indicated treatments. Processing of lysates, click-chemistry reaction, and pull-down of biotinylated protein was performed as detailed above.Cells were grown to 70% confluency in 10 cm dishes. Cells were washed once with 4°C PBS to prevent membrane internalization. Surface-exposed proteins were biotinylated with 0.1 mg/ml EZ-Link biotin (Invitrogen) in PBS at 4°C for 15 minutes. Excess biotin was quenched and removed with three 5-minute washes of 100 mM glycine in PBS at 4°C. All plates, except one complete strip control, were then incubated with 37°C prewarmed serum-free media containing the indicated treatments for chase periods to allow internalization. After indicated chase periods, cell were washed twice with 4°C PBS to halt the experiment. Remaining extracellular biotin was removed, from all but a no strip control, with MESNA buffer in PBS  for three 5-minute washes at 4°C. Residual MESNA was removed with six washes with 4°C PBS. Cells lysates were taken in NP-40 lysis buffer containing protease inhibitor cocktail (Roche) at 4°C. Processing of lysates and precipitation of biotinylated protein from equal protein samples was performed as detailed above. Equal protein was analyzed for c-Met by western blotting to indicate amount of c-Met internalized over time.Cells were grown to 60% confluency in 10 cm dishes. Cells were washed twice with room temperature PBS and then treated with 3.5 ml serum free media containing the indicated treatments  and incubated for the indicated time. Conditioned media was collected and washed. Cells were lysed in NP-40 lysis buffer. Conditioned media was centrifuged at 2100 rpm for five minutes at 4°C to pellet cell debris. Supernatant was transferred to a clean tube for TCA precipitation. Fresh 2% NaDeoxycholate was added 1:100, gently mixed and incubated at room temperature for 15 minutes. Fresh TCA was then added at 1:10, vortexed, and incubated at room temperature for 1 hour. The protein was centrifuged out at 5500 rpm for 10 minutes at 4°C. The supernatant was aspirated and the pellet was allowed to dry. The pellet was then washed in 200 μl ice cold acetone, vortexed, and left on ice for an additional 15 minutes, followed by a repeat centrifugation at 5500 rpm for 10 minutes at 4°C. Supernatants were then aspirated and pellets dried. 100 μl of Laemmli buffer with BME was added to each protein pellet. Equal volumes were loaded and analyzed by SDS-PAGE blotting with the DL-21 antibody to an extracellular c-Met epitope [c-Met isoform b (NM_00236.2) was restriction digested from pLenti-MetGFP  and ligated into the multi-cloning site of pCMV6-AC-HA (OriGene: PS100004) giving a c-terminal HA-tag. Fifteen separate site-directed mutants were engineered  using Agilent QuickChange II XL reagents . Each mutant was verified by sequencing . Transfections were performed using Continuum reagent (Gemini) according to manufacturer instructions.t-test (two tailed) with P-value <0.05 as significant.Data are expressed as means ± SEM. Statistical analysis was performed using Student's


The practical use of recycled concrete aggregate produced by crushing concrete waste reduces the consumption of natural aggregate and the amount of concrete waste that ends up in landfills. This study investigated two methods used in the production of fine recycled concrete aggregate: (1) a method that produces fine as well as coarse aggregate, and (2) a method that produces only fine aggregate. Mortar specimens were tested using a variety of mix proportions to determine how the characteristics of fine recycled concrete aggregate affect the physical and mechanical properties of the resulting mortars. Our results demonstrate the superiority of mortar produced using aggregate produced using the second of the two methods. Nonetheless, far more energy is required to render concrete into fine aggregate than is required to produce coarse as well as fine aggregate simultaneously. Thus, the performance benefits of using only fine recycled concrete aggregate must be balanced against the increased impact on the environment. The practical use of recycled concrete aggregate produced by crushing concrete waste reduces the consumption of natural aggregate as well as the amount of concrete waste that ends up in landfills. The crushing of concrete waste produces coarse recycled concrete aggregate (CRCA) and fine recycled concrete aggregate (FRCA), as defined by particle size. A number of studies ,9,10,11 Several studies ,17,18,19Lee  investigSim and Park  applied 3, which increased with the size of the particles. RC-3 particles between 2 mm and 4 mm in size had a density of 2.61 g/cm3. They concluded that optimizing the crushing method could enhance the quality of the resulting RCA.Florea and Brouwers  investiget al. [Ulsen et al.  producedSong and Ryou  introduc3 and water absorption of 2.52%. Their results demonstrate the efficacy of heat grinder systems in the production of high-quality FRCA suitable for the structure of new buildings.Koshiro and Ichise  employedClearly, the methods used in the processing of concrete waste influence the quality of the resulting FRCA. Most previous studies have obtained FRCA produced under laboratory conditions or using the methods typically employed in large-scale recycling facilities, in which CRCA and FRCA are produced simultaneously. In this study, we obtained FRCA from a recycling facility in Yilan, Taiwan, which using a crushing process that produces only fine recycled concrete aggregate. We then prepared and tested specimens using a variety of mix proportions to determine the influence of production process and FRCA proportion on the properties of the resulting mortar, which is a constituent of concrete.This study employed Type I Portland cement and FNA comprising clay slate and river sand, which was processed in a gravel plant. The other method employed in this study produces only fine aggregate using multiple processes. Concrete waste first undergoes crushing in a large jaw crusher, whereupon the resulting aggregate is separated using a vibrating screen. Fragments exceeding 50 mm are sent back to the large jaw crusher to undergo repeated crushing, and this cycle is repeated until all of the aggregate is less than 50 mm. The resulting material is then crushed using a small jaw crusher and a roll crusher and separated using a vibrating screen. Aggregate larger than 4.75 mm is returned to the roll crusher until it is all smaller than 4.75 mm. Aggregate between 600 μm and 4.75 mm is sent to a fine aggregate stockpile; whereas aggregate smaller than 600 μm is sent to a wheeled sand washer for the removal of sludge smaller than 150 μm. The remaining aggregate between 150 μm and 600 μm is sent to the fine aggregate stockpile. The second type of FRCA produced using the above-mentioned process is denoted as R2, the basic properties of which are also presented in The FRCA used in this study presents a higher water absorption than does FNA. We therefore applied pre-wetting to R1 and R2 sample for 24 h. We then adjusted the measurement of surface moisture (ASTM C70-13 ) prior tVarious tests were performed to characterize the attributes of the mortar, including flow tests, absorption tests, density tests, drying shrinkage tests, compressive strength tests, and ultrasonic pulse velocity (UPV) tests.0) on a flow table. The flow mold was then lifted off and the flow table was vibrated. The mean diameter (Da) of the resulting flow was calculated using results obtained after conducting the test four times, as follows:Flow testing was performed in accordance with ASTM C1437-13 . Freshlys). The specimens were weighed while suspended in boiling water 5 h (Wa) and again after being removed from the water that had cooled to 25 °C (Wb). The density of the specimens was calculated using the following formula.Density testing was performed using cylindrical specimens with a diameter of 100 mm and height of 50 mm in accordance with ASTM C642-13 . The sped). They were then soaked in water to achieve the SSD condition before being weighted (Ws). The water absorption was calculated using the following formula:Absorption testing was performed using cylindrical specimens with a diameter of 100 mm and height of 50 mm, in accordance with ASTM C642-13 . Specimei), which is the difference between the comparator reading of the specimen and the reference bar at 3 days. The specimens were then placed in a chamber under relative humidity of 50% ± 4% at a temperature of 23 ± 2 °C for curing. The length of the specimens was then measured at 7 days, 14 days, 21 days, and 28 days (Lx). The extent of drying shrinkage was calculated as follows:Drying shrinkage was tested on 285 × 25 × 25 mm prismatic specimens in accordance with ASTM C596-09 . We firsCompressive strength was tested on 50 × 50 × 50 mm cube specimens in accordance with ASTM C109-13 . SpecimeThe UPV test was performed on cylindrical specimens with a diameter of 100 mm and height of 200 mm in accordance with ASTM C597-09 . The meaThe attributes in i.e., (254 − 100)/100 × 100%). In addition, all of the mortar mixtures had a water/cement ratio of 0.55. Regardless of the amount of FRCA in the freshly mixed mortars, they all presented good flow values exceeding 154%.In accordance with the formulas in ASTM C1437-13 , we empli.e., the proportion of FNA substitution with FRCA. With the same replacement ratio, samples that included R1 presented lower flow values than did those that included R2. This can be attributed to the fact that R1 fragments have a rougher surface texture and greater angularity, which increases the friction among the particles. It should be noted that all of the flow values obtained from mixtures with a water/cement ratio of 0.55 presented flow values exceeding 154% (data not shown).The compressive strength of mortar specimens was tested at 7 days, 14 days, and 28 days. y = 0.0578x − 169.68, where x and y denote the UPV value and compressive strength, respectively. Regression analysis results indicate that compressive strength increases with an increase in UPV value.R2 has lower porosity, higher density, and lower water absorption than does R1, all of which are indicators of the superior quality of R2. This also demonstrates that the crushing process can significantly influence the quality of the resulting FRCA.In all of the mortars containing FRCA, an increase in the replacement ratio led to a reduction in flow values, density, compressive strength, and UPV values. This is a clear demonstration that the replacement ratio is an important factor influencing the physical and mechanical properties of the resulting mortar.When comparing mortars containing R1 or R2 at the same replacement ratio, the mortar containing R1 presented a lower flow value, lower density, higher absorption, higher drying shrinkage, lower compressive strength, and lower UPV values. This demonstrates that mortars containing R1 cannot match the physical or mechanical properties of R2, and further demonstrates the importance of the crushing process used in the production of FRCA.Our results demonstrate the superiority of mortars produced using aggregate processed from recycled concrete via multistage crushing to obtain only FRCA. Nonetheless, the performance benefits of using only fine recycled concrete aggregate must be balanced against the additional energy requirements and subsequent impact on the environment.This study investigated the use of two types of fine recycled concrete aggregate (R1 and R2) obtained using different production processes at a recycling facility in Yilan, Taiwan. R1 was produced using a process that simultaneously produces coarse as well as fine aggregate by crushing concrete waste with a large jaw crusher. R2 was produced using a process that produces only fine aggregate through repeated crushing of concrete waste. We then tested mortar specimens in which various proportions of FRCA were substituted for FNA. This led to the following conclusions:


Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has emerged as a novel therapy for acute liver failure (ALF). However, the homing efficiency of BMSCs to the injured liver sites appears to be poor. In this study, we aimed to determine if overexpression of c-Met in BMSCs could promote the homing ability of BMSCs to rat livers affected by ALF.Overexpression of c-Met in BMSCs (c-Met-BMSCs) was attained by transfection of naive BMSCs with the lenti-c-Met-GFP. The impact of transplanted c-Met-BMSCs on both homing and repair of ALF was evaluated and compared with lenti-GFP empty vector transfected BMSCs (control BMSCs).P < 0.05), and was dependent on hepatocyte growth factor (HGF). Furthermore, rats with ALF that received transplanted c-Met-BMSCs showed significantly improved homing ability to the injured liver; this was accompanied by elevated survival rates and liver function in the ALF rats. Parallel pathological examination further confirmed that transplantation of c-Met-BMSCs ameliorated liver injury with reduced hepatic activity index (HAI) scores, and that the effects of c-Met-BMSCs were more profound than those of control BMSCs.After cells were transfected with the lenti-c-Met-GFP vector, the BMSCs displayed very high expression of c-Met protein as demonstrated by Western blot. In addition, in vitro transwell migration assays showed that the migration ability of c-Met-BMSCs was significantly increased in comparison with that of control BMSCs (Overexpression of c-Met promotes the homing of BMSCs to injured hepatic sites in a rat model of ALF, thereby improving the efficacy of BMSC therapy for ALF repair. Acute liver failure (ALF) is the rapid loss of liver function due to severe damage to the liver, with common causative factors including viruses (particularly hepatitis B and C), toxins, prescribed drugs, and alcohol. ALF can lead to jaundice, coagulopathy, multiple organ failure, hepatic encephalopathy, and even death . CurrentMET gene and belongs to the tyrosine protein kinase family. The c-Met protein is a member of the transmembrane tyrosine kinase receptor superfamily and has independent phosphorylation activity [c-Met is encoded by the activity . HGF is activity .In this study, we aimed to establish c-Met-BMSCs by overexpression of c-Met, and to determine if c-Met-BMSCs could promote homing of BMSCs to rat livers, thereby improving their capability for repairing ALF.A total of seventy-two male Sprague-Dawley rats were purchased from the Animal Laboratory Center of Nanjing Medical University . Rats were aged 4 weeks, weighed 80–100 g, and were used to isolate BMSCs so that an ALF rat model could be generated. All rats were maintained according to the experimental animal care and research protocol, which was approved by the First Affiliated Hospital of Nanjing Medical University . All experiments on rats were carried out in compliance with the guidelines of the Chinese Ethical Council.2 culture dish with complete culture medium, and cultured in an incubator at 37 °C at an atmosphere of 5% CO2 with saturated humidity. BMSCs from the third passage were selected and then a total of 105/100 μL cells were labeled with different antibodies, including FITC-labeled anti-rat CD29, APC-labeled anti-rat CD45, PE-labeled anti-rat CD90, and anti-rat CD34 . The solution was subsequently stored on ice for 30 min in the dark. After washing with phosphate-buffered saline (PBS), the cells were subjected to analysis and validation of specific markers by flow cytometry.Fresh rat BMSCs were isolated from male rats. Briefly, male rats were initially anesthetized by administration of 10% chloral hydrate at a dosage of 0.3 mL/100 g. The ends of the femur, tibia, and soft connective tissues were carefully removed to expose the intact bone marrow cavity, which was washed twice with normal saline solution, and collected into a centrifuge tube. Bone marrow material was centrifuged for 10 min at 180 × g. The pellets were then dissolved in complete cell culture medium containing a low glucose solution of 89% Dulbecco’s modified Eagle’s medium , 10% fetal bovine serum , and 1% penicillin/streptomycin . The mixture was suspended in lymphocyte separation medium  and centrifuged for 20 min at 710 × g. The third layer was extracted, placed into a 10-cmd-galactosamine  at 950 mg/kg body weight  and lipopolysaccharide (LPS) at 10 μg/kg body weight .Male rats were selected to generate the animal model of ALF as previously described . In the d-GalN/LPS injection; 50 mg of each tissue was homogenized with 1.5 mL of lysis buffer  and the lysate was collected to measure the HGF levels of all tissue samples using the HGF ELISA kit , following the manufacturer’s instructions.The heart, liver, spleen, lung, and kidneys of rats were collected at different time points  post-8 TU/mL, which was measured using a fluorescence assay method. In parallel, the control lentivirus was produced by cotransfecting the lenti-GFP empty vector GV358 with GFP but without c-Met, and two lentiviral helper plasmids (Helper1.0 and Helper2.0) into 293 T cells, and an infectious viral titer of 1 × 108 TU/mL was obtained.For application of c-Met by polymerase chain reaction (PCR), a pair of primers were designed and synthesized as follows: Forward primer: 5’-GAGGATCCCCGGGTACCGGTCGCCACCATGAAGGCTCCCACCGCGCTGGCACCTGG-3’; Reverse primer: 5’-TCCTTGTAGTCCATACCTGTGTTCGCTTCGCCGTCAATGTTGTCTTG-3’. The resulting c-Met cDNA was inserted into the lentiviral vector GV358  to create the lentiviral vector, GV358-c-Met. We cotransfected the recombinant and two lentiviral helper plasmids  into 293 T cells to generate the target lentivirus with an infectious viral titer of 2 × 10To establish c-Met-BMSCs or control BMSCs, the lenti-c-Met-GFP or lenti-GFP empty vector was used to transfect naive BMSCs cells at passage 5 (MOI = 100), respectively. Three days after infection, c-Met-BMSCs or control BMSCs were selected and cultured in cell culture medium containing 9 μg/mL puromycin for 2 weeks to generate stable cell lines (Patent no.201610662140.4). c-Met-BMSCs or control BMSCs were visualized under fluorescence microscopy to confirm a 100% fluorescence positive rate, and cells were screened and selected by puromycin.Western blot (WB) analysis was performed to examine levels of c-Met protein, in which the same number of c-Met-BMSCs and control BMSCs (transfected with lenti-GFP empty vector) were used to extract total protein samples. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes as previously reported . The c-M5 cells/mL were added to the top of the chamber layer. In the bottom chamber, different concentrations of murine HGF  were used as a chemoattractant. The above cell transwell chambers were incubated in a humidified tissue culture incubator overnight at 37 °C and an atmosphere of 5% CO2. After 24 h, the cells were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet, respectively. The migrated cells were observed, imaged, and counted within three fields under an optical microscope.The cell migration assay was performed in a transwell chamber  which contains a polyethylene terephthalate (PET) track-etched membrane with an 8.0-μm pore; 0.5 mL suspensions of c-Met-BMSCs or control BMSCs at a concentration of 1 × 10d-GalN/LPS. After 24 h, rats in the c-Met-BMSC and control BMSC groups were given a transfusion of 1.0 × 107/kg cells, suspended in 1 mL normal saline by vena caudalis injection, respectively. In the NS group, animals were injected with 1 mL of normal saline. The survival of rats in the three groups were observed and recorded daily. The blood samples of all rats were collected at different time points  post-injection of d-GalN/LPS. The liver tissues of all the rats in the three groups were collected at 24, 48, or 72 h post-injection of d-GalN/LPS.A total of 36 rats were randomly divided into three experimental groups: c-Met-BMSC group, control BMSC group, and normal saline (NS) group, with twelve rats in each group. The rat model of ALF was induced and generated as previously described by co-injection of The amount of inflammation in the liver was quantified by assessing serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and hematoxylin-eosin (HE) staining. Serum levels of ALT, AST, and TBil were detected using a microplate test kit from Nanjing Jiancheng bioengineering institute . Rat liver tissues obtained from all three experimental groups were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced to a thickness of 4 μm. All slices were subsequently stained by HE. The pathological images were captured and grades of liver inflammation were assessed by hepatic activity index (HAI) grading following guidelines as previously described .6 cells from each group were transplanted into the rat with ALF through the vena caudalis. After 24 h, migration of cells into the liver was examined using an in vivo imaging system , and the homing efficiency was assessed by visualization of fluorescent intensity.To compare the homing efficiency of c-Met-BMSCs and control BMSCs to the injured rat liver, the dye DiR  was used to label both types of BMSCs, with or without c-Met overexpression; 1 × 10P values less than 0.05 were considered to be statistically significant.The experiments in this study were performed with triplicate samples in each group. Statistical analysis was conducted using IBM Statistics SPSS, version 16.0 . Data are expressed as means ± standard deviation (SD). The log rank test was used to compare survival rates among the three groups, and statistical analysis was carried out using one-way analysis of variance. Seven to ten days after isolated BMSCs were cultured the cell morphology was examined under an inverted microscope. Monolayers of BMSCs were formed with cells displaying a spindle shape and arranged in radial concentric circles or with broom-like growth (data not shown). Next, we characterized and validated the BMSCs at passage 3 by flow cytometry using specific cell surface markers for BMSCs. As shown in Fig. After naive BMSCs at passage 5 were transfected with the lenti-c-Met-GFP or lenti-GFP empty vector, and puromycin selection was completed, approximately 99% of these BMSCs were tested positive for green fluorescence as analyzed by fluorescence microscopy Fig. , indicatd-GalN/LPS injection. We also performed a parallel examination of HGF levels in other organs, including the heart, spleen, lung, and kidneys of the rat. The HGF concentrations at 0 h post-d-GalN/LPS injection were 4.89 ± 0.08 ng/mL, 4.16 ± 0.22 ng/mL, 1.29 ± 0.02 ng/mL, 1.17 ± 0.17 ng/mL, and 4.24 ± 0.145 ng/mL in the liver, heart, spleen, lung, and kidneys, respectively. We found that levels of HGF in the liver significantly increased after 24 h, with a peak level at 48 h post-d-GalN/LPS injection. However, there were no significant alterations in levels of HGF in the heart, spleen, lung, and kidneys of rats after d-GalN/LPS injection, which corroborates previous findings that HGF is induced by liver injury. As shown in Fig. d-GalN/LPS injection, whereas HGF levels in the liver were significantly higher compared with other organs at 24 h post-d-GalN/LPS injection (P < 0.001). These data indicate that there was induction of HGF through liver injury.As HGF is known to play an essential role in HGF/c-Met signaling-mediated repair of the injured liver, we examined the concentrations of HGF in the liver at 0, 24, and 48 h post-Next, we performed in vitro transwell migration assays to determine if the migration ability of c-Met-BMSCs could be improved in comparison with control BMSCs. The migrated BMSCs were visualized and counted under the microscope. The number of migrated cells in the c-Met-BMSC group were 43.0 ± 4.6, 100.7 ± 2.1, 127.3 ± 2.5, 80.3 ± 4.5, 66.0 ± 4.0, and 61.0 ± 3.6, respectively, while the number of migrated cells in the control BMSC group were 43.0 ± 2.0, 43.3 ± 5.0, 44.33 ± 1.2, 43.3 ± 2.1, 44.0 ± 3.0, and 43.3 ± 2.5, respectively. As shown in Fig. 9 in the liver of ALF rats transplanted with control BMSCs, and (3.22 ± 0.80) × 1010 in the liver of ALF rats transplanted with c-Met-BMSCs, which was significantly higher than that in the control BMSC group . These data demonstrated that c-Met-BMSCs could migrate more efficiently into the liver of ALF rats compared with control BMSCs.As described above, enhanced in vitro migration of c-Met-BMSCs may improve their homing ability to the injured liver. We therefore assessed effects of overexpressed c-Met protein on the homing capability of c-Met-BMSCs to the liver of rats with ALF. Control BMSCs and c-Met-BMSCs were labeled with the dye DiR and then transplanted into the liver of rats with ALF. Fluorescence was released from DiR-labeled cells in affected organs, and the fluorescent intensity was measured using an imaging system Fig. . As showP < 0.05). In addition, we tested the serum levels of ALT, AST, and TBil at 0, 24, 48, and 72 h post-d-GalN/LPS injection. As shown in Fig. d-GalN/LPS injection. However, there was no significant difference in serum levels of ALT, AST, and TBil among the three groups. The serum levels of ALT, AST, and TBil were significantly lower in the c-Met-BMSCs group compared with NS group, at 48 h or 72 h after d-GalN/LPS injection (P < 0.001). Moreover, significant differences in serum levels of ALT, AST, and TBil were observed between c-Met-BMSCs and control BMSCs groups at both 48 and 72 h post-d-GalN/LPS injection (P < 0.01).To evaluate the therapeutic effectiveness of c-Met-BMSCs for treatment of ALF in rats, we established a rat model of ALF. The rats with ALF were randomly divided into three groups: the c-Met-BMSC group, the control BMSC group, and the NS group. The survival rates were 83.3%, 50%, and 0% in the c-Met-BMSC, control BMSC, and NS groups, respectively Fig. . The ALFd-GalN/LPS injection. We observed moderate hepatic necrosis in the NS and control BMSC groups, whereas only mild hepatic necrosis was observed in the c-Met-BMSC group at 48 h post-d-GalN/LPS injection. At 72 h after d-GalN/LPS injection, moderate or severe hepatic necrosis was observed in the NS and control BMSC groups, whereas only sporadic (spotty) hepatic necrosis was observed in the c-Met-BMSC group . Interestingly, HAI scores in the c-Met-BMSC group were significantly lower than those in the control BMSC group at 48 and 72 h post-d-GalN/LPS injection (P < 0.01). These findings strongly suggest that c-Met-BMSCs improved the liver function in ALF induced by d-GalN/LPS and, in some of the cases, even resulted in reversal of the disease allowing rats to recover from ALF.We further examined whether c-Met-BMSC transplantation could improve repair of ALF as assessed by liver histology. Liver histology was conducted by HE staining at the indicated times. More than 10 low-power field microscopic examinations were carried out for each slide. We did not observe hepatic necrosis in any of the three groups at 24 h post-oup Fig. . FurtherTransplantation of mesenchymal stem cells (MSCs), liver progenitor cells (LPCs), and hematopoietic stem cells (HSCs) has emerged as a promising therapy to treat various forms of liver disease mainly due to the capacity of these cell types to promote liver regeneration , 11. Howd-GalN/LPS as previously reported [In this study, the ALF rat model was generated by intraperitoneal injection of reported . In recereported , 13. Indreported , 14, andreported , 16. Furreported . Furtherreported , 19, whireported  mainly dd-GalN/LPS injection in rats is consistent with previous results. Since HGF has been found to strongly inhibit apoptosis of hepatocytes, elevated levels of HGF may improve the survival rate of mice with ALF. Further studies have shown that HGF also plays an essential role in stimulating liver regeneration against ALF [HGF is a potent hepatic mitogen produced by liver mesenchymal cells. Zhu et al.  have repinst ALF , 22. c-Minst ALF , 24. Morinst ALF , 26. Recinst ALF , 28. TheExtensive studies have demonstrated that gene therapy is a promising approach for the treatment of many forms of diseases, and may improve patient care, survival, and outcomes , 30. VirNotably, the levels of HGF in liver tissue were significantly upregulated at 24 h and 48 h after induced liver injury in the ALF rat model. When the c-Met-BMSC and control BMSC transplantation groups were compared, we found that ALF rats in the c-Met-BMSC transplantation group experienced better therapeutic effects as assessed by survival rates, markers for liver function, and liver pathology. It is plausible that induction of the HGF/c-Met signaling pathway led to augmented c-Met-BMSC migration to the liver and, as a result, a greater number of c-Met-BMSCs were able to home into the rat liver. This theory is supported by our study results, which showed increased migration of BMSCs by in vitro transwell assays. Furthermore, we were able to demonstrate homing capabilities of c-Met-BMSCs to the injured liver compared with control BMSCs using an in vivo imaging system.Our study does have some limitations, as we could not exclude the possibility that cytokines such as SDF-1 might also be involved in the migration of c-Met-BMSCs –36, and In conclusion, our results indicated that overexpression of c-Met promotes homing of BMSCs to the injured liver of rats with ALF, thereby improving the efficacy of BMSC therapy for the repair of ALF.


Antibacterial coatings may prevent bacterial adhesion and biofilm formation, according to various preclinical studies. The aim of the present study is to report the first clinical trial on an antibiotic-loaded fast-resorbable hydrogel coating . Pre- and postoperative assessment of laboratory tests, wound healing, clinical scores and X-rays were performed at fixed time intervals.P < 0.03). No local or systemic side-effects related to the DAC hydrogel product were observed and no detectable interference with bone healing was noted.Overall, 253 patients were available with a mean follow-up of 18.1 ± 4.5 months (range 12–30). On average, wound healing, clinical scores, laboratory tests and radiographic findings did not show any significant difference between the two groups. Six surgical site infections (4.6%) were observed in the control group compared to none in the treated group (The use of a fast-resorbable antibiotic-loaded hydrogel implant coating provides a reduced rate of post-surgical site infections after internal osteosynthesis for closed fractures, without any detectable adverse event or side-effects.2. The Centers for Disease Control (CDC) healthcare-associated infection (HAI) prevalence survey estimated 157,500 surgical site infections (SSIs) associated with inpatient surgeries in 2011 in the USA [In spite of improved operating room, sterilization methods, barriers, surgical technique and routine systemic antimicrobial prophylaxis –5, SSIs After osteosynthesis for closed fractures, early SSI had a reported incidence of 3.9% in a large multicenter trial, with a median time to diagnosis of 30 days , while wIn this context, antibacterial coatings of implants may represent an attractive option to reduce post-surgical infections . A stron®; Novagenit Srl, Mezzolombardo, Italy) is composed of covalently linked hyaluronan and poly-d,l-lactide and is designed to undergo complete hydrolytic degradation in vivo within 48–72 h as well as being able to completely release a variety of different antibacterials at concentrations ranging from 2−10%. The hydrogel showed synergistic antibacterial and antibiofilm activity with various antibiotics and antibiofilm agents in vitro [Developing a new antibacterial coating appears challenging. Since bacterial colonization, from microbial adhesion to an established mature biofilm layer, only takes a few hours , any antin vitro , while iin vitro  and within vitro . Followiin vitro , 29, we From January 2014 to June 2015, 256 patients .Inclusion criteria were the presence of a fresh (<7 days) closed fracture requiring surgical reduction and internal fixation with either a metal plate and/or screws or with an intramedullary nail, in patients aged >18 years. Exclusion criteria were pregnancy, breast-feeding or planning to become pregnant during the study, the presence of a previous or active infection at site of fracture, severe malignancies with a life expectancy of <3 months, previous diagnosis of immune depression (including HIV) or immune suppressive treatment for organ transplantation, known allergy to the antibiotics or to DAC hydrogel constituents, patient not willing or not able to present for the follow-up consultations or if the patient did not sign the informed consent documents or was not able to do so.After routine preoperative work-out, all patients were treated according to the current principles of fracture reduction and internal osteosynthesis. The choice of the surgical approach and the type of osteosynthesis was left to each participating surgeon.Systemic antibiotic prophylaxis was performed with perioperative administration of a single dose of the antibiotic chosen at each center . All patAllowed fixation materials included plating, screw and intramedullary nailing systems from Stryker Inc. , Smith-Nephew  and DePuy-Synthes , respectively.Reconstitution of the DAC hydrogel was performed according to the manufacturer’s instructions. Briefly, the prefilled syringe, containing 300 mg sterile DAC powder, was filled at surgery with a solution of 5 mL sterile water for injection and the desired antibiotic. This allowed the antibiotic-loaded hydrogel with a DAC concentration of 6% (w/v) and an antibiotic concentration ranging from 20−50 mg/mL to be prepared in ~3–5 min, depending on the choice of the surgeon. The surgeons could choose the antibiotic from a list of antibacterials previously tested as being compatible with the hydrogel, including gentamicin, vancomycin, daptomycin, meropenem, rifampicin, and ciprofloxacin    were available with an average follow-up of 18.1 ± 4.5 months (range 12–30) and were considered for further analysis  of DAC hydrogel was needed to coat the implant. Gentamicin and vancomycin were the most used antibiotics, at concentrations of 4 or 2%, respectively.Early wound healing did not show any difference between groups, with an average ASEPSIS score at 7 and 14 days of 1.53 ± 3.94 and 1.93 ± 5.09 in the control group and 1.51 ± 4.14 and 1.33 ± 4.32 in the treated group, respectively. Delayed wound healing occurred in 7 (5.5%) and 5 (3.9%) in the control and treated group, respectively.P = 0.8).Unplanned antibiotic treatment during hospital stay, for reasons other than SSI (mainly urinary or respiratory tract infections), was reported in 12 (9.4%) and 10 (8%) patients in the control and treated groups, respectively  did not show any significant difference between groups Table .Table 4 At an average 12-month follow-up, average SF-12 clinical score did not differ significantly between groups Table .Table 5PP = 0.4).Delayed union was observed in 5 (3.9%) patients in the control group, compared to 2 (1.6%) in the treated group (P = 0.03). One patient in the control group underwent early plate removal for plate intolerance, without reported signs of infection. Detailed information regarding septic complications, including treatment and outcomes are provided in Table No adverse events attributable to the DAC hydrogel were reported. No detectable interaction was observed between the hydrogel and bone healing. Six SSIs were reported in the control group (4.7%), compared to none in the treated group  bone cement for local antibiotic prophylaxis in cemented total joint arthroplasty  and, altOther porous biodegradable materials for local antibiotic delivery, like collagen sponges , cancelld-poly-lactate acid/gentamycin intramedullary coated nails that, until now, only showed some positive results in a limited series of patients [Biodegradable polymers and sol–gel coatings have also been investigated to provide a controlled antibiotic release on titanium , 52 or hpatients .In this setting, an antibiotic-loaded fast-resorbable hydrogel coating may offer ease of use, versatility and large scale applications, opening the way to an affordable wide application of antibacterial implant protection, as recently shown in a multicenter trial focused on infection prevention in total hip and knee replacement .This study has some limitations. First, the follow-up is relatively short. Although the minimum 12-month monitoring appears adequate to detect early post-surgical septic complications, exceeding that defined in the IDSA Guidelines , and app


Neoadjuvant therapy may increase the rate of radical tumour resection in patients with pancreatic cancer. Its impact on tumour recurrence has not been investigated fully. This study aimed to assess the impact of neoadjuvant therapy on patterns of recurrence.A systematic review was performed of articles identified through the PubMed, Scopus, Embase, Ovid and Google Scholar databases that analysed the relationship between neoadjuvant therapy and recurrence published to January 2016. The main endpoint was overall tumour recurrence. Other endpoints included local recurrence, any kind of distant, hepatic, pulmonary or peritoneal metastasis.P < 0·001) and local  recurrence. Neoadjuvant therapy did not reduce the risk of any kind of distant , hepatic , pulmonary  or peritoneal  metastasis.A total of 4257 citations were reviewed. Twelve observational studies comprising 1365 patients were analysed. Neoadjuvant therapy significantly reduced the risk of overall (risk ratio (RR) 0·82, 95 per cent c.i. 0·74 to 0·90; Neoadjuvant therapy reduced the risk of local recurrence but not that of distant metastasis. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is dismal, with a 5‐year overall survival rate of approximately 5 per centversus 66·7 and 16·0 per cent in those who had no neoaduvant therapy. A further studyversus 18·9 per cent respectively), and decreased 2‐year (23·3 versus 42·6 per cent) and 5‐year (29·7 versus 45·9 per cent) local recurrence rates in patients with primary RPC.Several studies have investigated the impact of neoadjuvant treatment in resectable (RPC), borderline resectable (BRPC) and locally advanced (LAPC) pancreatic cancerversus upfront surgery on tumour recurrence in PDAC. In this study, a systematic review and meta‐analysis was performed of studies comparing patterns of recurrence in patients with RPC, BRPC and LAPC who either received neoadjuvant therapy or underwent primary surgery.No in‐depth meta‐analysis has been performed to compare the true impact of neoadjuvant therapy The decision was made to follow the PRISMA guidelinesversus primary surgery were excluded. Only studies containing numerical data regarding recurrent disease after curative surgery with or without previous chemo(radio)therapy were included. Systematic reviews and meta‐analyses were not considered, but were used to find additional studies.To minimize selection bias, two reviewers independently screened topics, abstracts and full‐text articles for the systematic review. Duplicates, all publications without clinical data, reviews and articles published in languages other than English were excluded. All articles that did not specifically identify PDAC or did not provide a comparison of recurrence rates after neoadjuvant therapy Full‐text articles were analysed, and data extracted to correlate the effect of neoadjuvant therapy on pattern of tumour recurrence. The primary endpoint of the meta‐analysis was the overall incidence of tumour recurrence after curative resection. Secondary endpoints included local recurrence, distant metastasis, hepatic metastasis, pulmonary metastasis and peritoneal metastasis. Patients with histologically proven PDAC were included. Other solid malignancies arising from or involving the pancreas  were excluded. Studies that contained patients who all received neoadjuvant or adjuvant therapy, single‐arm phase I/II trials, and all other studies comparing two different neoadjuvant models that did not include an arm with primary surgery patients, were also excluded. All studies in which no curative operation could be achieved after neoadjuvant therapy were excluded. In this context, studies with bypass operations after neoadjuvant therapy or those including a large proportion of patients undergoing R2 resection were excluded from further analysis.To minimize the risk of including doubled patients' data, only one study per study group was included in the meta‐analyses. When there were multiple publications by a group, data were presented to an independent reviewer, who decided to include the most recent paper or the study that had accrued most patients.I2). When heterogeneity was absent (I2 = 0) or low level (I2 less than 50 per cent) in the meta‐analysis, the Mantel–Haenszel fixed‐effect method was used to pool data. When I2 exceeded 50 per cent, a high level of heterogeneity was assumed and the Mantel–Haenszel random‐effects method was used, providing an estimate for an average risk ratio (RR). All results of meta‐analyses are expressed as the pooled RR with 95 per cent confidence intervals. A two‐sided P value was calculated, and a level of significance of α = 0·05 was used. Results of the STROBE items are expressed as median (range) values.RevMan™, version 5.3 software  was used for meta‐analysis. The exact numbers of patients with tumour recurrence, local recurrence, or any kind of distant, hepatic, pulmonary or peritoneal metastasis were pooled. Heterogeneity between the included studies of the different meta‐analysis was quantified using the inconsistency statistic ; these were pooled in a second subgroup analysis. The overall pooled effect of both subgroups was estimated, providing an adequate RR for tumour recurrence pattern.In addition, to provide a more accurate insight into the risk of overall tumour recurrence after neoadjuvant therapy, subgroup analyses were performed including only studies in which most patients received any kind of neoadjuvant radio(chemo)therapy. Consequently, studies that only included patients after neoadjuvant chemotherapy were excluded for this subgroup analysis.Fig. TableOf 4257 citations identified, 12 studies were included in the meta‐analyses  . Only a low level of heterogeneity was present in this meta‐analysis (I2 = 39 per cent), so the fixed‐effect model was used to perform a meta‐analysis that included 564 patients in the neoadjuvant therapy group and 674 in the primary surgery group. Recurrences were observed in 53·5 and 65·7 per cent of the patients respectively. Duration of follow‐up ranged from 11 monthsThe data of patients who had any kind of tumour recurrence following curative resection of PDAC were included. The pooled estimated RR of 11 studies01) Fig. a. Only P = 0·39) . In the subgroup of studies that included more advanced tumour stages, patients with RPC, BRPC or LAPC who received neoadjuvant therapy had a decreased risk of recurrence compared with patients who had surgery first  .The subgroup analyses indicated that, among patients with RPC, there was no statistically significant change in the risk of developing tumour recurrence after neoadjuvant treatment  Fig. a. In th01) Fig. a.Fig. a). The estimated heterogeneity between studies was low. In contrast to local recurrence, neoadjuvant therapy did not influence distant recurrence, which developed in 283 (50·2 per cent) of 564 patients in the neoadjuvant group versus 334 (49·6 per cent) of 674 in the surgery‐first group. The estimated RR of the meta‐analysis, which included 11 studiesP = 0·78) .To analyse the impact of neoadjuvant therapy on local tumour recurrence, 12 studiesP = 0·01) . No effect was seen in the subgroup of patients with RPC, BRPC or LAPC  .This effect was more pronounced in the subgroup analysis, in which patients with resectable disease showed a decreased risk of developing local recurrence. Distant metastasis in patients with RPC after neoadjuvant therapy was associated with an increased risk of developing distant recurrence compared with that in the primary surgery group .The overall risk of developing hepatic metastasis was not affected by neoadjuvant therapy: recurrence rate 27·3 per cent P = 0·98)  and peritoneal   recurrence was no different between neoadjuvant therapy and primary surgery groups.The risk of pulmonary  , predominantly by reducing local recurrence  . No statistically significant effect was observed on the overall risk of distant metastasis  , hepatic metastasis  , pulmonary metastasis   or peritoneal recurrence  .Neoadjuvant chemoradiotherapy reduced the risk of any kind of tumour recurrence , a clinically relevant increase in overall survival was observed. High rates of R0 resection and the more favourable postoperative outcome were in contrast to the preoperative radiological assessment, suggesting that this is unreliable in patients who have received neoadjuvant treatment.The use of preoperative chemotherapy alone (FOLFIRINOX – leucovorin (folinic acid), fluorouracil, irinotecan and oxaliplatin) was associated with less frequent perineural invasion and fewer tumour positive lymph nodes compared with primary surgeryP = 0·009). Recently, the results of the ESPAC‐4 studyversus 25·5 months for gemcitabine alone; HR 0·82, 0·68 to 0·98, P = 0·032). Data from the ESPAC trials underline the benefit of adjuvant chemotherapy in patients with pancreatic cancer.In contrast to neoadjuvant therapy, adjuvant chemotherapy for patients following curative resection is undebated. In the ESPAC‐1 trial, Neoptolemos and colleaguesThe major limitation of the present meta‐analysis is that all included studies were observational. No completed RCT has compared the effects of neoadjuvant chemotherapy or neoadjuvant chemoradiotherapy in PDAC. Thus bias, including selection bias, is present, as definitions of RPC, BRPC and LAPC varied, as did indications for administering neoadjuvant therapy. The type and timing of follow‐up after treatment differed considerably between institutions and clinicians. Compared with mortality, recurrence may be open to interpretation, as the disease may recur clinically, biochemically, radiologically or pathologically. The site of recurrence may be even misinterpreted more frequently, as there is no clinical need to rule out peritoneal metastases if liver metastases have been detected radiologically. For this purpose, included studies were screened for information on follow‐up; all but two studiesStudies were not clear about the numbers of dropouts during neoadjuvant treatment, owing to progressive disease or unresectable tumours at surgical exploration. The different effects of neoadjuvant therapy in patients with RPC, BRPC and LAPC should be stratified according to their radiological tumour stage to avoid any bias caused by mixed cohorts. Only well designed trials will further clarify the true effect of neoadjuvant therapy in pancreatic cancer.The authors declare no conflict of interest.Fig. S1 Forest plot comparing a hepatic, b pulmonary and c peritoneal metastasis following the use of neoadjuvant therapy versus primary surgery in patients with pancreatic cancer. Mantel–Haenszel (M‐H) models were used for meta‐analysis. Risk ratios are shown with 95 per cent confidence intervals. RPC, resectable pancreatic cancer; BRPC, borderline resectable pancreatic cancer; LAPC, locally advanced pancreatic cancerFig. S2 Forest plot comparing a tumour recurrence, b local recurrence and c distant metastasis following the use of neoadjuvant chemoradiotherapy (NCRTX) versus primary surgery in patients with pancreatic cancer. Mantel–Haenszel (M‐H) models were used for meta‐analysis. Risk ratios are shown with 95 per cent confidence intervals. RPC, resectable pancreatic cancer; BRPC, borderline resectable pancreatic cancer; LAPC, locally advanced pancreatic cancerFig. S2 Forest plot comparing a hepatic, b pulmonary and c peritoneal metastasis following the use of neoadjuvant chemoradiotherapy (NCRTX) versus primary surgery in patients with pancreatic cancer. Mantel–Haenszel (M‐H) models were used for meta‐analysis. Risk ratios are shown with 95 per cent confidence intervals. RPC, resectable pancreatic cancer; BRPC, borderline resectable pancreatic cancer; LAPC, locally advanced pancreatic cancerClick here for additional data file.