Dynamic changes in c-MET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of c-MET dynamics could therefore potentially guide c-MET-directed therapies. We investigated the feasibility of 89Zr-onartuzumab PET scans. The HCC827-xenografted mice underwent 89Zr-onartuzumab PET scans before treatment and while receiving biweekly intraperitoneal injections of 100 mg/kg NVP-AUY-922 or vehicle. Ex vivo, tumour c-MET immunohistochemistry was correlated with the imaging results.In vitro membrane c-MET levels were determined by flow cytometry. HCC827ErlRes, an erlotinib-resistant clone with c-MET upregulation, was generated from the exon-19 EGFR-mutant human NSCLC cell line HCC827. Mice bearing HCC827 and HCC827ErlRes tumours in opposite flanks underwent 89Zr-onartuzumab uptake was 26% higher (P < 0.05) in erlotinib-resistant HCC827ErlRes than in HCC827 xenografts, while HCC827 tumour uptake was 33% lower (P < 0.001) following NVP-AUY-922 treatment.In vitro, membrane c-MET was upregulated in HCC827ErlRes tumours by 213 ± 44% in relation to the level in HCC827 tumours, while c-MET was downregulated by 69 ± 9% in HCC827 tumours following treatment with NVP-AUY-922. In vivo, 89Zr-onartuzumab PET effectively discriminates relevant changes in c-MET levels and could potentially be used clinically to monitor c-MET status.The results show that The online version of this article (doi:10.1007/s00259-017-3672-x) contains supplementary material, which is available to authorized users. Dysregulation of c-MET is often found in solid cancers as a consequence of mutation, protein overexpression, gene amplification and auto/paracrine upregulation of its ligand hepatocyte growth factor (HGF). Aberrant activation of c-MET/HGF signalling leads to increased tumour proliferation, invasiveness and angiogenesis, resulting in poor prognosis in various human cancers, including non-small-cell lung cancer (NSCLC) and gastric cancer . NSCLC pSeveral c-MET-directed treatment strategies have been reported. NVP-AUY-922, a non-geldanamycin heat shock protein 90 (HSP90) inhibitor, can potently downregulate EGFR and c-MET, and can overcome c-MET-mediated resistance in an EGFR TKI-resistant clone of NSCLC cell line HCC827 . In a randomized phase II trial in patients with advanced NSCLC, the addition of onartuzumab to erlotinib resulted in an improvement in progression-free and overall survival in patients positive for c-MET on immunohistochemistry (IHC) . HoweverSelection of patients with c-MET amplification or overexpression could therefore potentially be beneficial for therapy decisions, due to their sensitivity to c-MET-directed agents. IHC is generally performed on archived tissues and therefore reflects c-MET status of the tissues at the time of retrieval. c-MET status, however, can vary over time and among lesions, indicating a need for biomarkers able to capture this plasticity for the body as a whole , 16. PET89Zr-onartuzumab, which has been reported to effectively discriminate c-MET expression in human gastric cancer xenografts [89Zr-labelled H2 cys-diabody, H2 minibody [89Zr-PRS110 [89Zr-onartuzumab, these tracers are not readily translatable to the clinic, as none of these tracer protein backbones has yet been administered to patients, and would therefore require extensive safety testing. Furthermore, administration of tracers based on the HGF ligand such as 64Cu-HGF, as well as the bivalent antibody DN30 [For ease of clinical translation, we selected the therapeutic antibody-based PET tracer nografts . Severalnografts . These snografts  or antibminibody , and antr-PRS110 . In contody DN30 , 22, mig89Zr-onartuzumab PET to assess c-MET upregulation-mediated erlotinib resistance, as well as c-MET downregulation after HSP90 inhibitor NVP-AUY-922 treatment in human NSCLC xenografts.We therefore aimed to validate the ability of 2. HCC827ErlRes, a stable erlotinib-resistant subclone, was generated by culturing the parental cell line HCC827 in the presence of 1 μM erlotinib and 50 ng/mL recombinant human HGF (PeproTech) for 2 weeks, followed by 2 weeks in 1 μM erlotinib, a method analogous to that described by Turke et al. [The human NSCLC cell line HCC827 was obtained from the American Type Culture Collection. Cells were authenticated in April 2015 by STR profiling using BaseClear and quarantined until screening showed that they were negative for microbial and mycoplasma contamination. Cells were subcultured twice weekly using Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% fetal calf serum (Bodinco BV) and incubated at 37 °C in a fully humidified atmosphere containing 5% COSurface expression of EGFR and c-MET was assessed using a BD Accuri™ C6 flow cytometer (BD Biosciences). Cetuximab  and onartuzumab  served as primary antibodies for EGFR and c-MET, respectively, and mouse anti-human Fc-specific FITC-conjugated secondary antibody  was used for readout of both EGFR and c-MET expression, with 10,000 events assessed per sample. The sensitivity of HCC827 and HCC827ErlRes cell lines to erlotinib and NVP-AUY-922 after 4 days of treatment was determined using the 3--2,5-diphenyltetrazolium bromide (MTT) assay .N-succinyldesferal-Fe3+ , in triplicate for each ratio, as described previously [2HPO4, 1.3 mmol/L NaH2PO4; pH 7.4) was used as the mobile phase. The ratio of conjugated tetrafluorphenol-N-succinyldesferal-Fe3+ to antibody was determined by the antibody-bound versus unbound 430 nm signal of Fe3+ on SE-HPLC.Onartuzumab was conjugated at five molar ratios  of antibody to tetrafluorophenol-eviously . Df-onar89Zr labelling was performed as described previously [89Zr-oxalate (Perkin Elmer). Maximum attainable specific activity was determined by radiolabelling Df-onartuzumab conjugates with specific activities between 50 and 1,000 MBq 89Zr per milligram of conjugate. The radiochemical purity (RCP) of 89Zr-labelling was assessed by the trichloroacetic acid precipitation test [89Zr-onartuzumab was tested by incubation for 7 days under the following conditions: 0.9% NaCl at 4 °C, human serum at 37 °C and 0.5 M HEPES buffer, pH 7.2, at 37 °C.eviously  using clion test . The sta2CO3 buffer, pH 9.6. 89Zr-Onartuzumab was diluted to 5,000 ng/mL in assay diluent which consisted of PBS, 0.5% bovine serum albumin fraction V (Bio-Connect) and 0.05% Tween 20 (Sigma-Aldrich). Plates were washed with washing buffer (PBS with 0.05% Tween 20) and blocked by incubation for 1 h at room temperature with 200 μL assay diluent, and washed again. Eight dilutions of unmodified onartuzumab at molar excesses between 0.004 and 156.25-fold were premixed 1:1 with diluted 89Zr-onartuzumab solution, and 100 μL aliquots of the mixtures were incubated in wells for 1 h at room temperature. After washing, the wells were broken apart and counted using a calibrated well-type 1282 Compugamma system . Counts were plotted against concentrations of competing unmodified onartuzumab and the half-maximum inhibitory concentration (IC50) was calculated using GraphPad 5.0 . The IC50 was divided by the final tracer concentration  to yield the immunoreactive fraction.The immunoreactivity of Df-onartuzumab conjugates was assessed by a competition assay as described previously. In brief, NuncBreakApart 96-well plates were coated overnight at 4 °C with 100 μL 100 ng/mL c-MET extracellular domain (Genentech) in 0.1 M Na111In labelling, OA-NBC was conjugated with p-SCN-Bn-DTPA (Macrocyclics) as described previously [111In-chloride . The RCP of 111In-OA-NBC labelling was checked by silica gel instant thin-layer chromatography (ITLC) using 0.1 M citrate buffer, pH 6.0, as eluent. Furthermore, OA-NBC was incubated with a 1:5 molar excess of OA-NBC to Df, in a procedure similar to that described for Df-onartuzumab, to yield Df-OA-NBC conjugate for 89Zr-OA-NBC experiments.To determine nonspecific tracer distribution in organs, as well nonspecific tumour uptake, a one-armed isotype control antibody OA-NBC (Genentech) was coinjected and used as control tracer in all experiments. For eviously . Radiolanu; Envigo) were inoculated subcutaneously with xenograft tumours. Mice bearing only HCC827 xenograft tumours were injected subcutaneously with 5 × 106 HCC827 cells into the right flank.Male nude mice  coinjected with an equal amount of 111In-OA-NBC (1 MBq) isotype control via a penile vein. Tumour sizes were 183 ± 63, 234 ± 46 and 180 ± 92 mm3 in the groups treated with 10, 25 and 100 μg, respectively. All tracer injections contained 10 μg radiolabelled 89Zr-onartuzumab (specific activity about 400–500 MBq/mg) and 10 μg 111In-OA-NBC (specific activity about 100 MBq/mg), with cold onartuzumab and OA-NBC added to reach the total stated protein doses. Another group of three mice received 10 μg 89Zr-OA-NBC (effective protein doses 9.7 ± 0.3 μg at 4.47 ± 0.13 MBq) coinjected with 10 μg 111In-OA-NBC (1 MBq) to validate the use of 111In-OA-NBC as a proxy for the 89Zr-labelled control molecule. MicroPET scans were performed 1, 3 and 6 days after injection, followed by ex vivo biodistribution analysis after the final scan.To determine the optimal tracer protein dose and imaging time-point, three groups each of four to six mice inoculated with HCC827 xenografts received 10, 25 or 100 μg 6 HCC827 cells into the right flank and 5 × 106 HCC827ErlRes cells into the opposite flank. The sizes of the HCC827 and HCC827ErlRes tumours were 457 ± 182 and 240 ± 74 mm3 (P < 0.01), respectively, at the time of biodistribution analysis (day 6). Mice received 10 μg 89Zr-onartuzumab (effective protein dose injected 9.7 ± 0.2 μg at 4.42 ± 0.09 MBq) coinjected with 10 μg 111In-OA-NBC (1 MBq) via a penile vein. MicroPET scans were performed 6 days after injection, followed by ex vivo biodistribution analysis.c-MET expression on HCC827ErlRes cells in comparison with that on HCC827 cells was evaluated by inoculating a group of ten mice with 5 × 103 received 10 μg 89Zr-onartuzumab  coinjected with 10 μg 111In-OA-NBC (1 MBq) via a penile vein on day 0. Scans were performed on day 6, after which the mice were treated by intraperitoneal injection with either 100 mg/kg NVP-AUY-922 in 5% glucose (seven mice) or 5% glucose vehicle (six mice) on days 6, 10, 13, 16 and 19. Mice received a second identical tracer injection on day 13 , followed by microPET scans on day 19 and ex vivo biodistribution analysis. Tumours measured 205 ± 127 mm3in NVP-AUY-992-treated mice and 313 ± 198 mm3 in vehicle-treated mice at the time they were euthanised.The effects of HSP90 inhibition on c-MET expression were evaluated in vivo using HCC827 xenograft-bearing mice. Mice bearing tumours of sizes 159 ± 65 and 208 ± 112 mm3 tissue density or measured tumour volume for longitudinal experiments. Scan data are presented as mean standardized uptake values (SUVmean), which were calculated from the mean activity in the ROI divided by the injected dose (corrected for decay) per gram body weight, as described previously [111In and 89Zr were counted in a single measurement at 311–500 keV and 758–1,144 keV, respectively, with crosstalk in the 111In channel corrected using a reference 89Zr dilution series. After decay correction, ex vivo tissue activities were expressed as the percentages of injected dose per gram tissue (%ID/g). Xenograft tumours were formalin-fixed and paraffin-embedded for IHC analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Groningen.All microPET scans were performed using a Focus 220 rodent scanner (CTI Siemens). MicroPET scans were reconstructed and in vivo quantification was performed using AMIDE v. 1.0.4 . Regionseviously . For bioFormalin fixed, paraffin-embedded tissue slices of thickness 4 μm were stained for c-MET using monoclonal rabbit anti-human c-MET antibody diluted 1:400  . Haematot test for paired data. P values ≤0.05 were considered significant.Data are presented as means ± SD. Statistical analyses were performed with GraphPad Prism 5.0 using the two-sided Mann–Whitney test for nonparametric data and the two-sided paired Student’s An erlotinib-resistant clone, HCC827ErlRes, was generated from the parental cell line HCC827 by culturing cells for 2 weeks with 50 ng/mL HGF and 1 μM erlotinib, followed by 2 weeks culture in the presence of 1 μM erlotinib. Surface expression of c-MET on HCC827ErlRes cells as measured by flow cytometry was upregulated to 213 ± 44%, while EGFR surface levels were downregulated to 35 ± 17% of levels in the parental HCC827 cells Fig. . HCC827E89Zr per milligram of Df-onartuzumab with RCP ≥95% at ratios above 1:2 onartuzumab bound to Df  at 23.5 ± 9.4%ID/g. SUVmean values in tumours of the 10, 25 and 100 μg 89Zr-onartuzumab dose groups were 2.7 ± 0.9, 2.9 ± 0.7 and 2.2 ± 0.8, respectively  than in HCC827 tumours . Biodistribution analysis revealed that 89Zr-onartuzumab uptake was 26% higher in HCC827ErlRes tumours (38.1 ± 8.4%ID/g) than in HCC827 tumours . Similar comparisons of tumour-to-muscle and tumour-to-blood ratios for the two groups showed similar trends, but the differences did not reach significance. IHC revealed similar levels of necrosis, as well as higher c-MET expression in HCC827ErlRes than HCC827 tumours, in accordance with the 89Zr-onartuzumab PET and biodistribution data , while uptake values in vehicle-treated mice before and during treatment were similar . Similar comparisons of the tumour-to-muscle and tumour-to-blood ratios for the two groups showed similar trends, but the differences did not reach significance. IHC showed a marked decrease in c-MET expression in the tumours, with similar levels of necrosis, in NVP-AUY-922-treated and in vehicle-treated animals , and insulin-like growth factor 1 receptor (IGF1R). The effect sizes of antibody tracer uptake reduction after target protein downregulation through HSP90 inhibition and everolimus treatment are comparable to the reduction in uptake of 89Zr-onartuzumab after c-MET downregulation found in the present study [89Zr-onartuzumab scans could potentially enlarge the patient population which might benefit from c-MET/HGF-targeted drugs [A limiting factor in the present study may have been the relatively small effect sizes observed for upregulation and downregulation of tracer tumour accumulation. Preclinical studies using nt study –39, and nt study , 41. Fured drugs .89Zr-onartuzumab c-MET PET provides a robust noninvasive and powerful tool that is easily translatable to the clinic for visualizing c-MET dynamics as a possible biomarker for c-MET-mediated resistance to erlotinib and for treatment response to HSP90 inhibition.Based on these promising preclinical results, we conclude that Supplementary Fig. 1a Efficiency of conjugation of Df to onartuzumab at various molar reaction ratios. b Radiochemical purity for 89Zr labelling at various Df to onartuzumab molar conjugation ratios, with specific activities between 50 and 1,000 MBq/mg conjugate. c Retained affinity of 89Zr-onartuzumab at various Df to onartuzumab molar conjugation ratios compared with that of naked onartuzumab in a competition assay. d Stability of 89Zr-onartuzumab in 0.9% NaCl at 4 °C, human serum at 37 °C and HEPES, pH 7.2 at 37° °C up to 7 days (PDF 38 kb)Supplementary Fig. 2a Ex vivo organ uptake in HCC827 xenograft-bearing mice of 89Zr-onartuzumab 6 days after injection at protein doses of 10, 25 and 100 μg  and uptake of 89Zr-OA-NBC 6 days after injection at a protein dose of 10 μg. b Corresponding ex vivo organ uptake in HCC827 xenograft-bearing mice of 111In-OA-NBC 6 days after injection. Data are expressed as %ID/g ± SD (PDF 48 kb)Supplementary Fig. 389Zr-onartuzumab and 111In-OA-NBC both 6 days after injection at a dose of 10 μg in ten HCC827 and HCC827ErlRes tumour-bearing mice. Data are expressed as %ID/g ± SD (PDF 35 kb)Ex vivo organ uptake of Supplementary Fig. 4a Ex vivo organ uptake of 89Zr-onartuzumab 6 days after injection in six vehicle-treated and seven NVP-AUY-922-treated (at a dose of 100 mg/kg) HCC827 xenograft-bearing mice. b Corresponding ex vivo organ uptake of 111In-OA-NBC 6 days after injection in HCC827 xenograft-bearing mice. Data are expressed as %ID/g ± SD (PDF 42 kb)Supplementary Fig. 5a) HCC827 and HCC827ErlRes tumours and (b) vehicle-treated and NVP-AUY-922-treated HCC827 tumours, where score 0+ represents 0–5% necrosis, 1+ 5–15% necrosis, 2+ 15–25% necrosis, 3+ 25–35% necrosis and 4+ >35% necrosis. (PDF 25 kb)Histological grading of necrosis (H&E staining) of  and in vivo PET tumour uptake (SUVmean). (PDF 31 kb)Correlation between ex vivo Below is the link to the electronic supplementary material.


Salmonella Typhimurium.The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.The potential lytic bacteriophages  were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens. Salmonella species is mainly acquired by the production of antibiotic-degrading enzymes, alteration in membrane permeability, and activation of multidrug efflux pumps [Salmonella serotypes has increased the failure in antibiotic treatments [Salmonella infection has become a global public health concern due to the annual increase in morbidity and mortality rates [Over the last few decades, the repeated misuse and overuse of antibiotics has accelerated the emergence of antibiotic-resistant bacteria, leading to serious clinical and public health problems , 2. The ux pumps . Recentleatments –7. The Mty rates . The theSalmonella bacteriophages  against Salmonella enterica serovar Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009, having different antibiotic resistance profiles.Bacteriophage has received much attention as a possible alternative due to the specificity and self-replicating property with no adverse effects on beneficial microflora and human cells . The speS. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009 were purchased from Korean Culture Center of Microorganism , American Type Culture Collection , and Culture Collection of Antibiotic Resistant Microbes , respectively. Escherichia coli ATCC 25922 was used as control strain to evaluate the antibiotic susceptibility. All strains were cultured in trypticase soy broth (TSB)  at 37 °C for 20 h. The cultured cells were harvested by centrifugation at 3000×g for 20 min at 4 °C, washed twice with phosphate-buffered saline , and diluted to 108 CFU/ml prior to use.Strains of S. Typhimurium ATCC 19585 was exposed to ciprofloxacin to induce antibiotic-resistant isogenic strain according to a serial passage assay [Salmonella Typhimurium ATCC 19585 strain was cultured repeatedly in TSB and TSA by increasing ciprofloxacin concentrations from 0.03 to 1 µg/ml. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 were stable for more than 10 passages in antibiotic-free TSB. The strain was cultured in ciprofloxacin-free TSB at 37 °C for 20 h prior to use.The strain of ge assay . SalmoneSalmonella bacteriophages, P22-B1 and P22, were purchased from ATCC and PBST10, PBST13, PBST32, and PBST 35 were obtained from Bacteriophage Bank at Hankuk University of Foreign Studies . The bacteriophages were propagated at 37 °C for 24 h in TSB containing S. Typhimurium KCCM 40253. The propagated bacteriophages were centrifuged at 5000×g for 10 min, filtered through a 0.2-μm filter to eliminate bacterial lysates, and further purified using a polyethylene glycol (PEG) precipitation assay. The bacteriophage titers were determined by using a soft-agar overlay method [7 CFU/ml) in TSB (0.5% agar). The mixture was poured over the pre-warmed base agar lawn. The plates were incubated at 37 °C for 24 h to enumerate the bacteriophages expressed as plaque-forming unit (PFU).y method . In brieSalmonella bacteriophages were determined by transmission electron microscope , located at the Korea Basic Science Institute . The bacteriophages were transferred to the surface of carbon-coated copper film and negatively stained with 5% aqueous uranyl acetate (pH 4.0). After air-drying, the stained bacteriophages were observed under TEM .The morphological properties of Salmonella bacteriophage (5 × 105 PFU/ml) was inoculated into a pre-heated tube containing 9.9 ml of TSB and then treated at each target temperature for 30 min. After heat treatment, each tube was immediately cooled in an ice-bath. The pH stability of bacteriophages was evaluated in phosphate buffered saline  adjusted to pHs ranged from 2 to 12. Each Salmonella bacteriophage (5 × 105 PFU/ml) was exposed to different pH levels at 37 °C for 30 min. The pH-treated bacteriophages were immediately diluted with PBS to avoid further inactivation. After heat or pH treatment, viable bacteriophages were enumerated by using a soft-agar overlay assay.The susceptibility of bacteriophages to heat was evaluated at the range of 30–80 °C for 30 min. For isothermal treatment, 0.1 ml of each Salmonella bacteriophages against S. Typhimurium KCCM 40253 (105 CFU/ml) was evaluated at different multiplicity of infections (MOIs) ranging from 0.01 to 10. The host cells infected by each Salmonella bacteriophage were incubated at 37 °C for 6 h. After incubation, the host cells were enumerated using an Autoplate® Spiral Plating System  and a QCount® Colony Counter . The reduction rate was estimated by comparing with the number of the control cells treated without bacteriophages.The lytic ability of S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009, was evaluated by using an agar disc diffusion assay. Each host strain was diluted to the turbidity of 0.5 McFarland standard and spread on Mueller–Hinton agar plate. Antibiotic discs used in this study were ampicillin (10 µg), cefotaxime (30 µg), cephalothin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), kanamycin (30 µg), levofloxacin (5 µg), meropenem (10 µg), nalidixic acid (30 µg), norfloxacin (10 µg), sulfamethoxazole/trimethoprim (1.25/23.75 µg), and tetracycline (30 µg). The antibiotic discs were placed on Mueller–Hinton agar plate and then incubated at 37 °C for 20 h. The clear zone was measured to evaluate the antibiotic susceptibility. The susceptibility (S) and resistant (R) strains were defined as compared with the susceptibility of the control strain, E. coli ATCC 25922.The susceptibility of host strains, Salmonella bacteriophages against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009 was determined using a spotting test. The serially (1:10) diluted bacteriophages (2 µl each) were spotted onto the soft-agar plates and incubated at 37 °C for 24 h. The formation of clear zone was determined to express the lytic capability.The lytic ability of Salmonella bacteriophages were used to evaluate the lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009. The host strains (105 CFU/ml each) were mixed with bacteriophage (105 PFU/ml) at MOI of 1 and incubated at 37 °C for 12 h. After 12-h incubation, the cultures were centrifuged at 3000×g for 20 min. The collected cells were serially diluted (1:10) with PBS. The proper dilutions were plated on TSA using an Autoplate® Spiral Plating System . After 24-h incubation, the viable cells were enumerated using a QCount® Colony Counter . The lytic activity was expressed as log Nc/Np; Nc and Np denote the counts of bacterial host cells treated without and with bacteriophages, respectively, after 12-h incubation.All analyses were performed in duplicate on three replicates. Data were analyzed using the Statistical Analysis System software . The general linear model (GLM) and least significant difference (LSD) procedures were used to compare means at p < 0.05.Salmonella bacteriophages  were evaluated using TEM  was evaluated at different MOIs of 0.01, 01, 1, and 10  was determined using a spotting assay Fig. . All bacS. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585. The clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 showed the highest resistance to Salmonella bacteriophages .The lytic activity of bacteriophages against various host strains was evaluated as shown in Fig. S. Typhimurium strains, including antibiotic-induced resistant and clinically isolated strains. In order to compare the host specificity depending on the interaction between bacteriophages and host receptors, the isogenic strain was used to induce the antibiotic resistance. The application of bacteriophages can be one possible approach as an alternative agent to control multidrug-resistant S. Typhimurium.This study demonstrates the lytic activity of selected bacteriophages against multidrug-resistant Salmonella bacteriophages tested in this study may not be changed at the broad ranges of temperature and pH, which are the important environmental factors for the lytic activity of bacteriophages [The heat stability of PBST10 and PBST13 was significantly decreased at 70 °C when compared to other bacteriophages Fig. a. PBST10iophages .The different lytic activities of P22 and P22-B1 were observed depending on MOI Fig. . The intS. Typhimurium ATCC 19585 was not different from that to ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (Fig. S. Typhimurium ATCC 19585 was not changed after the induction of antibiotic resistance. The observation implies that P22 can be used as biocontrol agent against multidrug-resistant S. Typhimurium. Bacteriophages have renewed attention to potential alternative over conventional antibiotics [S. Typhimurium CCARM 8009 might be due to the alteration in receptors, leading to the decrease in binding affinity [The specificity of P22 to antibiotic-sensitive affinity . The speaffinity , 24. Theaffinity , 25.S. Typhimurium. The most significant finding of this study was that the ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 was effectively inactivated by P22, but the clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 was resistant to P22 due to the alteration in bacteriophage-binding receptors. PBST10, PBST13, PBST32, and PBST35 showed the lytic activity against the clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009. The results would open the door for developing new bacteriophage control system against multidrug-resistant pathogens. However, in order to use bacteriophage as a potential alternative agent against multidrug-resistant S. Typhimurium, further study is needed to elucidate the alterations in bacteriophage-binding receptors in association with the lytic efficacy of bacteriophages.This study highlights the possibility of using bacteriophages for biocontrol of multidrug-resistant


Differences in lymph node yield and tumour-involved resection margins comparing neoadjuvant therapy plus surgery with surgery alone for oesophageal cancer are unclear. Patients who underwent oesophageal cancer surgery in Sweden in 1987–2010 were included. Patients treated with neoadjuvant therapy were compared with those who underwent surgery alone. Outcomes were the number of examined lymph nodes (main outcome), number metastatic lymph nodes, and resection margin status. Rate ratios (RRs) and 95% CIs of lymph node yield were calculated by Poisson regression, and odds ratios (ORs) and 95% CIs of resection margin status by multivariable logistic regression, both adjusted for confounders. Among 1818 patients, 587 (32%) had received neoadjuvant therapy and 1231 (68%) had not. Lymph node yield was lower in the neoadjuvant therapy group . Fewer metastatic nodes were identified following neoadjuvant therapy . Neoadjuvant therapy associated to decreased risk of tumour-involved resection margins when adjusted for confounders except T-stage , but the association did not remain after adjustment for T-stage . Neoadjuvant therapy seems to decrease the lymph node yield and decrease the risk of tumour-involved resection margins by shrinking primary tumour. In the recent decade, the routine addition of neoadjuvant chemotherapy or chemoradiotherapy to surgery and centralization of surgery to fewer centres have improved the 5-year survival in oesophageal cancer2. More extensive lymphadenectomy has been associated with better survival in some studies4, but the independent prognostic role of more extensive lymphadenectomy has been challenged by recent studies controlling for confounding by surgeon volume6. The debate whether the differences in opportunities of lymphadenectomy between the main surgical approaches, i.e. open transthoracic, transhiatal, or minimally invasive oesophagectomy, influence the survival is also ongoing, but no clear survival differences have been shown thus far10. The use of neoadjuvant therapy might complicate the assessment of lymphadenectomy and how it may influence survival. By shrinking lymph nodes, it is possible that neoadjuvant therapy makes the removal or detection of nodes more difficult. The few investigations examining the association between neoadjuvant therapy and the number of resected lymph nodes show contradictory findings, with some studies reporting a reduction in the number of nodes, while other studies do not17. A main aim of neoadjuvant therapy is to shrink the primary tumour. This in turn could facilitate radical (R0) resection, but this question requires more research18.The curatively intended treatment of most patients with oesophageal cancer includes oesophageal resection with lympadenectomyTaken together, there is a need to better understand how neoadjuvant therapy influences the lymph node yield and resection margins status in oesophageal cancer patients who undergo surgery. The aim of this study was to clarify these questions while taking confounding into account, including the potentially critical factors surgeon volume and tumour stage.In this population-based and nationwide Swedish cohort study from 1987 to 2010, the study exposure was the administration of neoadjuvant therapy (yes or no) before surgery for oesophageal cancer. The primary outcome was the number of removed and examined lymph nodes, while secondary outcomes were the number of removed and examined metastatic lymph nodes and the rate of radical resection without microscopic or macroscopic residual tumour in the resection margin (R0-resection).20, and the updated current version has also been described elsewhere21. Briefly, the cohort included 98% of all oesophageal cancer patients who underwent curatively intended treatment in Sweden between 1987 and 2010. The patients were selected by combining data from the Swedish Cancer Registry for identifying all patients with oesophageal cancer, and the Swedish Patient Registry for selecting only patients who had undergone oesophagectomy. The information from these registers was linked for all individual patients using the Swedish personal identity number, a unique 10-digit identifier assigned to each Swedish resident upon birth or immigration, which is a well-validated tool for research purposes22. For collection of more detailed clinical data, including the number of nodes, resection margin status, surgeon volume and tumour stage, surgery charts and pathology records were retrieved from all hospitals conducting oesophageal cancer surgery in Sweden during the study period. The data retrieved from the medical records followed a detailed predefined protocol, an assessment that has been validated for high concordance20. Comorbidity data were retrieved from the Patient Registry and were defined and categorized using the most recently updated and well-validated Charlson Comorbidity Index23. The study was approved by The Regional Ethical Review Board in Stockholm, Sweden. All methods were carried out in accordance with relevant guidelines and regulations. Individual informed consent was not acquired as this is not necessary for this type of study  according to Swedish law.Earlier versions of this cohort have been used for other clinical studies examining oesophageal cancer surgeryThe treatments, including neoadjuvant treatment, of the individual patients were agreed upon by surgeons and oncologists  together with the patients. The most frequently used neoadjuvant therapy was chemoradiotherapy, consisting of cisplatin- and fluorouracil-based chemotherapy supplemented by radiotherapy in 2Gy fractions for a total dose of up to 40Gy, but also radiotherapy and chemotherapy alone were used. Of those 1767 (97%) patients with data on surgical approach available, transthoracic resection with intrathoracic anastomosis was the dominating (96%) surgical procedure and a gastric tube which was pulled up and anastomosed to the proximal oesophagus was the preferred reconstruction. Only 4% of the patients underwent transhiatal oesophagectomy. No consensus on the extent of lymphadenectomy existed during the study period.a priori specified study protocol, defining and categorizing all exposures, outcomes and covariates. Because of the logarithmic distribution of the lymph node variables, Poisson regression was used to estimate ratios (RRs) with 95% confidence intervals (CIs) for associations between neoadjuvant therapy and number of removed and examined lymph nodes and metastatic lymph nodes. A stratified analysis was performed for T-stage groups. A subgroup analysis was conducted comparing patients receiving neoadjuvant chemoradiotherapy (excluding unknown neoadjuvant therapy and neoadjuvant chemotherapy or radiotherapy) to patients receiving surgery only. Multivariable logistic regression was used to calculate odds ratios (ORs) with 95% CIs for associations between neoadjuvant therapy and non-radical resection margins (R1/R2). In all models, the following covariates were selected as potential confounders: 1) age , 2) calendar year (continuous variable), 3) tumour histology (adenocarcinoma or squamous cell carcinoma), 4) comorbidity , 5) surgeon volume , and 6) pathological T-stage . Missing data covariate data were few  had neoadjuvant therapy prior to surgery and 1231 (68%) had surgery alone. The regimen of neoadjuvant therapy was available for 516 patients, of which 354 (69%) received neoadjuvant chemoradiotherapy, 113 (22%) received neoadjuvant radiation therapy, and 49 (9%) received neaodjuvant chemotherapy. Characteristics of the study participants are presented in Table Among all study participants, 1347 (74%) had information on the number of removed and examined lymph nodes and were thus included in this analysis. The distribution of number of removed and examined lymph nodes was skewed Fig. . In the The number of removed and examined metastatic lymph nodes was also lower among patients who had received neoadjuvant therapy , when compared to patients who had undergone surgery alone . This difference was confirmed in the fully adjusted model showing 24% decrease in number of metastatic lymph nodes after neoadjuvant therapy, compared to surgery only  patients had data on resection margins and were included in the analysis of this outcome. The proportion of patients with tumour-involved resection margins (R1/R2) was lower 14%) in the neoadjuvant therapy group compared to surgery alone group (20%). After adjustment for confounders, except for T-stage, neoadjuvant therapy was associated with a decreased risk of tumour-involved resection margins   nodes27. This might affect the number of removed and examined lymph nodes in the present and previous studies evaluating the association between neoadjuvant therapy and lymph node yield. A weakness in the present study is the low median number of removed and examined lymph nodes, which might be considered a poor oncological resection. This might reflect the lack of centralization and consensus regarding the extent of lymphadenectomy in Sweden during most of the long study period. However, it is unlikely that the extent of lymphadenectomy would differ in those undergoing neoadjuvant therapy or surgery alone after adjustments for calendar year and surgeon volume.Among methodological strengths of the study are the population-based nationwide design, the complete and validated data collection, and the large sample size. To counteract confounding, which is an inherent source if bias in observation studies, adjustments were made for several key covariates. Lymph node yield increases with surgical experience36. Two post-hoc analyses of randomized clinical trials from the Netherlands (n = 320) and France (n = 195), and one French hospital-based cohort study of R0-resected patients (n = 536), suggested a reduced lymph node yield after neoadjuvant therapy compared to surgery alone for oesophageal cancer17. A register-based study from the United States on patients undergoing oesophagectomy for gastroesophageal cancers  suggested that neoadjuvant therapy was associated with a decreased likelihood of obtaining 15 or more nodes, but did not assess specific numbers of nodes37. Another register-based study from the United States , a Taiwanese register-based study , and a hospital-based study (n = 111) from the United States found no association between neoadjuvant therapy and lymph node yield in oesophageal cancer38, but none of these studies adjusted the results for surgeon volume, or other potential confounders.The present study indicates that neoadjuvant therapy reduces the number of removed and examined lymph nodes. The available literature on this topic is limited. Randomized clinical trials have shown that neoadjuvant therapy increases the resectability of oesophageal cancer, but without reporting the lymph node yield11. The stratified analysis showed that the association is the strongest in the high-T-stage category, suggesting that neoadjuvant therapy could reduce the number of lymph node metastases even when no significant down-staging occurs in the primary tumour.Neoadjuvant therapy was also associated with a decreased number of removed and examined metastatic lymph nodes in this study, which has also been observed earlier and is one of the goals of neoadjuvant therapy39. The disappearance of the association after adjustment and stratification for T-stage suggests, in line with a previous study40, that the influence of neoadjuvant therapy on tumour-involved resection margins is mediated by shrinkage of the primary tumour.The risk of tumour-involved resection margins was decreased in the neoadjuvant therapy group of the present study, which is in line with some previous studies41. This study suggests that the expected lymph node yield should be 25% lower after neoadjuvant therapy (and 32% lower after neoadjuvant chemoradiotherapy) than after surgery alone, even when adjusting for important confounders surgeon experience and time. This might not be a concern because recent large studies have not found any survival benefit of more extensive lymphadenectomy after neoadjuvant therapy11, or altogether in oesophageal cancer6. On the other hand, a recent large study from the Netherlands showed better survival  after more extensive lymphadenectomy in oesophageal cancer after neoadjuvant chemoradiotherapy42. However, it is still unclear whether the previously suggested cut-offs for adequate lymph node yield are relevant in the neoadjuvant therapy era, or whether they are a proxy for skill and experience of the surgeon6. The association between neoadjuvant therapy and lymph node yield also indicates that future studies assessing lymphadenectomy should adjust for the use of neoadjuvant therapy.This study has some potential clinical and research implications. The number of removed lymph nodes during surgery is considered an indicator of the quality of the esophagectomyIn conclusion, this nationwide and population-based study with adjustment for several confounders indicates that neoadjuvant therapy reduces the number of removed and examined lymph nodes and the risk of tumour-involved resection margins in patients who undergo surgery for oesophageal cancer. These findings might contribute to changing the view regarding the need for a certain lymph node yield following neoadjuvant therapy and that neoadjuvant therapy should be taken into account in analyses of lymph node yield in future research.Supplementary Table S1


Recycling waste tire rubber by incorporating it into concrete has become the preferred solution to dispose of waste tires. In this study, the effect of the volume content of crumb rubber and pretreatment methods on the performances of concrete was evaluated. Firstly, the fine aggregate and mixture were partly replaced by crumb rubber to produce crumb rubber concrete. Secondly, the mechanical and durability properties of crumb rubber concrete with different replacement forms and volume contents had been investigated. Finally, the crumb rubber after pretreatment by six modifiers was introduced into the concrete mixture. Corresponding tests were conducted to verify the effectiveness of pretreatment methods as compared to the concrete containing untreated crumb rubber. It was observed that the mechanical strength of crumb rubber concrete was reduced, while durability was improved with the increasing of crumb rubber content. 20% replacement of fine aggregate and 5% replacement of the total mixture exhibited acceptable properties for practical applications. In addition, the results indicated that the modifiers had a positive impact on the mechanical and durability properties of crumb rubber concrete. It avoided the disadvantage of crumb rubber concrete having lower strength and provides a reference for the production of modified crumb rubber concrete. The number of waste tires is continually increasing, as a result of the growing use of transport vehicles . Almost Several methods of recycling waste tires have been proposed, including use as a fuel in cement kilns and to produce carbon black ,6. TheseConcerning the reuse of waste rubber in concrete, extensive studies had been conducted ,12,13. TThe properties of crumb rubber concrete were significantly affected by rubber content. Ghaly and Cahill  studied et al. [In order to minimize the loss in strength caused by introducing crumb rubber into concrete, prior surface treatment of rubber particles by modifiers was utilized . Mohammaet al.  pretreatet al.  adopted et al.  mentioneThis paper investigates the mechanical and durability properties of concrete containing waste crumb rubber. The rubberized concrete was produced by replacing the fine aggregate and mixture with crumb rubber at different volume ratios. Compressive strength, splitting tensile strength, axial compressive strength, the modulus of elasticity, freezing-thawing resistance and sulfate resistance were evaluated for concretes with different contents of crumb rubber. In addition, the crumb rubber was pretreated in six modifiers. Additionally, the effects of modifiers on the properties of crumb rubber concrete were comparatively analyzed and discussed.Composite Portland cement of Grade 32.5  with a specific gravity of 3.12, conforming to GB 175-2007 , was useCrumb rubber with the particle size (2–4 mm) specially used to pave a rubber runway was selected as the replacement material, as shown in Modifiers for the pretreatment of crumb rubber, including emulsion, ethoxyline resin, synthetic resin, amino-acrylate (contact glue), chloroprene adhesive and unsaturated resins (marble glue), were commonly used in decoration and construction engineering. They had satisfactory performance in improving adhesion and enhancing strength. The crumb rubbers after pretreatment by modifiers are shown in This study consisted of one plain concrete as the control and 9 crumb rubber concretes. All of the concretes were designed at a constant water-cement ratio of 0.42. Crumb rubber was used as the replacement for an equal part of fine aggregate and mixture. Considering the different specific gravities of crumb rubber and mineral materials, the replacement with crumb rubber was conducted based on the volume other than weight ,30. The Concrete specimens were produced as per the JTG E30-2005 standard . Mixing Compressive strength, splitting tensile strength, axial compressive strength and the modulus of elasticity of hardened concrete were measured as per GB/T 50081-2002 . Comprescnf is the compressive strength of concrete experiencing twenty-five cycles of freezing and thawing. Additionally, cf0 is the compressive strength of the control concrete.Concrete specimens of 150 mm in size cured for 90 days were used to test the durability (freezing-thawing resistance and sulfate resistance) according to GB/T 50082-2009 . The resfK is the anti-corrosion coefficient; cnf is the compressive strength of the tested concrete after fifteen cycles of drying and soaking; cf0 is the compressive strength of the control concrete.In the case of sulfate resistance, the specimens were immersed in sulfate solution. Then, the waterish specimens were dried at a constant temperature of 80 ± 5 °C. The anti-corrosion coefficient of concrete after fifteen cycles of drying and soaking was calculated as Equation (2).The mechanical properties and durability of concrete were significantly affected by introducing crumb rubber into concrete. In this paper, the variation of properties in concrete with the changing of rubber content was investigated. Additionally, the effect of modifiers was evaluated.The mechanical properties of crumb rubber concrete were tested and are listed in The splitting tensile strength of crumb rubber concrete cured at 28 days was measured, as shown in The reduction in axial compressive strength was similar to the compressive strength. It decreased with the increasing of the percentage content of crumb rubber. When the mixture was replaced by crumb rubber, the axial compressive strength significantly decreased as compared to the concrete with fine aggregate replacement. There was a strong correlation between the axial compressive strength and the replacement volume of crumb rubber.The modulus of elasticity represented the deformation capacity of concrete. The test photograph is shown in There are many reasons accounting for the lower strength of crumb rubber concrete . FirstlyFreezing-thawing resistance and sulfate resistance are the important aspects of the durability of concrete. In this paper, the freezing-thawing resistance and sulfate resistance of crumb rubber concrete were investigated. Additionally, the effect of crumb rubber content on freezing-thawing resistance and sulfate resistance was analyzed and discussed.Crumb rubber concrete cured for 90 days was set in the refrigerator for freezing, as shown in The crumb rubber concrete after sulfate corrosion is illustrated in The crumb rubber particles in the rubberized concrete can prevent the formation of cracks and material separation. This may be the reason for the good performance to resisting freezing-thawing and sulfate attack for crumb rubber concrete ,35.As can be seen from the above study about the mechanical properties and durability of crumb rubber concrete, 5% replacement of the total volume of mixture resulted in a high decrease of strength. From a practical viewpoint, the rubber content should not exceed 5% of the total volume of the mixture due to the negative effect on strength. Therefore, crumb rubber concrete with 5% replacement of total mixture was selected to investigate the effect of modifiers on the properties of rubberized concrete.Modified crumb rubber concrete was produced by introducing crumb rubber after pretreatment using modifiers. Compressive strength, splitting tensile strength and axial compressive strength were tested. Compared to the measured strength of unmodified crumb rubber concrete, the percentage was calculated, and corresponding results are listed in The effect of pretreatment methods on freezing-thawing resistance and sulfate resistance was investigated. Additionally, the testing results are listed in Crumb rubber concrete prepared with modified rubber had an obvious improvement in mechanical properties and durability compared to untreated rubber, which could be the result of better bonds between rubber particles and cement paste. Additionally, the treated rubber by modifiers could block the early initiation of internal cracks, which enhanced the freezing-thawing resistance and sulfate resistance . ComprehIn this paper, crumb rubber concretes with different replacement forms and replacement levels were produced. The effect of the volume content of crumb rubber and pretreatment methods on the performances of concrete was investigated. The following conclusions have been obtained.(1) Adding crumb rubber into concrete resulted in a significant decrease of the mechanical properties, but increased the durability. The effect caused by replacing the mixture with crumb rubber was higher than that caused by fine aggregate replacement.(2) Compressive strength, splitting tensile strength, axial compressive strength and the modulus of elasticity were reduced with the increasing percentage content of crumb rubber, while freezing-thawing resistance and sulfate resistance were improved. A 20% replacement of fine aggregate and a 5% replacement of the total mixture with crumb rubber met the safety strength requirements of concrete and had excellent durability.(3) The negative effect of crumb rubber on mechanical strength could be minimized and avoided by pretreatment of the crumb rubber using modifiers. These pretreatments enhanced the adherence between the rubber and cement paste and achieved the uniform distribution of rubber particles in mixture.(4) Synthetic resin significantly improved the mechanical and durability properties of crumb rubber concrete as compared to the other modifiers. The compressive strength, splitting tensile strength and axial compressive strength of crumb rubber concrete prepared with pretreated rubber using synthetic resin increased 12%, 40% and 5%, respectively. Additionally, the strength loss ratio after 25 cycles of freezing-thawing was reduced to 1.2%.


Mycobacterium tuberculosis and links antibiotic production to sporulation in the filamentous Streptomyces venezuelae. Chloramphenicol biosynthesis is directly regulated by MtrA in S. venezuelae and deletion of mtrB constitutively activates MtrA and results in constitutive over-production of chloramphenicol. Here we report that in Streptomyces coelicolor, MtrA binds to sites upstream of developmental genes and the genes encoding ActII-1, ActII-4 and RedZ, which are cluster-situated regulators of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red). Consistent with this, deletion of mtrB switches on the production of Act, Red and streptorubin B, a product of the Red pathway. Thus, we propose that MtrA is a key regulator that links antibiotic production to development and can be used to upregulate antibiotic production in distantly related streptomycetes.MtrAB is a highly conserved two-component system implicated in the regulation of cell division in the Actinobacteria. It coordinates DNA replication with cell division in the unicellular  Streptomyces have complex life cycles and make numerous specialized metabolites, including more than half of all known antibiotics . We . We S. c pathway . The proicidin A , althougS. coelicolor and S. venezuelae, and binds to developmental genes in both vegetatively and developmentally growing species. We have also shown that deletion of mtrB or manipulation of MtrA activity can be used to increase antibiotic production in these Streptomyces species. Given the fact that MtrAB is conserved in all Streptomyces species and in other filamentous actinomycetes, we suggest that manipulation of MtrA activity could be a general tool for upregulating antibiotic production in these bacteria.In conclusion, we have demonstrated that the MtrAB two-component system helps control antibiotic production in the distantly related


At first glance, it might surprise when an editorial series in a scientific journal asks how science will help to create companies and jobs. However, a recent ‘Nature’ editorial has encouraged ‘research leaders in the United States and elsewhere to address the needs and employment prospects of taxpayers who have seen little benefit from scientific advances’ , and a UK government report estimated that antibiotic‐resistant infections kill 700 000 people each year worldwide (https://amr-review.org/). An increase to worldwide 10 million deaths per year is projected for 2050 if antibiotic resistance continues unabated (https://amr-review.org/). Therefore, curbing antibiotic resistance is a major public health priority as also dramatically formulated by the World Health Organization (http://www.who.int/mediacentre/news/releases/2014/amr-report/en/). The media report cases of patients dying in local hospitals from hitherto relatively harmless pathogens if they turn out to be antibiotic resistant and drastic surgical interventions remain the only option. Therefore, ‘Alternatives to Antibiotics’ became research calls of major grant organizations and the title of international conferences.The public health incentive for alternatives is indeed high: the Centers for Disease Control and Prevention (CDC) calculated 23 000 deaths per year in the USA attributed to infections with antibiotic‐resistant bacterial pathogens  to limit antibiotic use in farm animals. FDA now bans the use of ‘medically important’ antibiotics  as antibiotic growth promoters (AGP) in animal husbandry. FDA has also launched discussions to change the status of antibiotics for disease prevention in farm animals from an over‐the‐counter to a veterinary prescription status. Until recently, a farmer could buy a 50‐kg bag of antibiotics in a veterinary feed supply store without a prescription while a personal use of 200 g antibiotic needed a prescription by a doctor. According to a recent FDA report, 62 per cent of antibiotics given to animals in 2015 were medically important for human health  and could thus contribute to the selection of antibiotic‐resistant pathogens particularly when used at subtherapeutic doses. The amounts of antibiotics used in agriculture are huge: 17 million kg of antibiotics were used in farm animals compared to an estimated 4 million kg in human per year in the USA. A major part of these antibiotics is given in small doses to healthy chicken, cattle and pigs to accelerate weight gain, or in slightly larger doses  to healthy animals for preventing infections. With the ban on antibiotics as growth promoters and the proposed regulation to put disease prevention under prescription, the FDA hopes to see less selection pressure for pathogens to develop antibiotic resistance. It is currently not clear whether the FDA ban is binding or only a recommendation. In the USA, change might also be encouraged by consumer groups who put pressure on fast food restaurants to use meat from chicken raised with minimal or no antibiotics and who publish reports about compliance by the different companies.In January 2017, the US Food and Drug Administration (FDA) set new rules . In the animal feed industry, nutritional supplements is a very active and growing field, many new products were launched, and these activities have created an increasing demand for microbiologists to develop alternative growth promoters for farm animals. One approach would be to study the impact of in‐feed antibiotics used at subtherapeutic (subinhibitory) concentrations on the gut microbiome of farm animals  and by the US Department of Agriculture and formulated in a conference report, microbiome research could play an important role in business development  and on the other hand probiotics that reduce the pathogenicity of opportunistic infectious agents . As the animal feed industry in Europe cannot claim for probiotics antimicrobial effects against pathogens – such products would fall under drug regulation – probiotics are currently marketed for growth‐enhancing benefits and as ‘gut flora stabilizer’. Adisseo, number three of the animal feed industry, has just launched a Campylobacter jejuni colonization and increased clostridia in the crop without lowering the pH of the intestinal digesta. A blend of organic acids and essential oils stimulated body weight gain and decreased E. coli, while lactic acid increased Lactobacillus in the chick ileum.Feed supplements are not limited to probiotics and prebiotics, other compounds are increasingly explored for growth promotion, particularly formic acid in protonated form as an ‘acidifier’ of the intestinal tract in chicken possibly reducing diarrhoea impact. Also chemical companies like BASF are active in that field investigating short‐chain fatty acids for nutrition and gut microbiota modulation. In chicken, formic acid reduced et al., et al., Beyond the animal feed supplement field, microbes and microbial compounds are investigated as antimicrobial agents. These approaches include bacteriophages, phage lysins and bacteriocins that directly kill bacterial pathogens by viral infection (phages), enzymatic action (phage lysins) or lytic action (bacteriocins). As treatment and prevention of bacterial infections with these products would represent medical activities, they will fall under drug legislation and need rigorous proof of efficacy, hence substantial financial investments including further job opportunities for microbiologists. As clinical trials have not yet provided a sound support for these concepts as antibiotic alternatives, investments are only increasing slowly and so far only small biotech companies are active in that medical field . Job opportunities will increase when the first successful clinical trials with phage products are published. Notably, a recent controlled clinical trial with phage therapy underlined the need for microbiome research to identify suitable bacterial target pathogens for phage therapy , different bifidobacteria or the yeast Saccharomyces boulardii. These probiotics generally do not re‐colonize the disturbed gut microbiome, but were designed to restore the physiological gut microbiome composition. The mechanism of probiotic action is frequently not clear and defining these processes with microbiome research could lead to ameliorated probiotic strains. Empirically, doctors in some European countries prescribe an Enterococcus faecium probiotic as adjunct to antibiotic treatment to prevent gut microbiome disturbances. Following the success of faecal transplantation in Clostridium difficile patients and building on earlier clinical trials with synthetic defined bacterial strain mixtures, bacterial cocktails for re‐colonization of a gut microbiome disturbed by intensive antibiotic treatment like in C. difficile infections are intensively explored. In fact, oral antibiotic treatment has not only disturbing effects on the gut, but also on other body sites like the vaginal microbiome which can potentially also be alleviated with probiotic approaches. The current trends in microbiome research reinforce the high potential of microbiological research for translation into practical application leading to new companies, extended activities in existing companies and job creation for microbiologists mastering microbiome research as well as classical microbiology. Bacteriology has thus maintained its eminent role as both a basic and applied branch of science, with important impacts on medicine and industry as predicted in a now 100‐year‐old editorial announcing the genesis of a new science . Meta‐analysis suggests that the consumption of specific probiotic strains is associated with lower rates of AAD in children and adults, but not in elderly patients. The studies were of heterogeneous quality, differed for antibiotic type used and probiotic applied. Most studies in that field relied only on a handful of probiotic bacteria, mainly lactobacilli (None declared.


Concrete science is a multidisciplinary area of research where nanotechnology potentially offers the opportunity to enhance the understanding of concrete behavior, to engineer its properties and to lower production and ecological cost of construction materials. Recent work at the National Research Council Canada in the area of concrete materials research has shown the potential of improving concrete properties by modifying the structure of cement hydrates, addition of nanoparticles and nanotubes and controlling the delivery of admixtures. This article will focus on a review of these innovative achievements. Typical concretes consist of ordinary Portland cement (OPC), fillers such as sand, coarse aggregates, admixtures and water. This combination of materials allows concrete to be produced in a fluid form that can be pumped and moulded. The complex chemistry and physical structure of cement hydrates in concrete however mean that issues of fundamental science still need to be resolved. Research at the nanoscale has the potential to contribute to these debates and questions. Analysis at the nanoscale may provide further insight into the nature of hydrated cement phases and their interaction with admixtures, nanofillers and nanofibers. These interactions offer the possibility of modifying cement reactions, creating new surface chemistries (referred to as nanoscience), developing new products for the concrete industry (referred to as nanotechnology), and allowing a more controlled and ecologically friendly manufacturing route to cement and concrete.2, and H2O are represented by C, S, and H respectively. The hyphens in C-S-H indicate indefinite stoichiometry and the hydrate is sometimes referred to as “C-S-H gel”. C-S-H is produced along with calcium hydroxide in the chemical reaction of the silicate phases  with water. C-S-H is the principal binding agent in the cement paste and is responsible for its important properties such as strength and shrinkage. Resolving the structure of this material at the nano scale is an essential part of understanding and predicting its behavior. It is also important in the context of modification and development of novel C-S-H systems discussed in the next section.The main product of the hydration of Portland cement is a nearly amorphous material − Calcium Silicate Hydrate (C-S-H) − that forms up to about 60% by volume of the paste. In cement chemistry, CaO, SiO2 (C/S ratio) in C-S-H is one of the main parameters in defining and controlling the properties of a calcium silicate hydrate system. This value varies from 1.2 to 2.1 in hydrated silicate phases and has an average of about 1.75 . Na. Na28]. Little information exists on the engineering performance of organic/inorganic C-S-H nanocomposites. Data indicating an apparent increase in the modulus of elasticity (40–100%) at low polymer content with respect to cement have been reported . QuantitCurrently, there is an extensive use of chemical admixtures mainly to control/modify the fresh and hardened properties of concrete. The most common admixtures for cement and concrete include accelerators, set retarders, air entraining agents, and superplasticizers. Their successful use requires a basic knowledge of concrete technology, standard construction procedures, and familiarity with cement-admixture interactions. A particular challenge of interest to the authors is to optimize the use of dispersing agents such as superplasticizers in high performance concretes containing high volumes of supplementary cementing materials (SCMs). Dispersing agents such as superplasticizers are commonly used in these concretes. There are, however, practical problems such as loss of workability with time that are controlled by interactions with cement components. Controlling the timing of the availability of an admixture in cement systems is essential for its optimal performance.Control release technology provides a route to prolonged delivery of chemicals while maintaining their concentration over a specific time period. In the field of medicine, a chemical is generally administered in a high dose at a given time, with dose repeated several hours or days later. This method is not economical and sometimes results in damaging side effects. As a consequence, increasing attention has been focused on methods of delivering drugs continually for prolonged time periods and in a controlled fashion. The primary method of accomplishing this controlled release has been through incorporating the chemicals within polymers , biodegr6Al2 (OH)16CO3·4H2O]. Most LDH minerals can be represented by the general formula: [M2+1-x M3+x (OH)2]x+[An-x/n. mH2O]x- where M2+ and M3+ are di- and trivalent metal cations , An- are interlayer anions of charge n, x is the molar ratio of M2+/( M2++ M3+), and m is the number of moles of co-intercalated water molecules per formula weight of the compound. Structurally, cationic brucite-like layers are bound together by the interlayer counter anions as well as water molecules  may create problems. Adsorption, co-precipitation, and also intercalation within the structure of the hydrates formed at the early stage could be possible sources of undesired interactions, leading possibly to a loss of workability [The extensive use of cement and concrete and the frequent occurrence of kuzelite (a sulfate-bearing AFm phase) in hardened concrete ,43 and ikability ,50.There have been a number of applications in cement and concrete where different means of controlling the effect of admixtures via a controlled release technique were used. A number of patents and research articles describe “encapsulation” procedures for delivery of liquids and solids. A corrosion inhibitor, such as calcium nitrite, was dispersed by encapsulation in coated hollow polypropylene fibers . This anetc.) and an inorganic anionic exchanger: hydrocalumite capable of exchanging anions  [Another method to control the release of chemicals in cement-based materials is by “intercalation-de-intercalation”. A cement additive for inhibiting concrete deterioration was developed with a mixture of an inorganic cationic exchanger: a calcium zeolite capable of absorbing alkali ions  . The resversus time characteristic was compared to that of the controlled release formulation CaDisal.More recently, work examined means to control the timing of the release of chemical admixtures through their incorporation in nanoscale composite materials ,56. MoreConcrete is a brittle material with a cement paste binder having a pore structure that contains micro (<2 nm in diameter) and fine mesoporosity (2–50 nm). Depending on its constituents, it can be very strong in compression (>200 MPa ultimate strength), but is generally weak in tension and flexure. It also has relatively low fracture toughness. These differences in strength are due as much or more to the internal pore structure of the concrete than the material itself. Typical concretes, made with mixtures of coarse rock aggregate, sand, OPC and/or other cementitious materials and water, have random packing of those materials that may produce a heterogeneous microstructure. In addition, the hydration reaction between the cementitious materials and the water does not produce an even growth of hydration products, which instead form a complex structure that becomes denser over time. This structure is generally less dense where it contacts aggregates, reducing the bond between the cement matrix and providing a route for cracks to propagate.In traditional concrete, weakness in tension and flexure is overcome by designing to minimize the effects of those forms of loading and by the addition of reinforcing steel to provide adequate strength when the loads do take place. The reinforcing steel rods serve to accommodate the tensile stresses in flexural members. Pre-stressing steel placed under tension keeps the surrounding concrete under compression to prevent cracking due to loading.Although these traditional approaches described above have been very successful, they are limited both in terms of preventing local cracking in the concrete matrix and in allowing the design of structures capable of dealing with high flexural loadings. Fiber reinforcements have been used in concrete to try to overcome these limitations . With tyThe use of carbon nanotubes (CNT) as a reinforcing material is intended to move the reinforcing behavior from the macroscopic to the nanoscopic level. In addition to the well known advantages of these materials as reinforcements, which include extremely high strengths  and YounWhile there has been considerable effort directed toward the development of a variety of CNT composites, OPC composites offer unique challenges with respect both to dispersion and formation of the composite itself. In polymeric composite materials the CNT may be fiber dispersed in a solvent suitable for the production of the composite, with or without functionalization. In metallic or ceramic composites, mechanical methods such as co-grinding may be employed. Neither approach is entirely applicable to OPC composites. Typical solvents used for polymer composites may have adverse effects on OPC hydration properties, while the relative softness of some OPC constituents such as gypsum means that CNT would be unlikely to become well dispersed using a grinding process. Instead, researchers have focused on methods of dispersal that are compatible with OPC chemistry. The primary approach has been to use commonly used admixtures such as water reducing admixtures and superplasticizers as dispersing agents. Research to date suggests that dispersion in polycarboxylate or in solutions of polyacrylic acid can be successfully achieved for multiwalled CNT (MWCNT) ,66,67,68Another dispersion method used in recent work on SWCNT ,70 was tAs with early work on other CNT composite materials, the results of mechanical testing of OPC/CNT composites have been highly variable, with some tests producing significant improvements in compressive strength, Young’s modulus and hardness, while others giving inconsequential changes in compressive strength or significant decreases in Young’s modulus. The best observed performances include a 50% increase in compressive strength in a MWCNT sample , over 60A survey of these results strongly suggests that improvements in composite performance require very specific combinations of admixtures, water to cement ratio and nanotube content. A study of fifteen different combinations of MWCNT, admixtures and w/c ratios found one that produced significant improvements in strength as compared to a control sample . SimilarWhile measurements on bulk samples have been inconsistent, investigations of microstructure have indicated that if the nanotubes are well dispersed there may be potential for improving the mechanical properties of cement-based materials in a more consistent way. Fourier Transform-Infrared Spectroscopy  has showIn addition to the mechanical studies, work has been undertaken on the electrical properties of MWCNT/cement composites. Although the researchers found significant improvements in DC electrical conductivity as compared to control samples, the measured values, on the order of 130–170 ohm-cm ,72, fallBoth the electrical behavior of the composite material and the observed microstructural properties can be understood through an examination of the effects of the CNT on the hydration processes of the OPC. Recent work  has showNanoparticles technology, which covers synthesis, process, characterization and application of nanoparticles, currently attracts considerable attention because of a wide variety of potential and practical application, including medicine, electronics and advanced ceramics. In general, a nanoparticle has a particle size in the range of 1 to 100 nm. The distinctive properties of nanoparticles are significantly different from those of the bulk materials. The application of nanotechnology became a key topic in the field of cement and concrete in the last decade, and consequently the nanoparticle technology started to play an important role as well.2 and Fe2O3 [2O3 and nano-SiO2 were also used to increase the abrasion resistance of concrete for pavement [2 particles have been prepared and their thermal properties were characterized to study the anomalous behaviors of Ca(OH)2 in cement paste [2. It has recently been reported that the TiO2 nanoparticles accelerated the rate of hydration and increased the degree of hydration [The most widely conducted studies on the use of nanoparticles in cement and concrete have been on nano-oxides, especially SiOnd Fe2O3 ,78,79,80nt paste . Also, ont paste . Anotherydration . Its phoydration . Synthetydration . The pos3 can have a significant, beneficial impact on the hydration of OPC/SCM blends [3, potentially solving this roadblock to reducing greenhouse gas production in the construction industry.The use of high volumes of SCMs such as fly ash and blast furnace slag as replacements for OPC in concrete can have environmental as well as economic benefits, through the reduction of green house gases from the production of OPC, diversion of the SCMs from landfills and reduced use of the natural resources used to manufacture OPC. The appropriate use of SCMs can also improve concrete properties and increase the service life of concrete structures. One drawback of using high volumes of SCMs is a resulting delay in initial setting time, which reduces early strength of concrete. However, recent work has shown that nano-CaCOM blends ,87 as co3 was first considered as a filler in cement to replace OPC. However, the results from a number of studies have shown positive effects of CaCO3 additions in terms of strength and acceleration of hydration rate. A study on the accelerating effect of finely ground CaCO3 addition on the hydration of C3S showed that the higher the CaCO3 addition, the greater was the accelerating effect [3 addition on the hydration of cement paste was also observed [The use of CaCOg effect . The accobserved .3 particles are shown in 3 was approximately 5 to 20 µm whereas that of nano-CaCO3 was about 50 to 120 nm. The surface area values of micro-and nano-CaCO3 were 0.35 m²/g and 20 m²/g, respectively. Different cement pastes with variable additions of fly ash and nano-CaCO3 were prepared and analyzed. 3 was added to the OPC/fly ash blend (sample 3), a slight acceleration above that of sample 2 was observed. When nano-CaCO3 particles were incorporated (sample 4), the rate of heat development was significantly accelerated.Scanning electron microscope (SEM) images of both types of CaCO3 is added to OPC/fly ash blend. The microhardness value at 1–day hydration for 100% OPC reached 56 MPa but dropped to 15 MPa for 50% OPC and 50% fly ash blend, with a similar decrease for the blend with micro-CaCO3. However, when 20% nano-CaCO3 was added to the OPC/fly ash blend, the microhardness value increased to 35 MPa. At 3–day hydration, the microhardness value for the OPC/fly ash blend was again increased with the 20% addition of nano-CaCO3 (sample 4) as compared to OPC/fly ash blend alone or with micro-CaCO3. These observations clearly indicate the efficacy of using nano-CaCO3 (as opposed to micro-CaCO3) in improving the initial strength development of OPC containing high volumes of SCMs.Microhardness tests were performed by indentation under static loading  on the sConcrete is the single most commonly used construction material in the world. More than 2.6 billion tons of OPC was produced worldwide in 2007 , correspIn addition, the nature of the construction industry is such that it is easier to implement process innovations, rather than disruptive product innovations. Construction integrates products from a wide range of suppliers and skills from a wide range of contractors, subcontractors and trades into a single finished structure. A change in the way a structure is built can be determined by the construction company itself, but a significantly new product needs to be created by a supplier, understood and approved by architects, engineers and the client and implemented by on-site workers who may need to be specifically trained in its use.All of these factors must be taken into consideration in developing nanotechnology for use in concrete. First, concrete and related products are bulk commodities. Even high value concrete structures require low materials costs and the ability to handle large quantities of material in a safe and environmentally acceptable manner. Second, innovations need to be thoroughly developed and field tested in order to build the knowledge and confidence in the construction community. Finally, concrete structures can be difficult to demolish, often requiring explosive or other high-energy approaches as an initial step to break up the major components of the structure. Nanotechnology used in concrete must therefore be compatible to these traditional practices.Given these constraints, the initial nanotechnology applications in construction are those that provide a clear benefit in terms of added functionality with relatively small amounts of nanomaterials that can be delivered in using standard construction practices and will not affect other aspects of the performance of the material. Novel products that improve the delivery of existing materials, such as the control released admixture work described here, are likely to be next to market. Other innovations, such as nanocalcium carbonate accelerators, will become more common as the price of the nanomaterial falls to the point where it can be used in bulk. Carbon nanotube/cement composites, in contrast, will likely take the longest time to implement as they will require further fundamental research, reductions in CNT prices, the development of specialized delivery techniques and equipment, greater understanding of the environmental impact of CNTs and specialized demolition methods before wide spread adoption can occur.29Si MAS NMR techniques [The emergence of nanotechnology applications in the area of concrete construction is relatively recent and many developments are still in the commercialization process. However, the research described here has economic implications ranging from the indirect impact of improved understanding of the performance of cement and concrete to new products that are in the process of becoming market ready. In terms of basic science, improvements in the understanding of the characteristics of the hydration products of Portland cement (particularly C-S-H) at the nanoscale should facilitate the more efficient manipulation of the nature of cement-based materials. For example, current research has demonstrated that the incorporation of nano-sized C-S-H particles as seeds into cement systems can be used to tailor the overall composition of the C-S-H and monitored using newly developed quantitative chniques . This maThe economic impact of carbon nanotube/cement composite materials is restricted by the high expense of the carbon nanotubes. Even at very low rates of addition, current prices of carbon nanotubes are high enough that the production of significant composite structures is cost prohibitive. Even with large reductions in nanotube prices, the composite material is most likely to be used in niche applications, where the requirements for ultra-high toughness and strength compensate for the nanotube cost and the potential difficulties associated with end of service life demolition. Bridges, nuclear reactor containment vessels, dams in high risk locations and military facilities are among the potential applications.The ability of nanoparticle additions to accelerate the rate of hydration of OPC blends opens the possibility of significantly lowering the content of cement in concrete. High supplementary cementing material (SCM) contents in blended cements can drastically slow hydration, but the effect can be compensated by the presence of a nanoparticle accelerator. A key factor to economic viability is the need for the cost of the nanoparticles to be lower than the cost of the cement that is replaced. This condition in turn implies that much lower nanoparticle contents should be used in the blend than the percentage of cement that was replaced. Providing this condition is met, significant economic benefits are possible through reduction of the amount of cement used in a structure. In addition, increasing the possible percentage of SCMs in OPC blends should produce strong environmental benefits due to the reuse of waste materials and reductions in the greenhouse gases associated with OPC production.Finally, commercial applications for the controlled release (CR) technology are numerous. In concrete construction, they involve in-situ, real-time delivery of accelerators, retarders, superplasticizers and other concrete admixtures used in oil well cements, cast in place, and precast concretes. The interaction of the commercial chemicals with cement minerals and hydration products can therefore be controlled during the hydration process, resulting in improved performance of both admixtures and the resulting concrete products.The examples discussed here show that the range of potential economic outcomes from the application of nanotechnology to cement and concrete reflects the diversity of those applications. The overall potential is, however, very high. Nanotechnology offers a route to precisely engineer concrete for specific applications, reducing costs and increasing performance. Entirely new applications for concrete are also possible. Developing cement and concrete related nanotechnology will therefore have a sustained and important impact on the future of the construction industry.


The medical emergency team (MET) can be activated anytime and anywhere in a hospital. We hypothesized the timing and location of MET activation are associated with seriousness of outcome.We tested for an association of clinical outcomes with timing and location using a university hospital cohort in Japan (n = 328). The primary outcome was short-term serious outcome (unplanned ICU admission after MET activation or death at scene).P = 0.010; night-time: adjusted OR = 2.45, 95% CI = 1.09–5.50, P = 0.030). Patients for whom the MET was activated in public space had decreased short-term serious outcome compared to medical spaces . Night-time (vs. daytime) and medical space (vs. public space) were significantly associated with higher risks of unexpected cardiac arrest and 28-day mortality.Patients for whom the MET was activated in the evening or night-time had significantly higher rates of short-term serious outcome than those for whom it was activated during the daytime (vs. evening: adjusted OR = 2. 53, 95% CI = 1.24–5.13, Patients for whom the MET was activated in the evening/night-time, or in medical space, had a higher rate of short-term serious outcomes. Taking measures against these risk factors may improve MET performance. The concept of a medical emergency team (MET) has been implemented worldwide to rapidly respond to patients in a deteriorating condition, as well as to prevent in-hospital cardiac arrest, unexpected intensive care unit (ICU) transfers, and mortality –3. The mMedical resources may differ between the various departments in a hospital; the baseline characteristics of patients in a deteriorating condition may also vary. These may in turn lead to variation among departments in the frequency of MET activation, as well as in subsequent clinical outcomes. However, evidence is limited to a few investigations comparing this frequency between monitored and unmonitored units , 7; it rTiming is the other important factor in the frequency and clinical outcomes of MET activation—temporal differences in the frequency of MET activation have been reported. For instance, MET activation is more common on Mondays , in the If investigators elucidate the relationships between timing/location and clinical outcome, healthcare providers at the scene may be more aware of at-risk patients. This would improve MET-based care through early recognition and activation.We hypothesized that the timing and location of MET activation are associated with seriousness of outcome. Therefore, using a university hospital cohort in Japan, we investigated whether clinical outcome after MET activation differed by timing and location. The primary outcome variable was short-term serious outcome (unplanned ICU admission or death at scene). The secondary outcome variables were unexpected cardiac arrest upon receipt of MET call and 28-day mortality after MET activation.We conducted an observational study at Chiba University Hospital, Japan. At the time of the study, the hospital had a total of 835 beds, including 22 ICU beds, for medical, surgical, and cardiovascular patients. There were 739 physicians treating an average of 2,064 outpatients and 759 inpatients daily in 2014. Prior to the implementation of our MET into the hospital, all hospital workers had been informed about inclusion and exclusion criteria described below. The notification was completed three months in advance of the MET implementation. The hospital workers were communicated orally and/or by document  in order that everyone working in the hospital knows how to activate our MET.The MET consisted of physicians from the department of Emergency and Critical Care Medicine, as well as nurses/clinical engineers from ICU. The MET can be activated for anyone within the hospital grounds: inpatients, outpatients, visitors, hospital staff, and vendors who require an emergency procedure. All hospital staff, including physicians, nurses, clinical engineers, medical assistants, and technologists can activate the MET in response to the following pre-defined MET activation criteria: obstructed airway, abnormal breathing, circulatory failure, disturbance of consciousness, trauma, unexpected cardiac arrest, a risk of any of these conditions, and any other deteriorating condition that the discoverer considers worthy of MET activation; exclusion criterion is: patients presenting the condition as a consequence of considerable terminal illness. There are no criteria based on the absolute value of vital signs .We screened all patients for whom MET activation was requested between February 2012 and January 2015 (n = 336), excluding eight patients who had data missing. Thus, 328 patients were included in the analysis. The Institutional Review Board at the Chiba University Graduate School of Medicine approved the current study. The review board waived the need for written informed consent.In the present study, short-term serious outcome was defined as unplanned ICU admission after MET implementation, or death at the scene where MET care was initially implemented  12]. Pa. Pa12]. Furthermore, patients were categorized according to the time of the MET call: daytime , evening (17:00–0:59) and night-time (1:00–8:59). We defined Saturdays and Sundays as “weekend”, and the remaining days as “weekdays”. We defined off-hours as during the evening, night-time, or weekend. In the same way, patients were categorized according to the location of the incident: “medical space”, “general wards”, and “public space”. “Medical space” comprised the outpatients’ department, examination rooms , and treatment rooms . “Public space” comprised the hall, stairs, lounge, and hospital parking lot. Sepsis was defined using the 2001 international sepsis definitions .Data was prospectively collected by both the requester and the MET, who completed designated MET data forms, which were then independently checked by data managers, who created the data set for the analysis. We made a report for each case after MET was activated regardless of patients’ severity or managements by MET. There are no data lost once our MET is activated.The primary outcome variable was short-term serious outcome (unplanned ICU admission after MET intervention or death at scene). The secondary outcome variables were unexpected cardiac arrest upon receipt of the MET call and 28-day mortality.U test (for continuous data).Differences among timing of MET calls in terms of the probability of short-term serious outcome, unexpected cardiac arrest, and 28-day mortality  were tested using the chi-square test. Differences in baseline characteristics and clinical outcomes between the Serious and Non-serious groups were analyzed using the chi-square test  or the Mann–Whitney P-value < 0.1 in the univariate analysis of baseline characteristics .As the primary analysis, we used multivariate logistic regression to test for differences in short-term serious outcome by timing  and location . Such an analysis involved adjustment for potential confounding factors based on biological plausibility and previous studies –19 usingP-values of < 0.05 were considered significant. Odds ratios (ORs) and 95% confidence intervals (CIs) were shown. Analyses were performed using SPSS  statistical software.To evaluate the effect of timing and location on the secondary outcomes, we used multivariate logistic regression analysis, which adjusts for confounding factors . Two-tailed Of the 328 study subjects, the MET was activated for the largest number during the daytime ; this was followed by the evening , and finally by the night-time . Furthermore, short-term serious outcome occurred more frequently during the weekend than on weekdays, although this was not a significant result . Similarly, in patients for whom the MET was activated on the general ward, the incidence of serious outcome was lower than in patients for whom it was activated in medical space, although this difference was not statistically significant  and medical space (vs. public space) were significantly associated with higher risks of unexpected cardiac arrest and 28-day mortality . Patients for whom the MET was activated in the evening or at night had a significantly higher incidence of short-term serious outcome than those for whom the MET was activated during the daytime; the same was true of patients for whom the MET was activated in medical space: they had a significantly higher incidence of short-term serious outcome than those for whom the MET was activated in public space. A non-significant trend was also found, whereby short-term serious outcome was more frequent in patients for whom the MET was activated in medical space than in those for whom it was activated on a general ward. Furthermore, night-time and medical space were significantly associated with higher risks of unexpected cardiac arrest and 28-day mortality.In the present study, the incidence of MET activation was higher during the daytime and on weekdays. Previous studies have consistently shown a high frequency of MET activation during the daytime , 10 or wP < 0.05; weekend P = 0.091). To the best of our knowledge, the current study was the first to demonstrate an association between the timing of MET activation and short-term serious outcome.Patients for whom the MET was activated during off-hours had a high probability of short-term serious outcome, unexpected cardiac arrest, and 28-day mortality in univariate analysis . The offP < 0.05). In accordance with this, Jones et al. previously reported a high incidence of in-hospital cardiac arrest during the night-time between 2:00 and 3:00 AM, and between 6:00 and 7:00 AM. They conducted a historical control study to compare the incidence of in-hospital cardiac arrest before and after the introduction of the MET service. They concluded that immediate MET activation might decrease the incidence of cardiac arrests during the night [We also found an association of night-time and weekend MET activation with unexpected cardiac arrest  timing and (2) location of MET activation; this was the strength of the study. However, these results were based on data from single center, and the sample was not large; these constitute the limitations of the study. Further studies using a larger sample from multiple centers may strengthen our study results.Patients for whom the MET was activated had higher rates of short-term serious outcome in the evening, at night-time, and in medical spaces. If we take measures against these differences by increasing the requesters’ and the MET’s awareness of the risk factors, we may improve the afferent and efferent limbs of the MET system, as well as clinical outcomes.S1 File(XLSX)Click here for additional data file.