Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities. Here, we report the capacity of the denitrifying biofilm to sustain environmental changes, and the impact of these changes on the co-occurrence of  H. nitrativorans and  M. nitratireducenticrescens.The biofilm of a methanol-fed denitrification system that treated a marine effluent is composed of multi-species microorganisms, among which 3−) and nitrite (NO2−) concentrations, and to different pHs and temperatures. In a second set of assays, the OB was cultivated in ASW medium for five weeks with (i) a range of NaCl concentrations, (ii) four combinations of NO3−/methanol concentrations and temperatures, (iii) NO2−, and (iv) under oxic conditions. Finally, the OB was cultivated for five weeks in the commercial Instant Ocean (IO) seawater. The growth of the biofilm and the dynamics of NO3− and NO2− were determined. The levels of  M. nitratireducenticrescens and  H. nitrativorans were measured by qPCR.In a first set of assays, the original biofilm (OB) was cultivated in an artificial seawater (ASW) medium under anoxic conditions to colonize new carriers. The new formed biofilm was then subjected to short exposures (1–5 days) of a range of NaCl, methanol, nitrate . In the biofilm cultures cultivated at low NaCl concentrations (0% to 1.0%), persistence of H. nitrativorans strain NL23 was observed, with the gradual increase in concentrations of M. nitratireducenticrescens strain GP59. High levels of H. nitrativorans strain NL23 were found in the IO biofilm cultures. The concentrations of M. nitratireducenticrescens strain JAM1 were lower in most of the biofilms cultures than in OB.In the first set of assays, the biofilm cultures had the capacity to sustain denitrifying activities in most of the tested conditions. Inhibition occurred when they were exposed to high pH (10) or to high methanol concentration (1.5%). In the second set of assays, the highest specific denitrification rates occurred with the biofilm cultures cultivated at 64.3 mM NOH. nitrativorans strain NL23, for which growth was impaired above 1% NaCl in the ASW-based biofilm cultures in favor of M. nitratireducenticrescens strain GP59.These results demonstrate the plasticity of the marine methylotrophic denitrifying biofilm in adapting to different environmental changes. The NaCl concentration is a crucial factor in the dynamics of  Most naturally-occurring microbial biofilms, such as those encountered in bioremediation processes, are composed of multiple microbial species. Studying such complex biofilms is a challenge, as each species can influence the biofilm development. The biofilm microbial community inside a bioremediation process adapts to the prescribed operating conditions and shapes the efficiency of the bioprocess to degrade the pollutant(s) . The mec2) for energy production when oxygen depletion occurs, leading to the production of gaseous nitrogen (N2). Four sequential reactions are strictly required for the reduction of NO3− to gaseous nitrogen, via nitrite (NO2−), nitric oxide and nitrous oxide, and each of these reactions is catalyzed by different enzymes, namely NO3− reductases (Nar and Nap), NO2− reductases (Nir), nitric oxide reductases (Nor) and nitrous oxide reductases (Nos) (3− in different wastewater treatment processes (3− that accumulated (up to 200 mg NO3−-N/L) in the 3 million-L seawater aquarium that contains fish, birds and invertebrates. The fluidized carriers in the denitrification reactor were colonized by naturally occurring multispecies bacteria to generate a marine methylotrophic denitrifying biofilm to be composed of 15–20 bacterial species  . These brocesses . The Mon species . Fluoresommunity .Hyphomicrobium spp. are methylotrophic bacteria that are ubiquitous in the environment . Both Nar systems contribute to the reduction of NO3− to NO2− during growth of strain JAM1 (2O by strain JAM1 (2− reductase gene (nirS or nirK) is absent, which correlates with accumulation of NO2− in the culture medium during NO3− reduction. The genome of H. nitrativorans strain NL23 contains the operons that encode for the four different nitrogen oxide reductases, among which a Nap-type NO3− reductase  on the overall microbial community. The fourth objective of the study was to determine whether denitrifying bacteria other than H. nitrativorans strain NL23 and M. nitratireducenticrescens strain JAM1 are present in the biofilm.We have initiated a study with the aim of assessing the performance of the Biodome denitrifying biofilm when exposed to environmental changes. The original biofilm (OB) taken from the Biodome denitrification system was cultivated in a homemade artificial seawater (ASW) medium, instead of the commercial Instant Ocean (IO) used by the Biodome, under batch mode and anoxic conditions at laboratory scale and exposed to a range of specific physico-chemical parameters. Thus, the objectives of this study were to determine the impact of these changes: (1) on the denitrification performance of the biofilm; (2) on the dynamics of the co-occurrence of the H. nitrativorans strain NL23 occurred without the loss of denitrifying activities. This decrease concurred with the occurrence of the new denitrifying bacteria M. nitratireducenticrescens strain GP59. The genome of strain GP59 has been sequenced and is highly similar to that of strain JAM1. Both strains have the same denitrification genes except that the genome of strain GP59 contains a nirK gene, which is missing in strain JAM1  under specific conditions based on the short exposure assays to develop new biofilm cultures. The impact of these changes on the denitrifying activities and on the population dynamics of M. nitratireducenticrescens strain JAM1 and strain GP59, and H. nitrativorans strain NL23 were measured. Results from the third and fourth objectives are presented by Methylophaga and Hyphomicrobium strains in a marine biofilm system.Here, we report the results for the first two objectives of our study. First, the OB was cultivated in ASW medium under anoxic conditions to derive the reference biofilm cultures. These cultures were then exposed for short periods (1 to 5 days) to different physico-chemical parameters. Such parameters included a range of NaCl, NO2*6H2O, 2 g MgSO4*7H2O, 1 g KCl, 0.5 g CaCl2, 456 µL of FeSO4*7H2O 4 g/L, five mL of KH2PO451.2 g/L, five mL of Na2HPO4 34 g/L. The Instant Ocean (IO) seawater medium was bought from Aquarium systems  and dissolved at 30 g/L. Estimation of its composition and comparison with the ASW medium is provided in 4*7H2O 0.9 g/L, CuSO4*5H2O 0.03 g/L and MnSO4*H2O 0.2 g/L) and with NaNO3  at various concentrations, depending on the experiment  medium was composed of (for 1 liter solution): 27.5 g NaCl, 10.68 g MgClperiment  and 2. Tperiment  and 2. TG1 3−-N/L (21.4 mM NO3−) and 0.15% (v/v) methanol (C/N = 1.5). The vials were incubated at 23 °C and shaken at 100 rpm .The carriers (Bioflow 9 mm) containing the denitrifying biofilm  were taken from the denitrification reactor of the Montreal Biodome and frozen at −20 °C in seawater with 20% glycerol  until us3− and NO2− in the growth medium. The residual suspended biomass was taken for DNA extraction. Methanol and NaNO3 were added when needed if NO3− was completely depleted during the week. During the fifth carrier-transfer cultures, multiple samples (500 µL) were collected at regular intervals for 1–3 days to determine the NO3− and NO2− concentrations. After this monitoring, total biomass in a whole vial was determined by protein quantification.On average once a week, the twenty carriers were taken out of the vial, gently washed to remove the excess medium and the free bacteria, transferred into a new vial containing fresh anaerobic medium, and then incubated again under the same conditions . Samples3− and NO2− concentrations in the vials were then monitored by collecting 500 µL medium samples at regular intervals for 1 to 3 days. Biomass was taken at the end of this monitoring to extract DNA and for protein quantification. All assays were performed in triplicates, except those with NO2−.The carriers containing the reference biofilm cultures were used as starting material to test the impact on the denitrification performance of the biofilm by varying specific physico-chemical parameters . Figure The reference biofilm cultures subsection. The formulation of the ASW medium was adjusted with the prescribed NaCl, NO3−, NO2− and methanol concentrations, and pHs, and the vials were incubated at prescribed temperatures  over time for each replicate (mM h−1). The specific denitrification rates were reported as the denitrification rates divided by the quantity of biomass (mg protein) in a vial (mM h−1 mg-protein−1). Ammonium concentration was measured using the colorimetric method described by Measurements of NOThe DNA was extracted from the suspended biomass and the biofilm that was scraped from the carriers as previously described . The PCRM. nitratireducenticrescens strains JAM1/GP59 (narG1), M. nitratireducenticrescens strain GP59 (nirK), M. nitratireducenticrescens strain JAM1 (tagH) and H. nitrativorans strain NL23 (napA) (Quantitative PCR assays (SYBR green) to measure the concentrations of 3 (napA)  in the b3 (napA) .Statistical significance in the denitrification rates between different types of cultures was performed with one-way ANOVA with Tukey’s Multiple Comparison Test.3−-N/L (21.4 mM NO3−) and 0.15% (v/v) methanol (C/N = 1.5) at 23 °C. The choice of ASW medium was based on our ability to change the composition of the medium, as opposed to the commercial seawater medium (Instant Ocean) used by the Biodome. Each week for five weeks, only the carriers were transferred in fresh medium . As its name implies, the conditions of these cultures were used as a reference to measure changes in the denitrifying activities resulting from changes in physico-chemical parameters in the culture medium.The original biofilm (OB) of the Biodome denitrification reactor was used to inoculate vials containing new carriers to allow the development of a fresh denitrifying biofilm on the carrier surface. The vials were incubated under batch-mode anoxic conditions in a homemade ASW medium (2.75% NaCl) with 300 mg NOh medium . The bio3− and NO2− concentrations were analyzed sporadically for the first four carrier-transfer cultures, and showed increasing rates of denitrifying activities with subsequent transfers. During the fifth carrier-transfer cultures, the concentrations of NO3− and NO2− were analyzed more thoroughly. NO3− was consumed in 4 to 6 h, with accumulation of NO2− that peaked at 10 mM after 4 h. NO2− was completely consumed after 12 h  mM-NOx h−1 mg-protein−1. The production of ammonia was negative in these conditions, which rules out dissimilatory NO3− reduction to ammonium. Further carrier transfers did not improve the specific denitrification rates.The NOter 12 h . The denM. nitratireducenticrescens strain JAM1 that was much stronger in all carrier-transfer cultures, whereas the DGGE band corresponding to H. nitrativorans strain NL23 was no longer visible after the second carrier-transfer cultures , but also its resilience to adverse conditions that could occur during the operation of a bioprocess .The colonized carriers from the Ref300N-23C biofilm cultures were exposed for a short period (few hours to few days) to a range of methanol, NOeratures . These c3− and methanol (fixed C/N at 1.5) showed increases in the denitrification rates compared to the original conditions of the Ref300N-23C biofilm cultures (300 mg-NO3−-N/L with 0.15% methanol), with the highest increases observed at 600 mg-NO3−-N/L with 0.3% methanol (58% increase), and at 900 mg-NO3−-N/L with 0.45% methanol (56% increase) (3−-N/L. This inhibition could have been caused by the methanol concentration (1.5%) in the medium. Variations in methanol concentrations (0% to 0.5% with fixed concentration of NO3− at 300 mg-NO3−-N/L) showed no improvement of the denitrification activities compared to the original conditions (3− concentrations (fixed concentration of methanol at 0.15%) showed the highest denitrification rates (74% increase) at 900 mg-NO3−-N/L  allowed activities to resume for one day with a further 20% NO3− consumption.Increasing the concentrations of NOncrease) . Inhibitnditions . As methNO3−-N/L . Denitri2− has a strong effect on the denitrification rates with 11-fold and 6.6-fold decreases with 400 mg-NO2−-N/L and 200 mg-NO2−-N/L, respectively in the medium, compared to the original conditions  . Howevernditions . MixtureM. nitratireducenticrescens strains JAM1/GP59 was around 105narG1 copies/ng DNA and of H. nitrativorans strain NL23 between 101 and 10−1napA copies/ng DNA.No significant differences in the quantity of biomass  was observed at the end of all these assays between vials. In all the tested conditions, the bacterial diversity profiles (PCR-DGGE profiles) showed no changes  or to 300 mg-NO3−-N/L  at 30 and 36 °C showed between 53% to 74% increases in the denitrification rates compared to the original conditions (3−-N/L and 0.45% methanol (C/N = 1.5), and incubated at either 23 °C or 30 °C , or at 300 mg-NO3−-N/L and 0.15% methanol (C/N = 1.5) at 30 °C (here named 300N-30C). In parallel, the Ref300N-23C biofilm cultures were derived by the same protocol (3− reduction), two-way ANOVA showed that temperature was the main factor affecting the specific denitrification rates (p < 0.001), not the concentrations of NO3− (p > 0.05).As described above, the Ref300N-23C biofilm cultures exposed for a short period to 900 mg-NOnditions . Based oprotocol , Fig. S3protocol . AssuminM. nitratireducenticrescens strains JAM1/GP59 and H. nitrativorans strain NL23. The concentrations of strains JAM1/GP59 increased by two orders of magnitude (1.9 to 2.3 × 105narG1 copies/ng DNA), whereas the concentrations of strain NL23 decreased by 2–3 orders of magnitude (6.1 to 18 × 101napA copies/ng DNA) in the four biofilm cultures compared to the OB . The concentrations of strain JAM1 in the four biofilm cultures ranged from 2.4 to 8.5  × 102tagH copies/ng DNA, which is 3 to 10 times lower than its concentration in the OB (2.5 ×103tagH copies/ng DNA)  of these four biofilm cultures were all similar (same profile illustrated in o the OB . After to the OB . The con/ng DNA) .In the first set of assays, we showed that the Ref300N-23C biofilm cultures could sustain high variations of NaCl concentrations for a short period without affecting the denitrification capacity. Based on these results, we tested the capacity of the OB to acclimate to a range of NaCl concentrations. The dispersed biomass of the OB was cultivated during five carrier-transfers under anoxic conditions in modified ASW medium with NaCl concentrations ranging from 0% to 8% , Fig. S33− took more than 24 h to be consumed and NO2− consumption was minimal. This result contradicts what was obtained in the first set of assays where the Ref300N-23C biofilm cultures subjected for 3 days to 5% and 8% NaCl did not affect the denitrification performance. To investigate this further, another Ref300N-23C biofilm cultures were derived, but after the fifth transfer cultures, the carriers were transferred in 5% NaCl ASW medium (here named 2.75–5% NaCl) and cultivated for another three carrier transfers (20 days). These biofilm cultures were able to sustain this high salt concentration. Compared to the Ref300N-23C biofilm cultures, the 2.75–5% NaCl biofilm cultures had a 74% increase in the denitrification rates. Because the biomass was twice the amount in these cultures, the specific denitrification rates were similar to those found in the Ref300N-23C biofilm cultures , the amounts of biomass developed were 1.3 to 2 times lower than in the Ref300N-23C biofilm cultures . This locultures .4napA cp/ng DNA) were at the same order of magnitude than in the OB (8.7 ×104napA cp/ng DNA) (1napA cp/ng DNA) compared to the OB (M. nitratireducenticrescens strains JAM1/GP59 increased by one order of magnitude in the 0% NaCl and 0.5% NaCl biofilm cultures (ca. 4 × 104narG1 cp/ng DNA) compared to the OB (5.6 × 103narG1 cp/ng DNA), at similar levels than strain NL23 (M. nitratireducenticrescens strains JAM1/GP59 increased by another order of magnitude (1.5 × 105narG1 cp/ng DNA), which is similar to that found in the Ref300N-23C biofilm cultures (2.3 × 105narG1 cp/ng DNA) (1tagH cp/ng DNA) when compared to the OB (2.5 × 103tagH cp/ng DNA) (2 nirK cp/ng DNA) were at similar level as in the OB (1.6 × 102 nirK cp/ng DNA). It increased by one order of magnitude in the 0.5% NaCl biofilm cultures (1.2 × 103 nirK cp/ng DNA) and by two orders of magnitude in the 1.0% NaCl biofilm cultures (2.5 ×105nirK cp/ng DNA), similar to the levels reached in the Ref300N-23C biofilm cultures (2.9 × 105nirK cp/ng DNA)  . As obseo the OB . The levain NL23 . However/ng DNA)  and 3B. /ng DNA) . The con/ng DNA)  and 3B. H. nitrativorans and M. nitratireducenticrescens populations. Another factor that could have influenced these changes is the ASW medium that we used instead of the Instant Ocean (IO) from Aquatic systems Inc. used by the Biodome  were 2.5 lower than in the Ref300N-23C biofilm cultures (x h−1 mg-protein−1) were the same as in the Ref300N-23C biofilm cultures.The important decrease of strain NL23 and the enrichment of strain GP59 in the Ref300N-23C biofilm cultures suggest that the switch from the continuous-operating mode that prevailed in the Biodome reactor to the batch mode in our biofilm cultures could have generated these specific changes in the  Biodome . The disrotein−1 . In the cultures . Howevercultures , its spe4napA cp/ng DNA) as in the OB (8.7 × 104napA copies/ng DNA). In the IO biofilm cultures, the concentrations of strain JAM1 and strain GP59 were both approximately one order of magnitude lower than those of strain NL23  generated biofilm cultures with denitrification rates 2.5 times lower than those in the Ref300N-23C biofilm cultures , NO2− (14.3 to 28.6 mM) and methanol (0% to 0.75%), and a range of pH (4 to 8) and temperature (4  °C to 36 °C). Denitrifying activities were inhibited when the biofilm cultures were exposed at pH 10 or with 1.5% methanol, and were strongly impacted by the presence of NO2−. Low pHs had a strong effect on the denitrification rates, with a 2.7-fold increase at pH 4.0 than at pH 8.0 in the original conditions. At the end of the assays (10–12 h), however, the pH in the medium increased to reach pH 8.0. Denitrification is an alkalization process that leads to the increase of pH in the medium , which allowed a 85% increase in the specific denitrification rates compared to the Ref300N-23C biofilm cultures. We did not try to cultivate the OB in low pHs because of the rapid fluctuations of the pH during the short exposure assays. The NaCl concentration was a critical factor for the persistence of H. nitrativorans strain NL23 in the biofilm cultures. In the ASW-modified media with low NaCl concentrations , high levels of H. nitrativorans strain NL23 were found. Methylophaga spp. requires Na+  and high methanol concentrations (1.5%). The biofilm cultures that resulted in the highest specific denitrification rates were those cultivated for 5 weeks with 64.3 mM NO3−, 0.45% methanol at 30 °C. The OB can sustain growth and denitrifying activities in ASW with low concentrations of NaCl, but showed poor developmental growth in ASW with 5% and 8% NaCl. Although lower biomass developed in the biofilm cultures cultivated in the commercial IO medium, specific denitrification rates similar to the ones of the reference biofilm cultures (ASW medium) were observed. Important changes in the co-occurrence of the populations of M. nitratireducenticrescens and H. nitrativorans were observed in these biofilm cultures when compared to the OB. In the ASW biofilm cultures, substantial growth of a subpopulation of M. nitratireducenticrescens (strain GP59 as representative) with full denitrification capacity displaced H. nitrativorans strain NL23. Strain NL23 however persisted in biofilm cultures cultivated in ASW with low concentration of NaCl, but also in the ones cultivated in the IO medium. This study shows the plasticity of the denitrifying biofilm to adapt to different conditions. Knowing the dynamics of the principal actors in the denitrifying biofilm in response to environmental changes will allow to better predict the behavior of a denitrification system in response to changes in the operating mode. For instance, as the mature biofilm cultures were able to denitrify under low or high NaCl concentrations, this suggests that the Biodome denitrification system could have sustained high variations in salt concentrations.The denitrifying biofilm taken from the Biodome denitrification system, which was frozen for many years, can be revived and cultivated at laboratory scale without the loss of denitrifying activities. The resulting reference biofilm cultures can sustain for a short period of time (2-5 days) changes in NaCl, NO10.7717/peerj.7497/supp-1Supplemental Information 1Figure 1. Nitrate, nitrite and NOx concentrations in the Ref300N-23C biofilm cultures. Concentrations in the original biofilm (OB) and in the Ref300N-23°C biofilm cultures of Methylophaga nitratireducenticrescens (strains JAM1/GP59) and Hyphomicrobium nitrativorans strain NL23 by qPCR.Figure 2. Denitrification rates determined in the Ref 300N-23°C biofilm cultures exposed for short period to different conditions, and the calculation of the relative denitrification rates with statistical analysis.Figure 3. Concentrations in the different biofilm cultures of Methylophaga nitratireducenticrescens  and Hyphomicrobium nitrativorans strain NL23 by qPCR with statistical analysis.Table 3. Denitrification rates and the concentrations of protein measured in the different biofilm cultures, and the calculation of the relative specific denitrification rates with statistical analysis.Click here for additional data file.10.7717/peerj.7497/supp-2Supplemental Information 2Figure S1. Development of the reference biofilm cultures (Ref300N-23C)Figure S2. Schematic of the conditions used to test the denitrification capacity of the Ref300N-23C biofilm culturesFigure S3. Schematic of the procedures used to derive the biofilm cultures cultivated under different environmental conditionsFigure S4. Bacterial community profiles derived by PCR-DGGE of the biofilm cultures cultivated in ASW with different NaCl concentrationsFigure S5. Bacterial community profiles of the Ref300N-23C biofilm cultures and the IO biofilm cultures by PCR-DGGEClick here for additional data file.10.7717/peerj.7497/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.7497/supp-4Supplemental Information 4Table S2. Primers used in this studyTable S3. Denitrifying activities of the Ref300N-23C biofilm cultures exposed for short period under specific conditionsClick here for additional data file.


In amperometric measurements, the sensitivity was more than 4 times higher with 0.39 µA·mM−1·cm−2 for 3D electrodes and 0.09 µA·mM−1·cm−2 for the 2D configuration. The stability analysis of the enzymes on the 3D carbon showed reproducible results over 7 days. The selectivity of the electrode was evaluated with solutions of glucose, uric acid, cholesterol and ascorbic acid, which showed a significantly higher response for glucose.An enzyme-based electrochemical biosensor has been developed with 3D pyrolytic carbon microelectrodes that have been coated with bio-functionalized reduced graphene oxide (RGO). The 3D carbon working electrode was microfabricated using the pyrolysis of photoresist precursor structures, which were subsequently functionalized with graphene oxide and enzymes. Glucose detection was used to compare the sensor performance achieved with the 3D carbon microelectrodes (3DCMEs) to the 2D electrode configuration. The 3DCMEs provided an approximately two-fold higher sensitivity of 23.56 µA·mM Electrochemical biosensors are one of the most successful sensor technologies in terms of real-world applications and commercial maturity ,2,3,4,5.In this work, microfabricated 3D carbon electrodes are evaluated for electrochemical biosensing and their biosensor performance is compared with the one of 2D carbon electrodes fabricated with a similar approach. For our study, one of the simplest and most cost efficient techniques, Carbon Microelectromechanical systems (C-MEMS) technology, is used to fabricate 3D pyrolytic carbon microelectrodes (3DCMEs). In C-MEMS, a patterned polymer precursor template is pyrolyzed at high temperatures (≥900 °C) to obtain well-defined 3DCMEs ,23. SimiGraphene is one of the most studied nanomaterials during the last decade due to its extraordinary properties . Althoug2 layer was deposited on 4-inch Si wafers by thermal oxidation. A first step of UV photolithography with a 17-µm thick layer of the negative epoxy-based photoresist SU-8 2035  was performed to define the pattern of the 2D working (WE) and counter electrode (CE)  A. For thent step D. The SUn the 2D B and 3D n the 2D E carbon The bio-functionalized graphene was synthesized according to a method that was reported previously in reference . Graphen® solution by sonication. The electrode chips were pre-cleaned in O2 plasma for 3 min. The solution was drop casted on the WE area of the 2D  with the working electrode potential fixed at 0.15 V and 0.1 V for the 2D/RGO-PEI/GOx and 3D/RGO-PEI/GOx carbon electrodes, respectively. The operational stability of the bio-functionalized graphene-based 3D/RGO-PEI/GOx electrodes was tested. For this purpose, amperometric measurements were performed every day for seven days using the 3D electrodes stored at 4 °C. The selectivity of the biosensor was evaluated in the presence of other coexisting common easily oxidizable analytes, such as cholesterol, ascorbic acid and uric acid. The glucose level in real human blood serum samples obtained from a local Danish hospital was measured both with the 3D/RGO-PEI/GOx carbon electrodes and a commercially available blood glucose measurement device (Aviva Accu-chek). The samples were handled according to the guidelines of the hospital. For evaluation of the glucose level, human blood serum (3–9 µL) was directly added to the electrolyte solution  on the 3D electrode surface and the steady state current was measured. Three individual measurements were performed for two different samples.All electrochemical measurements in this present work were obtained at room temperature (23 ± 2 °C) using an Autolab System  operated by the NOVA 1.10 software or by a CHI 760C electrochemical workstation equipped with a Faradaic cage. The electrochemical behavior of the bio-functionalized 2D/RGO-PEI/GOx and 3D/RGO-PEI/GOx electrodes was studied by cyclic voltammetry (CV) in 50 µL of 10 mM PBS electrolyte (pH 7). The WE potential varied from −0.2 V to 0.5 V at a scan rate of 20 mVsThe 2D and 3D carbon microelectrodes for enzyme-based electrochemical biosensing were fabricated as reported previously in references ,30. FiguThe electrochemical behavior of the bio-functionalized 3D electrode and glucose biosensing was first studied by cyclic voltammetry. −1·cm−2 and 23.56 µA·mM−1·cm−2, respectively. As clearly seen in −1·cm−2 and 0.09 µA·mM−1·cm−2 for 3D/RGO-PEI/GOx and 2D/RGO-PEI/GOx electrodes, respectively. This means that the LOD in amperometric glucose sensing is 2.5 times and the sensitivity is around 4.3 times higher for the 3D electrode due to the larger active surface area.Furthermore, chronoamperometric glucose biosensing was performed by using both the 2D/RGO-PEI/GOx and 3D/RGO-PEI/GOx electrodes . The amp®) improves selectivity by repelling the uric acid and ascorbic acid through electrostatic repulsion.In order to evaluate a realistic application of the 3DCMEs coated with RGO, the selectivity of the sensor towards some common electroactive species existing in human blood samples, such as cholesterol, ascorbic acid and uric acid, was studied . As seenThe stability of the bio-functionalized graphene-based 3D electrodes during storage was tested. The 3D/RGO-PEI/GOx carbon electrodes were further tested as sensors to evaluate the glucose level in real human blood serum samples. The obtained results were comparable with data from the commercially available self-monitoring blood glucose device for the same samples . The rel−1·cm−2) and lower limit of detection (1.2 µM) for amperometric glucose sensing compared to the 2D/RGO-PEI/GOx electrodes. The lower limit of detection and sensitivity of 3D electrodes were 2.5 times and 4.3 times higher than that of the 2D electrodes, respectively. The developed enzymatic biosensor was highly selective and stable over time for glucose sensing. The electrodes were successfully applied for glucose detection in real blood serum. With the more sensitive 3D carbon microelectrodes, the developed sensor platform could be ideal for highly sensitive electrochemical biosensors.An enzyme-based electrochemical biosensor was developed with bio-functionalized 3D carbon as the working electrode. The 3D/RGO-PEI/GOx pyrolytic carbon electrodes provide a higher sensitivity (0.39 µA mM


The maximum tolerance dose (MTD) of oral administration in mice was 2400 mg·kg−1. The main pharmacokinetics parameters were as following: Cmax of 1.38 μg·mL−1, Tmax of 8 h, a half-life (t1/2) of 6.97 h, an apparent volume of mean residual time (MRT) of 8.76 h, and an area under the curve (AUC) of 10.50 h·μg·mL−1. MHO7 displayed a wide tissue distribution in mice, with most of the compound in liver (3.01 ± 1.53 μg·g−1) at 1 h, then in fat (5.20 ± 3.47 μg·g−1) at 4 h, and followed by reproductive organs with the concentrations of 23.90 ± 11.33 μg·g−1,13.69 ± 10.29 μg·g−1, 1.46 ± 1.23 μg·g−1, and 0.36 ± 0.46 μg·g−1 at 8, 12, 20 and 30 h, respectively. The most influenced genera of gut microbiome belonged to phylum Firmicutes (21 of 28), among which 18 genera originated from the order Clostridiales, class Clostridia, and families of Ruminococcaceae (11 of 18) and Lachnospiraceae (4 of 18). These results provide that MHO7 is suitable for oral administration in the treatment of breast cancer with the target organs of reproductive organs and regulation on Ruminococcaceae and Lachnospiraceae.Sesterterpene MHO7 derived from mangrove fungus is a novel estrogen receptor degrader for the treatment of breast cancer. To explore its safety and pharmacokinetics in vivo, Log P/D values, stability in simulated gastric/intestinal (SGF/SIF), toxicity, and pharmacokinetics studies were carried mainly by liquid chromatography technique coupled with tandem mass spectrometry (LC–MS/MS) method in mice, and the effect of MHO7 on mice gut microbiota at different time points was revealed by 16S rRNA sequencing. Log P/D values ranged 0.93–2.48, and the compound in SGF and SIF is stable under the concentration of 5 mM·L Fusarium sp. and ophiobolins from marine-derived fungus Aspergillus spp. [50 values ranging from 0.08 μM to 85 ± 12 μM [Of the more than 80,000 identified terpenoids , sestertlus spp. ,5,6. Ophlus spp. ,8,9. TheAspergillus ustus 094102 [In our previous study, MHO7  and simulated intestinal fluid (SIF) of MHO7. Then, we investigated acute toxicity of MHO7 in mice by oral administration. Based on these results, the pharmacokinetics and tissue distribution study of MHO7 in mice were carried out. Moreover, the effect of oral administration of MHO7 on gut microbiota at different time points was studied.p values were 1.29 ± 0.05 and 2.48 ± 0.03 at 25 °C and 37 °C, respectively. The Log D values were 1.12 ± 0.03, 0.93 ± 0.02, and 1.26 ± 0.01, respectively. Proudfoot et al. (2005) reviewed the oil–water distribution coefficients of 1791 oral medicines on the market in the past 60 years and there were 37% oral medicines with the Log p values of 1–3 and 22.6% of 3–5 [Log P value of MHO7 was carried out by measuring the solubility of MHO7 in n-octanol and water system at 25 and 37 °C, respectively. The Log D values of MHO7 at physiological conditions of stomach, intestine, and plasma were examined under three pH values of 1.5, 5.0, and 7.4, respectively. The result of HPLC methodological validation is shown in % of 3–5 , illustr−1. At 8 h, the percentage concentrations were 76.19 ± 0.99% in SGF and 94.83 ± 1.14% in SIF after adding MHO7 at 0.05 mM·L−1; however, when the initial addition concentration was 0.5 mM·L−1, the percentage concentrations of MHO7 were 80.2 ± 3.15% in SGF and 89.74 ± 0.86% in SIF, which revealed that MHO7 is more stable in SIF than in SGF. −1, which was also observed in SIF of trypsin precipitation.Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) were prepared in the in vitro incubation system mainly containing pepsin and trypsin, respectively. Concentrations of MHO7 from each time point were obtained by liquid chromatography technique coupled with tandem mass spectrometry (LC–MS/MS) and the stability of MHO7 in SGF and SIF is shown in −1 by oral administration. Within 14 days after oral administration of MHO7, no animals died, and their weight increased with time  of MHO7. The MTD of MHO7 in both male and female KM mice was determined to be 2400 mg·kgith time . There wY) of MHO7 to IS versus the nominal concentration (X) of MHO7 with the standard curves Y = 0.0486392 + 1.8329 × X, Correlation coefficient R2 = 0.9982 for plasma and Y = 0.0608319 + 1.39753 × X, R2 = 0.9976 for tissues. The limits of quantification of MHO7 in mice plasma and tissues were 0.5 and 2.0 nM·L−1, respectively. Then, the relative standard deviations (RSDs) were measured to be in the range of 4.23–6.94% for inter-day precision and 2.02–4.87% for intra-day precision. Moreover, the accuracy ranged 93.57–100.26% for inter-day and 92.50–101.15% for intra-day. In addition, the matrix effects and extraction recoveries of MHO7 in plasma and tissues were 90.05–100.63% and 90.09–93.97%, respectively. Finally, the stability under a variety of storage and handling conditions of MHO7 was stable during the routine analysis for the pharmacokinetics and tissue distribution study. Other results of methodological validation are shown in A HPLC-MS/MS method for the analysis of MHO7 in mice biological samples was developed. Calibration curves of MHO7 in plasma and different tissues were constructed by plotting the peak area ratio  was 479.02 L·kg−1. The elimination half-life (t1/2) was 6.97 h, indicating that MHO7 was absorbed and cleared slowly from the mice plasma. The clearance (CL/F) and mean residence time (MRT) were 476.08 L·h−1·kg−1 and 8.76 h, respectively.The pharmacokinetics and tissue distribution following single oral administration of MHO7 at doses of 500 mg·kgter 16 h A. The phmax of MHO7 in plasma was still low. The potential mechanism of low bioavailability might not be associated with the absorption in the gastrointestinal tract but plasma, such as a high plasma protein combination rate.The percentage of MHO7 in the stomach and intestine at different time points after oral administration of MHO7 is illustrated in −1, as shown in The MHO7 concentrations in other tissues were determined at 1, 4, 8, 12, 20 and 30 h after oral administration at a dose of 500 mg·kg−1), followed by brain (0.95 ± 0.80 μg·g−1) and reproductive organs (0.88 ± 0.68 μg·g−1). At 4 h, the MHO7 was concentrated in fat (5.20 ± 3.47 μg·g−1), reproductive organs (2.29 ± 1.80 μg·g−1) and liver (1.64 ± 1.09 μg·g−1) in sequence. At 8 h, the highest level of MHO7 was detected in reproductive organs (23.90 ± 11.33 μg·g−1), followed by fat (11.15 ± 8.87 μg·g−1) and kidney (1.66 ± 0.74 μg·g−1). However, at 12 h, the levels of MHO7 from high to low were reproductive organs (13.69 ± 10.29 μg·g−1), kidney (8.16 ± 6.23 μg·g−1), and lung (2.34 ± 1.91 μg·g−1). Furthermore, the highest level was exhibited in reproductive organs with the concentration of 1.46 ± 1.23 μg·g−1 and 0.36 ± 0.46 μg·g−1 at 20 h and 30 h, respectively.The pie chart of concentrations of MHO7 in tissues is exhibited in The results indicate that MHO7 was widely distributed in most tissues and decreased obviously after 20 h. In our previous study, MHO7 exhibited antitumor activity against breast cancer cells (MCF-7) by the mechanism of down regulating ERα . Breast Furthermore, the concentration of MHO7 in liver at 1 h was obviously higher than in other tissues, illustrating that MHO7 metabolite in liver occurred firstly. In addition, the concentrations of MHO7 in kidney at 12 h were markedly higher than in other tissues except reproductive organs, which demonstrated the accumulation of MHO7 in kidney and suggested that renal excretion might be a main elimination route for MHO7. Finally, MHO7 exhibited a low concentration in the brain, spleen, and heart, which indicates that blood flow and the perfusion rate of the organ did not play a key role in the distribution of MHO7.To identify the effects in the compositional distribution of cecal microbiota induced by MHO7, the variable regions V3–V4 of 16S rRNA gene of the cecal samples from different time groups after oral MHO7 was sequenced by Illumina HiSeq/MiSeq platforms. In total, 760 million 400-bp paired-end reads were generated, with an average length of 437 bp of 1,262,525 sequences for each sample. To determine whether the sequencing adequately captured the diversity of the gut microbiota, rarefaction and Shannon index analysis was performed indicating that the sequencing depth was sufficient to cover most of the diversity . The resp < 0.05). A remarkable reduction of gut community richness measured by the Sobs index (p < 0.01) and the amount of OTUs (p < 0.001) appeared in MHO7-8 h and MHO7-30 h groups, compared to the control group. However, there was no significant difference in richness, diversity, or OUT number of the gut microbiome between control and MHO7-1 h group, indicating that the effect of oral MHO7 to gut microbiome of mice was influenced over time.Principal coordinates analysis (PCoA) and Hierarchical clustering analysis revealed that a distinct clustering of the microbiota composition for each group A,B. AfteTo analyze the differences among control and treatment groups, a supervised comparison of the microbiota between control and treatment groups was performed by linear discriminant analysis (LDA) and effect size analysis (LEfSe) without any adjustments, which are often used to identify the specific bacterial taxa among different groups. The greatest differences in taxa between each treatment time and the identified key phylotypes as microbiological markers at various phylogenetic levels are shown in Taxonomic representations of the significantly different taxa (cladogram) between control and treatment groups are shown in To profile specific changes after oral MHO7 at different times in gut microbiota community of mice from a taxonomic perspective, the taxon tree from phylum to genus is constructed in At the phylum level, Firmicutes and Bacteroidetes were the dominant bacterial communities in all samples  and therAt the class level, MHO7 markedly diminished the level of Clostridia at 1 h (38.58 ± 11.1%) and 8 h (28.98 ± 20.11%) after oral administration when compared to control group (51.23 ± 18.09%), but the reduction was recovered at 30 h (47.82 ± 20.66%) ; in contAt the family level, the relative abundance of Ruminococcaceae, Christensenellaceae, and Rikenellaceae displayed a sustained reduction at 1, 8 and 30 h after oral administration of MHO7 comparing with control group. The level of Lachnospiraceae and Family_XIII was declined in MHO7-1 h and -8 h groups but raised again in MHO7-30 h group when compared with control group. To the contrary, the relative abundance of Lactobacillaceae, Streptococcaceae, and Bacteroidales_S24-7_group was increased in MHO7-1 h and -8 h groups and decreased in MHO7-30 h group. The level of Peptococcaceae, Prevotellaceae, Alcaligenaceae, and Verrucomicrobiaceae was reduced at 1 h and increased at 8 and 30 h after oral administration of MHO7. On the contrary, norank_o__Mollicutes_RF9 and Anaeroplasmataceae were increased in MHO7-1 h group and decreased in MHO7-8 h and -30 h groups.The relative abundance of Ruminococcaceae was enriched in breast cancer case patients, while the Lachnospiraceae were relatively less abundant ,24. The g__norank_f__Bacteroidales_S24-7_group, g__norank_f__Lachnospiraceae, Lactobacillus, and Rikenellaceae_RC9_gut_group in all groups , inflammatory bowel disease (IBD), and chronic kidney disease [Ruminococcaceae_UCG-010 and Ruminococcaceae_UCG-014 had a negative correlation with SCFA [Ruminococcaceae NK4A214 group and Ruminococcaceae UCG-005 had a positive correlation with the SCFA [Coprococcus_1 genus can reduce the production of propionic acid [In addition, the amounts of  disease ,38, illu disease . The corith SCFA  while ththe SCFA ,41. Bactthe SCFA  and Coprnic acid . In concAspergillusustus isolated from mangrove rhizosphere. The compound was isolated by column chromatography and the purity was controlled by HPLC using the standard curves for quantitative detection. Progesterone (IS), acetonitrile and methanol (HPLC grade), n-octanol, pepsin, trypsin, sodium dihydrogen phosphate, disodium hydrogen phosphate, dilute hydrochloric acid, sodium hydroxide, potassium dihydrogenphosphate, and dipotassium hydrogenphosphate were purchased from Sigma-Aldrich . Ultrapure water was obtained from distilled water using a Milli-Q system .MHO7 (purity ≥ 98%) was obtained from the fermentation products of −1. The inject volume of the sample solution was 10 μL.An HPLC system  was equipped with a LC-20AD binary pump system, a SIL-20AD auto sampler, a CBM-20A system controller, and a CTO-20A oven. Separation of MHO7 and IS from endogenous substances was carried out on an Agilent Zorbax XDB C18 column , which was kept at 30 °C. The mobile phase was composed of 30% phase A (water) and 70% phase B (acetonitrile) with a flow rate of 1 mL·min−1. The gradient elution used to achieve chromatographic separation was as follows: 0–5 min, 10% B; 5–8 min, 10–70% B; 8–12 min, 70% B; 12–18 min, 70–95% B; 18–27 min, 95% B; and 27–30 min, 95–10% B. The inject volume of the sample solution was 10 μL and the autosampler temperature was maintained at 10 °C.Chromatographic separation was achieved using the HPLC system , equipped with a quaternary pump system, a de-gasser, a diode-array detector, an autosampler, and a column oven. Separation of MHO7 and IS from endogenous substances was carried out on a Waters CORTECS C18 column , which was kept at 30 °C. The mobile phase is composed of phase A (water) and phase B (acetonitrile) with a flow rate of 0.4 mL·minMass spectrometric analysis was carried out on a Thermo Scientific TSQ Quantum MS/MS system equipped with an electrospray ionization interface (ESI). Compound dependent parameters and instrumental parameters were optimized by infusing neat solutions of MHO7 and the IS separately by using a syringe pump. A selective-reaction monitoring (SRM) mode was applied for the detection of the transition of m/z 367.3 → 349.2, 307.2 m/z for MHO7 and m/z 315.4 → 109.2, 97.0 m/z for progesterone (IS), respectively. The typical operating source conditions for MS scan in positive ion ESI mode were optimized as follows: sheath gas flow rate, 20; Aux gas flow rate, 5; spray voltage, 3.50 kV; capillary temperature, 320 °C; tube lens, 75.2 V of MHO7 and 89.1 V of IS; and collision energy, 13 V and 11 V of MHO7  and 28 V and 20 V of IS , respectively.The LC–MS/MS method was validated according to the U.S. Food and Drug Administration (FDA) guidance for bioanalytical method validation .−1 were also determined for interference. Each blank sample was tested for exclusion of endogenous interference at the retention times of MHO7 and IS.The specificity and selectivity of the method were investigated by the screening analysis of incubation system solutions and six individual blank mice plasma and tissues samples. Two other incubation system solutions and blank plasma and tissues samples containing an IS concentration of 5.0 mM·L−1 in oil–water system, 0.01–5 μM L−1 in the in vitro incubation system (SGF), 0.01–5 μM·L−1 in plasma, 0.005–5 and 0.05–50 μM·L−1 in gastrointestinal contents, and 0.005–5 and 0.5–50 μM·L−1 in mice liver. LLOQ was determined as the lowest concentration of analytes with %CV (coefficient of variation) not exceeding 20% and accuracy in the range of 80–120%.The linearity of the method was evaluated by using six calibration standards over a calibration range of 0.01–2 mM·LIntra- and inter-batch precision and accuracy of the developed method were investigated by analyzing QC samples at four different concentrations for six replicates. The precision of the method was determined by %CV and accuracy was evaluated by recovery. As per US-FDA guidelines the acceptable limit of %CV was < 20% for LLOQ and ≤ 15% for HQC, MQC, and LQC. The accuracy was calculated as percent difference in mean value of the observed concentration and nominal concentrations of QC samples.n = 6) to the standard analytes solution of same concentration. To study matrix effect, initially, blank SGF, plasma, and tissue samples were processed followed by spiking of the analyte MHO7 and IS to the post processed samples. Further, aqueous solutions of analyte and IS of the same concentrations were prepared and analyzed. The matrix effect was calculated using the equation in the range of 0.8–1.2.The extraction recovery and matrix effects were investigated at HQC, MQC, and LQC concentration levels. Extraction recovery of MHO7 in SGF, mice plasma, and tissues was evaluated by comparing peak area ratios of MHO7 to IS of an extracted sample  and determined by HPLC with the detection wavelength of 234 nm. Each set of solvent systems for experimental logP/D value was the mean of the three determinations.Log P value of MHO7 between n-octanol and water was determined at 25 and 37 °C, respectively. Log D value of MHO7 between n-octanol and phosphate buffer under three different pH  systems were determined at 37 °C. The phases of distilled water and n-octanol were saturated with each other by shaking for 48 h with 250 rpm in a shaking water bath prior to use. Solutions of MHO7 was prepared in concentration of 10 mM·LSGF was prepared with modifications according to Lee et al. (2012) by mixing 100 mL of distilled water with a pH value set to 2.0 with hydrochloric acid and 1 g pepsin solution (800–2000 U/mg of protein). SIF was produced according to USP specifications . Monobasic potassium phosphate (0.68 g) was dissolved in 25 mL of water. Then, 7.7 mL of 0.2 M NaOH was added to adjust the pH to 6.8. To this, 1 g of pancreatin was added and shaken gently until dissolved and the volume adjusted to 100 mL with water. Pancreatin was added after adjusting the pH of the solution to 6.8 to avoid precipitation of the enzyme.−1 for HQC, MQC and LQC, respectively.Aliquots (199 µL) of SGF and SIF were placed in 1.5 mL microcentrifuge tubes and incubated at 37 °C for 10 min in a water bath, respectively. Different concentrations of MHO7 (1 µL) were added to each of the above microcentrifuge tubes to start the reaction with being incubated at 37 °C at 250 rpm in a shaking water bath. The reaction was stopped by adding ice ACN (800 µL) to each tube at intervals of 0, 0.5, 1, 2, 4, 6, and 8 h. The samples were vortexed for 15 min and centrifuged at 4 °C and 15,000 rpm for 15 min. After centrifuging the supernatant layer was taken directly for LC-MS analysis. Each experiment was carried out in triplicate, and average values were plotted. QC samples were prepared in the same way of inactivating pepsin in SGF incubation system at the concentrations of 1, 0.1 and 0.01 μM·LMale and female KM mice of 8–10 weeks of age (20–22 g) were used in acute toxicity experiment and only female KM mice were subjected in other animal experiments. All mice were purchased from the Vital River Laboratory Animal Technology Co. Ltd. . The mice were bred in a standard environment  with a 12-h light/dark cycle and food and water were provided ad libitum. All animal protocols were approved by the Animal Care and Use Committee of Wuhan University Center for Animal Experiment ABSL-Ⅲ Laboratory and complied with the Guide for the Care and Use of Laboratory Animals . In total, 130 mice were used for in vivo studies under the ethical approved protocols.−1, bw) diluted in corn oil the next morning. Then, mice were placed with free access to chow and water. The mice were observed for two weeks in all groups, and the weight was recorded at 0, 1, 7, and 14 days. The number surviving and their behaviors was also recorded every day.Male and female KM mice of 8–10 weeks of age (20–22 g) were used in acute toxicity experiment. After animals were acclimatized for 1 week, 20 female and 20 male mice were randomly and evenly divided into four groups. The mice of acute toxicity experiment were fasted overnight and administered by the oral dose of MHO7  linear regression analysis. The regression equation of the calibration curve was then used to calculate the concentration of MHO7 in the plasma. QC samples and standard curve series solutions were prepared in the same way of blank plasma. The concentrations of HQC, MQC, and LQC were 5, 0.5, and 0.05 μM·L−1, respectively, and the concentrations of standard curve series solutions were 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, and 0.01 μM·L−1.Ninety female KM mice of 8–10 weeks of age (20–22 g) were divided randomly into 15 groups, and, after fasting for 12 h, the mice in other experiments were oral administrated at the dose of 500 mg·kgn = 6 per group) were collected at 1, 4, 8, 12, 20, and 30 h, and the tissues including heart, liver, spleen, lung, kidney, stomach, intestine, brain, muscle (hind limb), fat (white adipose tissue ), reproductive organs  stomach and its contents, and intestine and its contents, and the blood samples were simultaneously collected. All the tissues were washed with cold physiological saline (4 °C), excess fluid blotted, accurately weighed, and subjected to the processing as described in the sample preparation. QC samples and standard curve series solutions were prepared in the same way of blank gastrointestinal contents and livers, respectively. The concentrations of HQC, MQC, and LQC in mice tissues were 10, 1, and 0.1 μM·L−1, respectively, and the concentrations of standard curve series solutions were 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, and 0.005 μM·L−1 for high and low concentrations of standard curves.The animals and treatment methods were the same as in the pharmacokinetic studies. Afterwards, the biological samples (n = 6 per group) mice were collected at 1, 8 and 30 h after oral administration of 500 mg·kg−1 MHO7 meanwhile control group was collected at 8 h after oral the same volume corn oil, respectively. All of the cecal samples were collected in sterile environments and containers, snap-frozen in liquid nitrogen, and then stored at −80 °C.Fresh cecal samples from 24 , 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit  and quantified using QuantiFluor ™ -ST  according to the manufacturer’s protocol.Microbial DNA was extracted from 24 cecal samples using the E.Z.N.A.® soil DNA Kit  according to manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer , and DNA quality was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5’- ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) by thermocycler PCR system . The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C; 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C; and a final extension at 72 °C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM·LPurified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. .http://drive5.com/uparse/) at a 97% similarity level with a novel “greedy” algorithm that performs chimera filtering and OTU clustering simultaneously. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using confidence threshold of 70%.Raw fastq files were quality-filtered by Trimmomatic and merged by FLASH according to the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Sequences whose overlap was longer than 10 bp were merged according to their overlap with mismatch no more than 2 bp. (iii) Sequences of each sample were separated according to barcodes (exactly matching) and Primers , and reads containing ambiguous bases were removed. Operational taxonomic units (OTUs) were clustered using UPARSE .Data for each group are presented as mean ± SD. Statistical significance of different formulations were measured by one-way analysis of variance (ANOVA) followed by the Tukey Kramer multiple comparison test, using SPSS software version 14.0 , and the statistical significance was expressed by a p-value of less than 0.05. The main pharmacokinetic parameters of MHO7 was fitted by the average mean of a group in each time point with the Phoenix WinNonLin software version 6.3 . The gut microbiome analysis was performed using the free online platform of Majorbio Cloud Platform  of MHO7 was 2400 mg·kgAfter oral administration of MHO7 for 8–30 h, the diversity and richness of gut community were significantly reduced. Although MHO7 had little influence on the level of dominant microbes at the phylum level, we observed that the relative abundance of 28 genera showed significant change among control and treatment groups. Most of the genera influenced belong to Firmicutes (21 of 28), most of these genera (18 of 21) under the order Clostridiales, class Clostridia. The remarkably changed genera classified to clostridia came from family Ruminococcaceae (11 of 18) and Lachnospiraceae (4 of 18). The downregulation of Ruminococcaceae and upregulation of Lachnospiraceae at family level of MHO7 has potential in the treatment of breast cancer ,24, but


Escherichia coli can cause fatal diarrheal diseases in both animals and humans. However, no antibiotics or antimicrobial peptides (AMPs) can adequately kill resistant bacteria and clear bacterial endotoxin, lipopolysaccharide (LPS) which leads to inflammation and sepsis. Here, the LPS-targeted smart chimeric peptides (SCPs)-A6 and G6 are generated by connecting LPS-targeting peptide-LBP14 and killing domain-N6 via different linkers. Rigid and flexible linkers retain the independent biological activities from each component. SCPs-A6 and G6 exert low toxicity and no bacterial resistance, and they more rapidly kill multiple-drug-resistant E. coli and more effectively neutralize LPS toxicity than N6 alone. The SCPs can enhance mouse survival more effectively than N6 or polymyxin B and alleviate lung injuries by blocking mitogen-activated protein kinase and nuclear factor kappa-B p65 activation. These findings uniquely show that SCPs-A6 and G6 may be promising dual-function candidates as improved antibacterial and anti-endotoxin agents to treat bacterial infection and sepsis.Pathogenic  Wang ZL and Wang XM design bactericidal peptides in which an antimicrobial domain is fused to a domain that facilitates the neutralisation of lipoplysaccaride (LPS) to prevent inflammation associated with the targeting of Gram-negative bacteria. They characterise their properties and structures, and show their efficiency in vitro and in vivo. Escherichia coli can cause outbreaks of diarrheal diseases in both animals and humans1. Globally, ~1.7 billion cases of diarrheal disease occur, killing 760,000 children every year2 and US$ 6.9 billion in losses for farmers and industries. E. coli that cannot be killed by the last resort antibiotic–colistin has been found in samples from animals, meat products and patients in China3. Lipopolysaccharides (LPSs), also termed endotoxins, are a major component of the outer membranes of Gram-negative bacteria and are released from the cell wall during bacterial growth4. LPS plays a key role in the pathophysiology of sepsis and shock6. Simultaneously, LPS is also a prime natural barrier that can protect bacteria from attack by drugs7. Although antibiotics have a rapid antibacterial effect, they have some shortcomings, including the development of bacterial resistance, weak LPS-neutralizing capacity and stimulating a 3–20-fold acceleration in the release of LPS into the bloodstream, which can induce various pro-inflammatory responses9. To date, no antibiotics can adequately treat sepsis10. Therefore, it is very necessary to find novel candidates that can clean the battlefield after killing the bacteria, including neutralizing the LPS toxicity and antagonizing the downstream cascade.Gram-negative pathogenic 12. However, these broad-spectrum AMPs may disrupt the normal flora of the body and can lead to numerous adverse side effects13. Therefore, the activities against the desired bacterium of some AMPs have been specifically improved by attaching a targeting region to generate novel, specifically targeted chimeric peptides (CPs) with little impact on the normal flora; these can contain functionally independent targeting and killing domains14. It has been demonstrated that some CPs such as G10KHc, M8(KH)-20, M8-33, S6L3-33, and Syn-GNU7 can enhance selectivity and improve in vitro killing activity against targeted bacteria16. However, these studies only provide a basis for the technology in which target-specific CPs were generated against some limited bacterial species, and little attention has been given to their toxicity, resistance, in vivo antibacterial/anti-endotoxic activity.Recently, increasing attention has been given to antimicrobial peptides (AMPs) due to their broad-spectrum antimicrobial activity and low level of induced bacterial resistance17. Empirical linkers are generally classified into in vivo cleavable, flexible, and rigid linkers. Cleavable linkers, cleaved by proteases under certain physiological conditions, are commonly applied in fusion proteins to target tumor sites19. Flexible linkers ((GS)n or (G)n) are most commonly used in CPs such as Syn-GNU7 and LHP7 to increase the spatial separation between two domains due to their flexibility20. Comparably, rigid linkers ((EA3K)n or (XP)n) have also been successfully applied to construct fusion proteins, to retain a fixed distance between the functional domains, which may be more efficient than the flexible linkers22. However, to our knowledge, thus far, no study has been reported for the rigid linkers used in AMPs.The successful construction of CPs requires indispensable functional elements and linkers that play a vital role in improving the folding, stability and intrinsic biological activities24. Moreover, a marine AMP-N6 displays potent bactericidal activity and can neutralize LPS25. Meanwhile, bacterial resistance is not developed against N6, but it exhibits some cytotoxicity25. Here, the smart CPs (SCPs)-A6 (pdb ID: 6K4W) and G6 (pdb ID: 6K4V) are generated by connecting LBP14 (targeting domain) with N6 (killing domain) via a rigid linker ((EA3K)2) and a flexible linker (G4S), respectively ) can retain significant binding ability to LPS and inhibit the binding of LPS to LBP3K)2 or a flexible linker G4S 2 and G4S linker retained a helical conformation in A6 (ranging from E15-K24) and random coil in G6 (G15-S19)  analysis showed that the   of A6 and G6 0.88–3.52 μΜ) did not show improvement when compared to N6 (0.4–1.6 μM) against  μΜ did nE. coli CVCC195 within 1 h, which is more rapid than N6 (4 h) , a 1 × or 2 × MIC of A6 and G6 could completely kill  h) Fig. , indicatE. coli in the mixed E. coli-S. aureus cultures. G6 displayed higher activity than A6 . This indicates that SCPs-A6 and G6 can preferentially kill the targeted bacteria in the mixed species cultures due to the attachment of the LPS-targeting domain LBP14.The selectivity killing result showed that both A6 and G6 selectively killed E. coli CVCC195  for 1 h and at different pH values (2–10) for 3 h, both A6 and G6 retained their intrinsic antibacterial activity against MDR 195 Fig. , c, indi195 Fig. . The ant195 Fig. .25, indicating that A6 had markedly less cytotoxicity than G6 and N6.After a 1–2 h incubation with serum, only 27–7% and 68.5–29.5% of A6 and G6, respectively, remained intact in native patterns, which was lower than for N6 (93–87.5%) Fig. . Both A6E. coli CVCC195 cells  images revealed that a normal cell morphology and intact cell surface were observed in the untreated lls Fig. . Howeverlls Fig. . For MDRE. coli CVCC195 cells remained a normal cellular shape and uniform cytoplasmic electron density in transmission electron microscopy (TEM)  Fig. . After t30. As shown in Fig. 31.LPS can form large micellar aggregates in water, and fluorescein isothiocyanate (FITC) fluorescence is quenched in FITC-labeled LPS micelles; peptide binding to LPS leads to the dissociation of LPS aggregates and an increase in fluorescence33. However, A6 had no positive peak at 195 nm. After binding to LPS, the antiparallel structure of A6 and G6 was decreased by 12.23% and 11.46%, lower than N6 (45.21%). In contrast, random coils of A6, G6, and N6 were increased by ~7%, 6.97%, and 26.7%, and their helices were elevated by 3.76%, 3.06%, and 19.84%, respectively , α-helices (10.97–13.46%), and β-turns (15.09–17.18%), with a characteristic positive peak at 225 nm and a negative peak at 200 nm –DPO2000:O2, Arg1 (HH21)–DPO2000:O2, Lys7–GMH1004:O7, Lys14–DPO2004:O6, Lys14–DPO2004:O5, Arg37–PO42004:O2, Arg37–GMH1005:O6, and Arg37–PO42004:O4. Comparably, fewer (two or three) hydrogen bonds were formed between LPS and LBP14 (Lys7 (HZ1)–GMH1005:O6 and Arg9 (HH21)–MYR1014:O2), LBPN6 (Lys14 (HZ1)–FTT1013:O2, Ala34 (HN)–KDO1003:O8 and Asn35 (HD22)–KDO1003:O5), A6CK (Arg5 (HH11)–PA11000:O4 and Arg5 (HH22)–KDO1003:O1A), and G6CK (Arg9 (HH21)–GLC1007:O6 and Arg9 (HH22)–GLC1007:O4) . Supplementary Fig. 25. As shown in Fig. 24.The BODIPY-TR-cadaverine (BC) displacement assay was used to further determine the ability of peptides to interact with LPS3 1 per Ms, 1.20 × 10‒3 1 per s, and 7.68 × 10‒7 M, respectively, which were superior to those of G6  and N6   was used to monitor their real-time interactions. All peptides except N6CK could bind to LPS or lipid A in a concentration-dependent manner , interleukin-6 (IL-6), and interleukin-10 (IL-10) to ~1065, 558, and 246 pg per ml, respectively After intraperitoneal injection of FITC-labeled peptides or free FITC, in the control group, free FITC was rapidly distributed and slowly cleared from the mouse body Fig. . At 0.5 E. coli (LD100 of 5 × 108 CFU) or LPS (LD100 of 13.5 mg per kg), followed by treatment with peptides or antibiotic. The untreated mice in the control group died within 2 d (for E. coli) and 4 d (for LPS) . After treatment with 0.125–0.25 μmol per kg PMB, the survival rates of the mice were 20–100%.To investigate the in vivo efficacy of the SCPs, mice were intraperitoneally injected with MDR PS) Fig. . In the 36. The effects of peptides on cytokines were determined by ELISA. A significant increase in TNF-α (348.3 pg per ml) and IL-6 (538.3 pg per ml) was observed in LPS-injected mice, as compared to a blank control group  Fig. . The suroup Fig. . At a do39. The effects of the SCPs on the IAP level were shown in Fig. Intestinal alkaline phosphatase (IAP) can dephosphorylate LPS and thus relieve the LPS toxicityTo investigate whether the SCPs protect mice from LPS-induced lung injury, the lungs were dissected and examined at 1 d and 6 d after treatment with peptides. The lungs from mice injected with LPS alone were significantly swollen and damaged, with non-uniformly distributed alveoli and a large number of inflammatory cells Fig. . There wThe effects of the SCPs on LPS-induced signal pathways involved in inflammation were detected by western blotting. As shown in Fig. E. coli remain one of the major severe health threats1. LPS, covers >70% of the outer leaflet of Gram-negative bacteria40, is spontaneously released from the outer membrane during bacterial growth or after exposure to antibiotics, and it is responsible for sepsis41. Thus, LPS can be a valid drug target to develop anti-endotoxic and antimicrobial compounds. In this study, SCPs-A6 and G6 were designed by connecting LBP14 (a LPS-targeting domain) with N6 (a killing domain) via linkers, to retain the desired bioactivity of each domain.Diarrheal diseases induced by Gram-negative bacteria such as MDR 43 thus, a proper linker is needed to connect the two functional domains to reduce their interference with each other, improve folding and retain the domains’ bioactivity17. Rigid and flexible linkers have been successfully applied to construct some CPs or proteins, such as Syn-GNU7, LHP7, and LL-37-haFGF44. Similarly, in our study, the higher MIC values of LBPN6 without a linker compared to SCPs indicated its very weak antibacterial activity, which may be related to a relatively limited spatial distance between the two domains , is often observed in some LPS-binding molecules45, suggesting that A6 and G6 are likely to have more LPS-binding sites than N6. This was also precisely confirmed by MD and NMR analysis of the peptide-LPS interaction . The Boman index, hydrophobicity and alpha helix content of the peptides were analyzed, predicted and calculated by the Antimicrobial Peptide Calculator and Predictor ,All peptides, including the SCPs and their scrambles, were synthesized by Mimotopes , and their purity was greater than 90%. The physicochemical properties  of A6, G6, and their parents were calculated by ProtParam (http://crdd.osdd.net/raghava/antibp2/submit.html), and HeliQuest (http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParamsV3.py), respectively.AntiBP2  to make the NMR samples. A series of 1D and 2D spectra, including 1H, 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC, and 1H-15N HSQC, were acquired for resonance assignment and structure analysis. All the TOCSY experiments were carried out using an 80 ms MLEV-17 spin-lock with a field strength of 6839 Hz. The mixing time for each NOESY experiment was 150 ms. The spectra widths were 7396.4 × 7396.4 Hz for 1H–1H, 7396.4 × 21124.9 Hz for 1H–13C, and 7396.4 × 1945.6 Hz for the 1H-15N correlation spectra. A total of 256, 140, and 128 complex points were collected for the indirect proton, carbon and nitrogen dimensions, respectively, and 512 complex points were collected for the direct observation dimension. The chemical shifts were referenced to the DSS at 0.00 ppm for the proton and indirectly referenced for the carbon and nitrogen dimension57. All NMR data were processed using NMRPipe and analyzed with the CcpNmr program suite58. Signal assignment and inter-proton distance restraints were derived from the NOESY spectrum. Backbone dihedral angle restraints (Φ and Ψ angles) were derived using the program DANGLE incorporated in the CcpNmr suite, and thus the secondary structure prediction was made. Structure calculation and NOE assignment were performed simultaneously by using the program CNS and ARIA260. A total of 100 structures were calculated and 20 (for A6) or 10 (for G6) structures with the lowest total energy were selected to perform a refinement procedure in water. The atomic coordinates of the peptides and NMR data had been deposited in the Protein Data Bank and the Biological Magnetic Resonance Data Bank (see ‘data availability’ for detail). The dihedral angle distribution was analyzed by Procheck-NMR61 and the ramachandran statistics for the ordered residues of A6 (residues 3–23 and 29–43) in the most favoured regions, additionally allow regions, generously allowed regions and disallowed regions were 88.7%, 11.3%, 0.0%, and 0.0%, respectively. And those for G6 (residues 9–12 and 24–38) were 88.3%, 11.7%, 0.0%, and 0.0%, respectively.The structures of the SCPs in aqueous solvent were analyzed by solution state NMR at 298 K on an Agilent DD2 600 MHz spectrometer equipped with a cold probe. For each peptide, 5 mg was dissolved in 500 µl of aqueous solvent . Peptides or antibiotic were diluted with PBS by 2-fold dilution and were added to each well (10 μl per well). Polymyxin B (PMB) and PBS were used as the positive and negative control, respectively. The plates were incubated at 37 °C for 16–18 h until visible turbidity was observed in the negative control. The lowest concentration that could completely inhibit bacterial growth was the MIC value of the peptides or antibiotic against the tested strains. All experiments were repeated three times.The MIC values of A6, G6, the parental peptides (N6 and LBP14), LBPN6, scramble controls , and PMB were determined by the microtiter broth dilution method25. MDR E. coli CVCC195 cells were cultured to logarithmic phase, diluted in Mueller Hinton broth (MHB) (1 × 105 CFU per ml) containing 1 × or 2 × MIC of A6, G6, N6, or PMB, and cultured at 37 °C (250 rpm). Physiological saline was added as a control. Bacterial samples (100 μl) were removed at different time points  and were counted on MH solid plates. The time-killing curve of A6 and G6 was plotted.The bactericidal effect of peptides was investigated according to previous methodsThe selectivity of A6, G6, and N6 against Gram-negative and Gram-positive bacteria was determined using the mixed-culture bactericidal kinetics assay, as described in the Supplementary Information.E. coli CVCC195 using a previous method with minor revisions25. The N6, A6, and G6 solutions were treated at different temperatures (20–100 °C) for 1 h, dissolved and incubated at different pH values (2–10) for 3 h, or incubated with trypsin or pepsin for 4 h. The inhibition zone diameters were measured after incubation at 37 °C for 18–24 h. The peptides were dissolved in H2O or mice serum at 37 °C to detect their serum stability and samples were removed at different time points to determine their residual activity by the inhibition zone or reverse-phase high-performance liquid chromatography (RP-HPLC) methods63.The thermal, pH and enzyme stability of A6 and G6 were evaluated against MDR 25. Mid-log phase E. coli CVCC195 (1 × 105 CFU per ml) was added into 96-well plates and incubated with A6, G6 or antibiotic (from 32 × to 0.25 × MIC). After 16–18 h of incubation at 37 °C, cells from the second highest concentration showing visible growth were used to inoculate the subsequent cultures. The MIC values for drug passage cultures were obtained as described above. This process was repeated for 30 d.Bacterial resistance against the peptides was assessed via the MIC assays25 hemolysis (%) = [/] × 100%.To determine the hemolysis of the peptides, eyeball blood was collected from 6-week-old specific pathogen-free (SPF) ICR mice. The blood was centrifuged at 4 °C for 10 min (1500 rpm), washed three times with 0.9% physiological saline, and diluted to an 8% suspension. The peptides were dissolved in 0.9% physiological saline (0.5–256 μg per ml). Red blood cell suspensions and peptide solutions (100 μl) were mixed in 96-well plates, centrifuged at 4 °C for 5 min (1500 rpm), and incubated at 37 °C for 1 h. The supernatant was added into a 96-well plate, and the UV absorbance was measured at 540 nm using a microplate reader. Physiological saline and 0.1% Triton X-100 were used as 0% and 100% hemolytic controls, respectively. The hemolytic ratio was calculated as follows:64. RAW 264.7 cells (2.5 × 105 cells per ml) were added into a 96-well plate (100 μl per well) and cultured at 37 °C  for 24 h. The medium was then removed and the cells were washed twice with PBS. The peptide solutions (from 0.5 to 128 μg per ml) were then added to the plates (100 μl per well), incubated for 24 h and washed twice with PBS. Next, 5 mg per ml MTT (100 μl per well) was added and incubated for 4 h, followed by the addition of 150 μl per well dimethyl sulfoxide (DMSO). After a few minutes of shaking in a shaker, the crystals in the bottom of wells were completely dissolved, and the absorbance of each well was measured at 570 nm. The cell survival was calculated according to the following formula: survival (%) = (Abs peptide/Abs control) × 100%.The cytotoxicity of A6 and G6 was evaluated using the 3--2, 5-diphenyltetrazolium bromide (MTT) methodE. coli CVCC 195 and S. aureus CVCC43300 cells and were incubated at 37 °C for 2 h. PBS was used as the negative control. After centrifugation, the bacterial precipitation was added with 2.5% glutaraldehyde and the cells were fixed at 4 °C for 24 h. After ethanol dehydration, drying and coating, samples were visualized by a QUANTA200 SEM 65.SEM was used to examine the ultrastructural changes in bacteria treated with peptides. Peptides  or PMB (4 × MIC) were added into mid-log phage MDR 65.The processing and preparation of TEM samples was similar to that of SEM until glutaraldehyde fixation. After washing with PBS, bacterial cells were fixed in 1% citric acid buffer for 1 h, dehydrated in a series of acetone (50–100%) and embedded, followed by preparation into ultra-thin sections and staining with 1% uranyl acetate. The samples were observed in a JEM1400 . At least 200 bacterial cells were visualized and scored for the observed characteristicsexcitation = 490 nm, λemission = 515 nm). FITC-labled LPS without peptides was used as a control66.To evaluate whether peptides can depolymerize LPS multimers, 180 μl of FITC-labeled LPS (0.5 μg per ml) and 20 μl of different concentrations of peptides  or PMB were added into a 96-well plate, respectively. The fluorescence intensity was detected in a microplate reader (λE. coli 0111:B4 LPS solution were determined at room temperature using a Pistar π-180 spectrometer. A6 (200 μg per ml), G6 (200 μg per ml), PMB (200 μg per ml), or N6 (25 μg per ml) were mixed with LPS (400 mg per ml) (v:v = 1:1). The mixture was added into a 1-mm diameter quartz cuvette for CD scanning with a scanning range of 190–260 nm, a step resolution of 2 nm and a speed of 10 nm per min. Samples without LPS were used as controls.Secondary structures of peptides  in 67. Briefly, the SCPs (A6 and G6) and LPS were used as ligands and a receptor, respectively. A grid map (70 × 80 × 80 points) was constructed with a grid spacing of 0.375 Å and center of the glucosamine II (GlcN II) in lipid A between peptides and LPS.MD was performed by using Autodock4.2 to predict the LPS-binding sites on A6 and G6 as described in the Supplementary InformationE. coli LPS was detected by the fluorescent BC probe68. The binding of fluorescent probe with LPS can cause fluorescence quenching. When peptides bind to LPS, BC probe can be replaced, which can result in an increase in fluorescence. Equal volumes of LPS (40 μg per ml) and BC probe (10 μM) in 50 mM Tris buffer (pH 7.4) were mixed and added into a black 96-well microtiter plate (180 μl mixture per well), followed by the addition of different concentrations of A6, G6, or N6 (20 μl per well). Ampicillin and PMB were used as the negative and positive control, respectively. Changes in fluorescence were measured by a fluorescence spectrophotometry at room temperature  and the BC displacement rate was calculated.The affinity of SCPs  to 69. The immobilization of ligands onto the carboxymethylated dextran (CM5) sensor chips was carried out with A6 (20 μg per ml), G6 (20 μg per ml), N6 (10 μg per ml), or PMB (400 μg per ml) at 10 mM in sodium acetate (pH = 5.5). After immobilization, 20 mM NaOH was added into the flow cells to remove unbound ligands. The direct detection of ligands was performed for 120 s at a flow rate of 30 μl per min, followed by dissociation for 600 s. All Biacore experiments were carried out at 25 °C. The affinity of peptides or PMB to LPS or lipid A was calculated by fitting the sensorgrams of kinetic injections to the bivalent binding model with Biacore evaluation software version 3.0 and the Biacore Evaluation Software (a 1:1 binding model), respectively.The real-time interaction between ligands  and analytes (LPS or lipid A) was observed by the SPR technology (GE Biacore™ 8K)1H and tr-NOESY were used as NMR probes to investigate the interaction between the SCPs with LPS70. Stock LPS solutions were titrated into peptides samples to see the line broadening effect which indicated interaction between molecules. The tr-NOESY spectra for A6 and G6 were collected with samples containing ca. 2 mM peptides and ca. 0.2 mM LPS.2CO3 and 20 mM EDTA, pH 9.6, 50 μl per well) overnight at 4 °C, washed three times with PBST, and blocked with PBS (containing 1% BSA) for 2 h. After washing with PBST again, the plates were incubated at 37 °C for 1 h with different concentrations of peptides , which were dissolved in Dulbecco's Modified Eagle's (DMEM) medium (containing 10% FBS). After washing, cells were incubated with anti-LBP mAb 6G3 (ThermoFisher) for 1 h at 37 °C and HRP-conjugated rabbit anti-mouse IgG for 1 h at room temperature, respectively. Tetramethylbenzidine (TMB) solution was then added and incubated for 25–30 min. Finally, the reaction was stopped by addition of 0.5 M H2SO4, and the absorbance was measured at 450 nm50.The 96-well microtiter plates were coated with 4 ng per ml LPS (0.1 M Na5 cells per well) were incubated with FITC-labeled LPS (100 μg per ml) or rhodamine-labeled A6 or G6 (0.1–10 μM) for 30 min at 37 °C and were washed with DMEM medium; the cells were then analyzed by a confocal laser scanning microscope (CLSM) and a FACS Calibur Flow Cytometer , respectively. Similarly, after an incubation with FITC-labeled LPS and washing with medium, cells were incubated with rhodamine-labeled A6 or G6 (0.1 μM) at 37 °C for 1 h. After washing again, the effects of peptides on the binding of FITC-labeled LPS to the cells were analyzed by CLSM and flow cytometry, respectively.To determine the location of peptides in cells, mouse peritoneal macrophages . The cells incubated with serum-free DMEM and LPS were used as a control. After treatment for 2 h, the supernatants were collected and assessed for TNF-α, IL-6, and IL-10 by using the ELISA kits in the Jiaxuan Biotech. Co. Ltd. .Macrophages RAW 264.7 were incubated with LPS (0.1 and 1 μg per ml) that was dissolved in serum-free DMEM medium, and then were treated with A6, G6, N6, or PMB (20 μM) alone in 24-well plates  and protocols were approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).The nude mice were intraperitoneally injected with 5 mg per kg FITC-labeled A6, G6, and N6, respectively. Free FITC was used as a negative control. The real-time fluorescence in mice was observed at 0.5, 1, 2, 4, 8, 24, 48, and 72 h, respectively, with the excitation wavelength at 488 nm using a Maestro 2 IVIS® Spectrum CT .E. coli CVCC195  or E. coli LPS  in PBS and were continuously observed for 7 d to determine the absolute lethal dose (LD100) in mice25.The 6-week-old SPF ICR mice  (5 mice per group) were intraperitoneally injected with MDR E. coli or LPS at a concentration of LD100, the mice were treated with a series of different concentrations of N6, A6, G6, or PMB at 0.5 h and 8 h, respectively. The mouse survival was recorded for 7 d. The mice injected with LPS or saline were used as negative or blank controls, respectively.After the intraperitoneal injection with MDR 100 and then were treated with 0.25 μmol per kg peptides  or 10 μmol per kg PMB at 0.5 h after challenge. The mice were sacrificed at 2 h and the sera were collected to detect inflammatory cytokines  using ELISA kits. To visualize IAP activity, the duodenum was rapidly excised from the mice and was homogenized; the IAP levels in the superpants were detected as described in the The mice were intraperitoneally injected with LPS at an absolute LD100. The lung tissues were taken at 1 d and 6 d, respectively, washed with PBS and placed in 4% paraformaldehyde for 24 h at 4 °C. After washing with PBS, the tissues were dehydrated by a series of different concentrations of ethanol  and were immersed in xylene for paraffin embedding. After sectioning, the samples were stained with hematoxylin and were observed by a light microscope. The mice challenged with LPS and PBS served as the negative and blank control, respectively.The mice were treated with N6 (0.25 μmol per kg), A6 (0.25 μmol per kg), G6 (0.25 μmol per kg), and PMB (10 μmol per kg), respectively after the intraperitoneal injection of LPS at a concentration of LD40. After centrifugation at 4 °C for 20 min , the protein levels of supernatants were quantified using a Bradford protein assay kit. A total of 30 μg proteins were analyzed on an 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and were immunoblotted onto PVDF membranes, followed by an incubation with the primary antibodies of p65, p-p65, extracellular signal-regulated kinase (ERK)1/2, p-ERK1/2, and IκBα overnight at 4 °C. After washing, the blots were incubated with the peroxidase conjugated goat-rabbit or goat-mouse antibodies. Relative protein expression levels were quantified by a densitometric measurement of chemiluminescence (ECL) reaction band. β-actin was used as the internal reference.Additionally, the mice were sacrificed at 8 h and the lung tissues were homogenized and suspended in 5 M HEPES buffer for 20 min according to standard proceduresp-value of <0.05 was considered statistically significant.Unless otherwise noted, experiments were repeated at least three times. All data are presented as means ± standard deviation (SD). Statistical analyses between treatments or groups were determined using one-way analysis of variance (ANOVA) models in SAS 9.2 , followed by Dunnett's multiple comparisons test. A Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryPeer Review File