We first demonstrated H2O2-mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H2O2. Then, using a 2D cell tracking system, we quantified bacterial motility descriptors including velocity, % running (of tumble/run motions), and a dynamic net directionality towards the molecular cue. In CheZ mutants, we found that increased H2O2 concentration (0–200 μM) and induction time resulted in increased running speeds, ultimately reaching the native E. coli wild-type speed of ~22 μm/s with a ~45–65% ratio of running to tumbling. Finally, using a microfluidic device with stable H2O2 gradients, we characterized responses and the potential for “programmed” directionality towards H2O2 in quiescent fluids. Overall, the synthetic biology framework and tracking analysis in this work will provide a framework for investigating controlled motility of E. coli and other ‘smart’ probiotics for signal-directed treatment.Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as ‘smart’ devices with programmable function. Building on the latter, we developed a genetic construct to control and direct bacterial motility towards hydrogen peroxide, one of the body’s immune response signaling molecules. A motivation for this work is the creation of cells that can target and autonomously treat disease, the latter signaled by hydrogen peroxide release. Bacteria naturally move towards a variety of molecular cues  in the process of chemotaxis. In this work, we engineered bacteria to recognize and move towards hydrogen peroxide, a non-native chemoattractant and potential toxin. Our system exploits  This structural change, involving oxidation and reduction reactions, occurs extremely quickly, at a rate of 9.7 s-1 [Bacteria naturally respond to oxidative stressors such as hydrogen peroxide and other reactive oxygen species (ROS) that are released by eukaryotic cells upon insult such as pathogen infection or wound generation. They possess several mechanisms, including the OxyR/S-mediated response triggered by hydrogen peroxide –3, for pia coli. , 6. OxySia coli. , 7. In tide bond , 8, 9. Tnd [oxyS  .oxyS promoter and its OxyR regulator within the native GI bacterium E. coli, as a ‘smart’ device for the eventual autonomous treatment of GI diseases based on wound-specific elicitation of ROS in general, and specifically, hydrogen peroxide.In the gastrointestinal (GI) tract, mammalian cells produce elevated levels of reactive oxygen species in response to various wounds and diseases, such as intestinal bowel disease (IBD), ulcerative colitis (UC), and Crohn’s disease (CD) , 11. We 2O2 should allow it to be used as both a monitor of E. coli’s response to H2O2 and as a controller of gene expression [2O2 concentrations investigated here are within the reported physiological ranges for eventual deployment at wound sites, including those in the GI tract [in vivo studies conducted in zebrafish, a popular vertebrate model system, demonstrated a burst release of H2O2 (0.1–0.2 mM) to recruit immune cells that serve to prevent infection [in vivo mice studies reported H2O2 levels of dermal wounds persisted during the immediate inflammatory days  as well as post-inflammatory days  [oxyRS promoter system, bacterial cell growth upon H2O2 insult, and our desired H2O2-mediated swimming phenotype.OxyR’s sensitivity to Hpression , 12–14. GI tract . Notablynfection . Additioounding) . Given t2O2 –programmed “smart” probiotic therapy . Even the most basic issues, such as the maintenance of the “programmable” functions of engineered bacteria that are deployed in noisy environments, should be considered [et al. noted the benefits of directing the therapeutic bacteria towards the site of action. As such, we and others have showed how exogenous triggers such as applied voltage from a microelectronic device [Naturally, there are many considerations that will need to be addressed prior to an Hnsidered , 18. Nonnsidered –25. Alsoc device , pathogec device  could be2O2, as a directional cue; we fully recognize the potential conflict in that induced directionality of therapeutic strains could, in turn, lead to their own damage. In order to attract our engineered bacteria toward to a localized injury marked by the presence of hydrogen peroxide, we developed a system that guides E. coli swimming towards H2O2 by controlling the (i) ratio of run to tumble, and (ii) cell velocity in the presence of an H2O2 gradient. Bacterial motility regulator, CheZ, is a phosphatase responsible for controlling the level of phosphorylated CheY, which, in turn, regulates the tumbling mode of bacterial swimming [Fig 1). For this, cheZ was expressed under control of the oxyRS gene-promoter system induced by the presence of H2O2 in cheZ-oxyRS+ mutants; we retained the native oxyRS genes to prevent additional oxidative stress to the bacteria and deleted genomic cheZ to examine how exogenous H2O2 affects motility.In this work, we turn to the endogenous, wound-indicating signal molecule, Hswimming , 9 . As noted above, elevated H2O2 activates OxyR [2–103 μM range causes toxicity to E. coli and decreases E. coli survival [2O2 was generated at the site of the ROS burst [2O2 without suffering significant oxidative stress. Thus, such engineered H2O2-controlled bacteria may allow for a wide range of application for future therapies or applications.The CheY and CheZ phosphorylation and dephosphorylation cascade (in conjunction with methylation and demethylation cascades) activates the bacterium’s tumbling and running motions , 9, resptes OxyR  which thsurvival . In physOS burst –31. HenccheZ mRNA levels and performed various phenotypic experiments. We measured induced swarming using motility agar plates. Motility experiments were also conducted with both transwell plates and a microfluidic device; results were analyzed using Matlab [2O2 (0–50 μM) or very short H2O2 exposure times (e.g. < 5–10 min.), our engineered E. coli had no ability to swim, only tumble; however, with higher concentrations of H2O2 and/or longer induction times (>10–15 min.), we found cells actively swimming with long runs and with less tumbling. Over time, when cells swim in the presence of a CheZ-inducing molecular stimulant and do not swim in the absence of the same stimulant, there will be a net propensity for the persistence of cells in the presence of the stimulant. This is a process of pseudotaxis [E. coli motility based on H2O2 control. Engineered bacteria were found to rapidly (within 5 to 15 min) express CheZ and swim in a dose-dependent manner to H2O2. While demonstrated using quiescent fluids, these studies which show rapid bacterial responses at physiologically relevant hydrogen peroxide levels, suggest that engineered commensal strains may prove beneficial in GI therapies using “smart” probiotics.In order to characterize the system, we measured g Matlab  and Imagudotaxis , 33–35 aE. coli K-12 W3110-ΔcheZ strain (HCW01) was constructed using a one-step inactivation method [oxyR and oxyS gene-promoter sequence from E. coli corresponding to NC_010473:4256210–4257127 (+ strand) and NC_010473:42560054256114 (- strand), respectively and the genomic cheZ gene were amplified using the primers specified in S1 Table and subsequently digested using the appropriate site restriction enzymes. The sequences relative to oxyR and oxyS promoter or cheZ were ligated using T4 Ligase (New England Technologies). Ligations were transformed in E. coli Top10 cells (Invitrogen) and plated on LB agar plates supplemented with ampicillin (50 μg/mL) and incubated at 37°C. Plasmid DNA  from each selected clone was isolated for sequencing using Qiagen miniprep and analyzed via restriction digestion. The same protocol was followed to insert eGFP into pET200 under the T5 promoter . E. coli K-12 wild type (W3110) and cheZ knockout strains were transformed with pFZY1 and pFZY1-oxyR-poxyS-cheZ (pHW02) or pFZY1, respectively.The n method . The genn method . Briefly600 0.05. Except for motility plates, transwell assays and microfluidic assays, H2O2 induced experiments were conducted at 24°C at 250 rpm. The strain-plasmid nomenclature and shortened names are listed in S1 Table.Bacteria were grown in LB media supplemented with ampicillin (50 μg/mL) and incubated at 37°C in a shaker at 250 rpm for all growth experiments. For all overnight inoculations, bacteria were grown from glycerol frozen stock; all morning re-inoculations were adjusted with sterile media to ODE. coli W3110-pFZY1 (WT-pFZY1), W3110-ΔcheZ-pFZY1 (HCW01-pFZY1), and W3110-ΔcheZ-pFZY1-oxyR-poxyS-cheZ (HCW01-pHW02) were grown to OD600 ~1.5, diluted to OD600 0.1, and one 2μL droplet of cells (2x105 cells) was added onto each motility plate. In turn, H2O2  was added to warm motility agar  and poured into Petri dishes  to yield concentrations of H2O2 (0–1 mM). Plates were incubated at 30°C for 18 hours. Negative and positive controls were performed in the absence and presence of 100 μM H2O2, respectively, added to the plates. For data analysis, technical and biological triplet data were obtained.E. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 were inoculated into 25 mL of LB in 125 mL flasks. Bacteria were shaken at 37°C and sampled every 30 minutes until OD600 ~0.5. After induction, bacteria were shaken at either 24°C or 37°C and sampled every 15 minutes for two hours. For data analysis, technical and biological triplicate data were obtained.E. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 were inoculated into 10 mL of LB in 50 mL flasks, and shaken at 37°C until OD600 ~0.5. Cells were pelleted and resuspended in fresh LB at OD600 0.1 and 0.4. The bacteria were induced with 0–200 μM H2O2 in clear 96 well plates  at 24°C, 250rpm for 5–15 minutes. All samples were assayed for H2O2 consumption with standards using the Quantitative Peroxide Assay Kit . For data analysis, technical and biological triplet data were obtained.E. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 were inoculated into 5 mL of LB in culture test tubes, and shaken at 37°C until OD600 ~0.5. Cells were induced with 0.125–1 mM H2O2 at 24°C, 250rpm for 5–15 minutes and pelleted by centrifugation. RNA extraction was performed using TRIzol (Fisher Scientific) and samples were treated with Dnase I (New England BioLabs) to eliminate possible DNA contamination.cheZ expression ΔCT values were calculated by the following equation: ΔCT = CT Target-CT Reference. The ΔΔCT value was calculated as ΔΔCT = ΔCT,sample- ΔCT,Wt where each ΔCT are represented by the difference between the Target and Reference (16srRNA) values, as above. Also, the relative quantification (RQ) is calculated as 2-ΔΔCT. Error bars represent the standard deviation of each RQ (2-ΔΔCT+s and 2-ΔΔCT-s). The relative quantification was based on the relative expression of cheZ versus 16S rRNA. Wild type E. coli CT values for cheZ were used as reference for all samples.Quantitative PCR conditions were carried out on an Applied Biosystems 7300 Real-Time PCR system using a 2-step cycling protocol. Primers were used at a final concentration of 400 nM, and 10 ng of RNA was used as template in each 20-μl reaction. Each reaction was performed in triplicate, with outlying data removed for select samples. 16s rRNA was used as the endogenous housekeeping gene. To calculate the levels of E. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 were grown overnight, reinoculated into 25 mL of LB in 125 mL flasks, and shaken at 37°C until OD600 ~0.5. Cells were induced with 0–300 μM H2O2 at 24°C, 250rpm for 5, 10, 15, and 60 min and then centrifuged at 4°C, 12,000 rcf for 10 minutes. Bacterial pellets were resuspended with 200 μL BugBuster  and protease inhibitor (HALT Protease Inhibitor Cocktail (100x), Fisher Scientific). Cell suspensions were shaken at 24°C, 150 rpm for 40 minutes. Insoluble cell debris were removed by centrifugation at 4°C, 12,000 rcf for 20 minutes and soluble fractions were transferred to new tubes.6-CheZ protein, and 25 μL of boiled samples  were loaded into 12% SDS-PAGE gels (Bio-Rad). A Semi-Dry Transfer Apparatus (Bio-Rad) was used to transfer proteins to nitrocellulose membranes (Thermo Scientific Pierce). The membranes were blocked with 10% milk  overnight at 4°C. Membrane was washed three times using TBS-T buffer and incubated for 1h 30 min with 1:10,000 anti-CheZ polyclonal antibody (produced by New England Peptide). Before incubation with anti-CheZ, it was adsorbed in 25% lysed E. coli cheZ knockout strain extract, 5% Bovine serum albumin (BSA), Sigma Aldrich, St. Louis, MO) to decrease nonspecific binding. Membrane was washed three times using TBS-T buffer and incubated with 1:15,000 of anti-rabbit alkaline phosphatase antibody (Sigma Aldrich) solution 5% BSA, TBS-T). The membranes were developed for 1 hour at 24°C in development buffer (with BCIP/NBT), and the reaction was stopped using deionized H2O.Total protein concentration  was calculated using the microplate procedure with BugBuster-BSA standards. Pre-stained ladder , HisE. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 (with pET200-T5-eGFP for trajectory images only) were inoculated into 5 mL of LB in 25 mL flasks, and shaken at 37°C until OD600 ~0.5. Bacteria were split into 1 mL cultures in culture test tubes and induced with 0 μM H2O2 (control) and 12.5–100 μM H2O2 at 24°C, 250 rpm for 5–15 minutes. The bacteria were centrifuged at 1,000 rpm and 4°C, washed twice, and resuspended in DPBS for brightfield and fluorescence motility videos (CellSense). Videos were recorded for 100 frames for subsequent Tumble Score [le Score  Matlab , 2.5 mL of cell suspension was added. In the top transwell, 1.5 mL of DPBS ± 0–300 μM glucose or 0–300 μM H2O2. Transwells were incubated at 37°C for two hours for the DPBS buffer and 25–300 μM glucose transwells; based on the H2O2 diffusivity properties dependent on temperature [2O2 transwells were incubated at 37°C for 45 minutes to ensure that the H2O2 gradient across the transwell membrane persisted throughout the experimental time course. For data analysis, technical duplicate and biological triplicate data were obtained.perature , the H2OE. coli WT-pFZY1, HCW01-pFZY1 and HCW01-pHW02 were inoculated into 5 mL of LB in 25 mL flasks, and shaken at 37°C until OD600 ~0.5. Cells were centrifuged at 1,000 rpm and 4°C, washed twice, and resuspended in DPBS buffer. The bottom channel of the motility device [-1. Bacteria were grown until OD600 ~0.6–0.8 and then introduced into the bottom channel at the cell inlet. This initially loads bacteria for subsequent filming and study. Both ends of the bottom channel were then wiped and taped to stop flow. They are then exposed to a gradient introduced by providing fluids in the upper source and sink channels, each having different concentrations of the gradient solute. For traditional chemotaxis experiments, a glucose solution (1 mM in DPBS) was then introduced at a rate of 50 μl hr-1 to replace DPBS in the source channel and to establish a maximum gradient concentration of 100 μM. The sink channel was maintained with 1 mM DPBS. This methodology rapidly generates a glucose gradient within the bottom channel. Note that there is no flow experienced by the cells in the lower channel . The time when glucose was added to the source channel was set to be t = 0. Bright-field images and videos were taken in the middle of the bottom channel at 0, 10, 20 and 30 minutes by a 20X Olympus objective. Then, for pseudotaxis experiments, H2O2 solutions  were introduced using the same method as the glucose solution, with final maximum H2O2 concentration gradients of 50 and 300 μM. Bright-field images were taken at 0, 5, 10, 12, 16, 18, and 20 minutes by a 20X Olympus objective.y device  was pretFor most experiments, one-way ANOVA using a multiple comparisons' Tukey-Kramer post-test were performed using Matlab (version R2015a). α values of 0.05, 0.01, and 0.001 were used to indicate statistical significance. Data are reported as mean values and standard deviation of the error, unless otherwise stated. ANCOVA linear regression analysis was performed using Prism.2O2. In addition to evaluating whether a potential dose response could be obtained, we were interested to find at what concentration H2O2 would prove too toxic so as to permit enhanced motility. In Fig 2A, control experiments using wildtype W3110 cells with empty pFZY1 vector (WT-pFZY1) exposed to 0 or 100 μM H2O2 demonstrated a base case cell motility exhibited by ~7 cm rings with no obvious adverse reaction to the H2O2. Similarly, isogenic cheZ null mutants (HCW01) with empty pFZY1 vector exhibited no spreading in either case, with 0 or 100 μM H2O2. Then, using the same null mutants transformed with the oxyRS induced CheZ vector, pZY1-oxyR-poxyS-cheZ, (HCW01-pHW02) an increasing ring size was observed with increasing H2O2 from 0 to 300 μM (2.5–6.5 cm). Interestingly, we found that bacterial spreading decreased dramatically at 500 μM H2O2, presumably due to peroxide toxicity. Results from biological triplicates are indicated in Fig 2B. In addition, 1 mM H2O2 motility plates resulted in no center colony or spreading (data not shown). We note that the 0 μM plate, which had a minimal but non-zero spread, suggested that some CheZ activity enabled minimal motility, perhaps due to small levels of H2O2 through metabolic activity or simple read-through CheZ transcription and translation from our plasmid vector. Interestingly, the 0, 0.5 and 1 mM observations are consistent with previously published data [2O2-induced CheZ enables motility and that the induced motility rescues the wildtype swarming phenotype.Initial screening studies were carried out to test whether engineered cells exhibited enhanced motility in the presence of H300 μM 2.–6.5 cm. hed data –41. In s2O2-mediated oxyS transcription of CheZ). In Fig 3A and 3B, we grew cells at 37°C until 120 min when we added H2O2. At induction, the cultures were split to grow at 24°C and 37°C to examine how the temperature and H2O2 concentrations affect bacterial growth and H2O2 consumption. Regression analysis indicated that all preinduction growth rates were similar (data not shown). Then, analogously, after a short transient phase, cultures continuing after OD600 ~0.45 all grew at similar rates irrespective H2O2 for each temperature level , S2A and S2B Fig (37°C)) until H2O2 concentrations above 500 μM. We note that swarming experiments indicated reduced motility at concentrations above 300 μM, perhaps suggesting that swarming was more sensitive than cell growth to H2O2 at elevated peroxide levels. We note however, that swarming and growth experiments are quite different, particularly in that exposure to H2O2 occurs under different time scales.We conducted growth experiments using WT-pFZY1, HCW01-pFZY1, and HCW01-pHW02 (H2O2 consumption experiments using the engineered bacteria (HCW01-pHW02) induced with 0 (control) to 200 μM H2O2. Specifically, cultures were induced at either OD600 0.1 or 0.4 to gauge consumption levels at different cell concentrations after 5, 10, and 15 minutes of incubation with H2O2. At each timepoint, unconsumed H2O2 present in the cell media was measured. All cultures induced at OD 0.4 consumed the H2O2 very quickly, within 5 minutes . When induced at OD 0.1, cells consumed high doses of H2O2 but more slowly. For example, in Fig 4B, roughly ~40 μM of 200 μM (1/5th) H2O2 had remained after 5 min, while ~5 μM remained at 5 min when dosed with 50 μM (1/10th). We approximated degradation kinetics using a first order rate law and estimated first order rate constants , qPCR data exhibited similar trends to our previous motility plate studies–a rise in gene expression from 0 to 300 μM and a decrease at 500 μM. In this case, to obtain greater resolution, we conducted additional tests at 350 and 400 μM, finding maximal cheZ mRNA levels at 400 μM 15 min post induction. Statistical analyses were performed by lumping tests above 100 μM and demonstrating significance relative to each of the lower levels . Importantly, cheZ mRNA increased monotonically with H2O2 over time for all ranges tested (2O2 showed similar increasing trends (cheZ expression and this was observed at the first non-zero time point (5 min).We conducted studies using qPCR to quantify s tested . A more g trends . InteresFig 6A and 6B, we used Western blotting to correlate H2O2 with CheZ protein levels in the HCW01-pHW02 cells. At 15 min, CheZ levels increased with H2O2. We found peak levels at 50–100 μM; this tended to decrease at the higher H2O2 levels  . Interestingly, the CheZ from uninduced wildtype cells was not revealed, presumably below detection in these blots. Moreover, we found no CheZ bands in the uninduced HCW01-pHW02 cells. Subsequent studies were carried out at shorter induction times and at additional H2O2 concentrations . Results were consistent and reveal that at 12.5 μM and 5 min H2O2 induction, there was significant CheZ present (significantly more than the wildtype cells). This level increased in time and in peroxide concentration until 100 μM above which perhaps H2O2 toxicity prevented further increases. These findings directly corroborated the relatively fast mRNA responses.In 2O2 were striking, as were the wildtype cells without H2O2 and engineered cells with 100 μM H2O2.In order to analyze bacterial movements and to differentiate chemotaxis, pseudotaxis, and importantly, swimming from swarming , we carrFig 7B, for cells without the GFP plasmid, Rose plots are depicted wherein many traces are superimposed with the initial points set at the origin of the coordinate axes. First, trajectories were observed in every direction as expected, illustrating random movement. Then, the E. coli HCW01-pFZY1 strain without H2O2 and E. coli WT-pFZY1 behaved as expected; E. coli WT-pFZY1 exhibited smooth running trajectories with random, interspersed tumbling while the E. coli HCW01-pFZY1 displayed increased tumbling with a few random spurts of running. Notably, the HCW01-pFZY1 cells moved far less from the origin than the WT-pFZY1 cells owing to the cheZ deletion and subsequent paucity of runs. Similarly, uninduced (0 μM H2O2) HCW01-pHW02 exhibited a phenotypic profile similar to HCW01-pFZY1. When induced with 100 μM H2O2, HCW01-pHW02  showed a phenotype more similar to WT-pFZY1, indicating that motility and CheZ running were recovered to wildtype levels.In 2O2  had statistically higher (α = 0.001) velocities than the HCW01-pFZY1 control (the two control bars represent 5 & 15 min samples). Also, CheZ induction with 75–200 μM H2O2 for both 5 and 15 minutes showed significant (α = 0.05) increases compared to the WT-pFZY1 cells with velocities reaching ~23 μm/s and 19 μm/s, respectively. HCW01-pHW02 with 0 μM H2O2 were observed to have similar velocities to the HCW01-pFZY1 cells . Velocities were observed to increase monotonically from 12.5 μM to 75 μM H2O2. In S3B Fig, we demonstrate that the same conclusions were drawn using both mean and median velocities.To more directly compare the phenotypic responses, motility movies were again taken also without GFP-yielding plasmids (we had suspected increased burden due to the GFP expression). We used 5-sec trajectories and similar computational analyses, except that all stationary particles were discounted instead of just non-fluorescing particles. Quantified videos thus represent only moving cells. For statistical analysis, mean velocities were analyzed. We found HCW01-pHW02 induced with 50–200 μM HS3A Fig). Swimming percentages, which we denote the fraction of time running versus tumbling, were calculated to be ~20–25% for the HCW01-pFZY1 cells and the uninduced HCW01-pHW02. The percent swimming observed for the HCW01-pHW02 induced with 12.5 to 25 μM H2O2 were notably higher (~35%). At concentrations above 50 μM H2O2, cells were observed to have increased running . Conversely, the increase in percent running per trajectory was inversely proportional to the average angle change per trajectory . The increased velocity observed with H2O2 is consistent with the increased fraction of running within a trajectory.Another quantity used to characterize swimming is the net angle change as a cell moves from frame to frame. For 5 second trajectories, the percentage of trajectories that exhibited a net angle change of < 1 radian/s and > 1 radian/s  were mea2O2 concentrations until ~100 μM, which mirrored the calculated swimming velocity and percentage of run versus tumble. At H2O2 levels above ~100 μM, the CheZ level appeared to drop while the swimming parameters appeared to plateau. Also, the swimming parameter levels appeared to ultimately match the WT phenotype. This was contra-indicated by the cheZ mRNA levels which increased monotonically to ~ 400 μM, suggesting other cellular control mechanisms override continued increases in cheZ mRNA.Interestingly, we observed that CheZ protein expression increased nearly monotonically with H2O2 yielded higher variability in cheZ phenotypic expression within the population. For example, we found greater ranges in the standard deviations (25 and 75% quartiles) and extremums in the ratio of path length to Euclidean distance at the lower levels . This measure is the ratio of the sum of all discrete distances travelled and the net distance from the beginning to end of a trajectory. It had significant variability and thus was an interesting measure to characterize the population distribution and its evolution with H2O2. In general, with increasing H2O2 concentration and induction time, the heterogeneity among phenotypes within the overall population decreased towards the observed wildtype values. Similar phenotypic focusing phenomena were observed in response to addition of quorum sensing signaling molecules [We did note, however, that the lower concentrations of Holecules .2O2 concentration gradients over time at 37°C for different initial H2O2 levels. At time zero, H2O2 was assumed to be uniform within the upper chamber, which in turn was assumed quiescent (no flow). In 2O2 gradients persisted over the course of the subsequent swimming experiments . Additionally, we found that HCW01-pHW02 induced with H2O2 migrated to the upper chamber in an apparent dose-dependent manner . Importantly, while the pseudotaxic response to H2O2 was notably lower than the chemotaxic response to glucose, the result that cells swam to the upper chamber was statistically relevant . This is particularly noteworthy in that previous findings have shown that bacteria swim away from H2O2 [2O2. When considering our results from Fig 3, we suspect that these cells metabolize or dissimilate the potential toxin at the same times they were found to swim towards it. To our knowledge, this is the first such report, and this suggests that pseudotaxis can be controlled by internally regulating the CheZ level via externally applied H2O2.We suspended exponentially growing bacteria in the bottom of a transwell apparatus containing Dulbecco's phosphate-buffered saline (DPBS) and at time zero, introduced either Hous work  wherein ous work , 34. In rom H2O2 ; however2O2, we performed additional studies using a microfluidic motility device [2O2–supplemented DPBS solution was added to the source channel (2O2) is established in the area indicated by the y-axis arrow . As we presume there may have been more cells at the source end of the device (closest to the inlet), this represents the natural spreading of the population to unpopulated regions. Then, when the cells were exposed to a glucose gradient, we observed a far greater increase in population at the source. This increase was observed to be sustained for over 20 min. Analogously, in Fig 9C, we examined swimming when bacteria were exposed to H2O2. When pHW02 were exposed to increasing amounts of H2O2, we observed an increase in the number of bacteria found at the source . Moreover, we found that in only 5 minutes exposure to a 300 mM H2O2 gradient, a notable increase in bacteria was found. Consistent with our previous results, the cells exposed to lower H2O2 concentrations (50 μM) responded more slowly and to a lesser extent. These results again demonstrate that CheZ-engineered bacterial cells can be “programmed” to swim towards H2O2. We presume that as cells moved towards the H2O2, they also helped to dissipate the level, enabling additional cells behind to swim towards the increasing peroxide level. We did not test, however, such spatially resolved H2O2 levels.At appropriate time intervals, images near the sink, middle, and source were taken to represent the prevailing population and cell density. In 2O2.In the current construct, because cells do not swim (or swim less) in the absence of the hydrogen peroxide, there should be a greater propensity for the cells to accumulate in the desired locale. An advantage to this system is that there is minimal competition with native chemotaxis processes . That is, that upon actuation of CheZ expression, the CheZ mutant should have fully restored motility and will swim according to the prevailing cues until the cell leaves the region of elevated H2O2-responsive promoter system and used the system to guide bacterial swimming. Complementary studies [We constructed a simple synthetic H studies , 45, 46 E. coli to sense and pseudotax towards H2O2 in highly-controlled and defined manners. Studies of CheZ expression and motility revealed a practical threshold level of ~12.5 μM was suitable for rapid and sustained induction of CheZ and cell swimming. We noted also that the bacteria were able to quickly consume or otherwise dissipate H2O2, diminishing its effects so that over a putative therapeutic range, bacteria exhibited little to no growth inhibition.We employed population based and single cell based analytical methods to characterize the system that enable 2O2 concentrations in mice studies were > 200 μM during the inflammatory phase and ~150 μM during the post-inflammatory phase in dermal wounds [2O2 was generated at the site of the ROS burst release [2O2 levels up to the 100–300 μM range, experiencing diminished CheZ expression, growth and motility at much higher levels (~500 μM). Thus, we suggest that the synthetic biology framework and tracking analysis in this work could be applied for the detailed design of engineered probiotics that would be deployed for directed treatment in the GI tract.In physiologically relevant settings , reported Hl wounds ; additio release –31. CorrS1 Figa. Plasmid design of pFZY1-oxyR-poxyS-cheZ. b. Plasmid design of pET200-t5-eGFP.(TIF)Click here for additional data file.S2 Figa. Post-induction growth curves. WT-pFZY1 and HCW01-pFZY1 with and without 100 μM hydrogen peroxide were controls. b. Tabulated specific growth rates. HCW01-pHW02 growth with 0–1000 μM hydrogen peroxide induction concentrations. All linear regression analyses for HCW01-pHW02 were compared to 0 μM hydrogen peroxide.(TIF)Click here for additional data file.S3 Figa. Average angle change. The average angle change in degrees per 5 second trajectory of the control bacteria  versus the engineered bacteria . These values are inversely proportional to the percent running per trajectory. b. Mean vs. median velocity. Quantification and comparison of reported mean vs. median velocities for all bacteria. Slightly lower velocity and higher variability are associated with the median velocity. Median and mean velocities follow similar trends. ϯ (α = 0.05) indicates the samples differed significantly from HCW01-pFZY1.(TIF)Click here for additional data file.S4 Figa. Comsol model heat maps. Four points (A-D) were recorded to estimate various hydrogen peroxide concentration gradients over time. Heat gradient maps were constructed for 45 minute (left) and 90 minute (right) gradients. Heat maps were generated using 50, 100, and 200 μM hydrogen peroxide starting concentrations. b. Hydrogen peroxide gradient curves over time. For each point , the hydrogen peroxide gradients were calculated over time to determine dynamics, steady-state conditions, and optimal measurement times (45 minutes).(TIF)Click here for additional data file.S5 Figa. Hydrogen peroxide induction for 5 minutes. b. Hydrogen peroxide induction for 10 minutes. c. Hydrogen peroxide induction for 15 and 60 minutes. His6-CheZ and WT-pFZY1 were controls.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


The experimental procedure includes the preliminary synthesis of Fe2+ and/or Mn2+ phenylphosphonates with the general formula Fe(1−x)Mnx[(C6H5PO3)(H2O)] (with 0 < x < 1), which are then mixed at different molar ratios with lithium carbonate. In this way the carbon, obtained from in situ partial oxidation of the precursor organic part, coats the LiMnxFe(1−x)PO4 particles. After a structural and morphological characterization, the electrochemical behavior of lithium iron manganese phosphates has been compared to the one of pristine LiFePO4 and LiMnPO4, in order to evaluate the doping influence on the material.The synthesis of organic-inorganic hybrid compounds based on phenylphosphonate and their use as precursors to form LiMn Moreover, in order to verify possible differences in the electrochemical performances, one of the lithium iron manganese phosphates, i.e., LiFe0.9Mn0.1PO4, has been also synthesized by using the precursor Fe0.9Mn0.1[(C6H5PO3)(H2O)]. Thermal, structural, and morphological analyses have been performed on both precursors and final materials; finally, an electrochemical characterization has been carried out on all prepared samples to evaluate if the synthesis process and the hetero-metal adding degree can influence their specific capacity.Nowadays, lithium-ion batteries are the most developed energy sources for modern portable electronics and their use in automotive application is also increasing ,5,6,7,8.ion cost ,12,13,14tibility ,16,17, w carbons , or by a carbons ,22,23,24stressed . Moreove2C6H5PO3), ammonium hydroxide (NH4OH), iron(II) sulphateheptahydrate (FeSO4·7H2O), and manganese(II) sulphatemonohydrate (MnSO4·H2O) were used for the synthesis without further purification. HPLC (High Pressure Liquid Chromatography) water  was used as a solvent. Usual Schlenck techniques were used to prepare the phenylphosphonate precursor materials.Analytical grade  phenylphosphonic acid (H0.9Mn0.1(C6H5PO3)(H2O) (P1), Fe[(C6H5PO3)H2O] (P2) and Mn(C6H5PO3)(H2O) (P3),were obtained following the synthetic procedure described previously for iron(II) phenylphosphonate monohydrate Fe[(C6H5PO3)H2O]  formed instantaneously as a white flaked precipitate. The white colloidal suspension thus obtained was maintained under continuous stirring under flowing nitrogen for approx. 2 h (pHfin = 6.14). The precipitate was then filtered in air, washed with water to neutrality with acetone, and finally air-dried. Three different metal(II) phenylphosphonate precursors, reported in All metal(II) phosphonate precursors, i.e., FePO3)H2O] ,22: 10 g0.1Fe0.9PO4 was synthesized starting from different precursors following different procedures, and the final products obtained were compared in terms of morphology and electrochemical performances. In detail, LiMn0.1Fe0.9PO4 was synthesized by mixing in a mechanical mill Li2CO3  and either the precursor P1 Fe0.9Mn0.1(C6H5PO3)·H2O (sample S1) or the precursors P2 Fe(C6H5PO3)·H2O and P3 Mn(C6H5PO3)·H2O in molar ratio 0.9/0.1 (sample S2). After the grinding process the light-grey powder homogeneous mixture of the reagents was placed in an alumina crucible and transferred into the central zone of a tubular furnace for calcination. In order to maintain an inert environment, the mixture of reagents was degassed for 1 h at room temperature under nitrogen flowing. Successively the powder underwent a calcination at 600 °C for 16 h under nitrogen flowing. The calcined product was then cooled under inert gas to room temperature, thus obtaining a fine black powder.In particular, LiMn2CO3, P2 and P3 precursors, as described in the previous paragraph. In order to obtain all the desired compounds, P2 and P3 were mechanically mixed with a molar ratio (1−x):x respectively, where x = 0.05, 0.1, 0.5, 0.9, and 0.95, followed by thermal treatment under inert atmosphere as reported above.The synthesis was performed starting from Li−1 and the band at 1604 cm−1 correspond respectively to the stretching and bending vibrations of the water molecule of M(II) phenylphosphonate monohydrate . Other characteristic bands of this compounds are located between 3074 and 3054 cm−1 and are associated with the stretching vibrations of the C-H bond of the phenyl group, while the band at 1438 cm−1 corresponds to C-C bond stretching of the same group. Finally, in the region between 1200–970 cm−1, the characteristic stretching vibrations of the P-O bond of the anion (PO3)2− are observed. The complete conversion of phenylphosphonic acid to metal(II) phosphonate is confirmed by the absence of the typical OH-binding strain vibrations of the P-OH group, generally observed as wide bands between 2900 and 2300 cm−1.Infrared spectra of the different monohydrate precursors appear quite similar and present several bands, as shown in 0.9Mn0.1) [1. In In .9Mn0.1) ,27. All 0.9Mn0.1(C6H5PO3)·H2O (P1)/Li2CO3 mixture (sample S1) and for Fe(C6H5PO3)·H2O (P2) and Mn(C6H5PO3)·H2O (P3) (P2:P3 = 0.9:0.1)/Li2CO3 mixture (sample S2) are reported, respectively, in DSC-TG  curves obtained under nitrogen flow for FePart of the water, probably physically adsorbed on the sample, is lost at temperatures lower than 100 °C. The remaining part, i.e., the crystallization water, is lost at about 200 °C: this phenomenon is clearly highlighted by the end thermal peak observable in the DSC curves at 180 °C. The weight loss up to 180 °C is about 11–15%. At higher temperatures two exothermic effects are displayed in the DSC curves, namely at 400 and 550 °C, which are accompanied by a weight loss in the TG curves of about 25–30% and 8–10%, respectively.0.1Fe0.9PO4 prepared from either P1 and Li2CO3 or P2, P3, and Li2CO3 precursors are very alike as well, as shown in Pnma (olivine like structure) [These effects are related to the decomposition of carbonate and organo-phosphonates and to the formation of lithium metal(II) phosphate. Such experimental evidences are in good agreement with literature, according with the dehydration of some hydrate metal phosphates which proceeds by both anion disproportion and condensation. The X-ray powder diffraction patterns of LiMnructure) . The sim0.1Fe0.9PO4) is characterized by larger and less regular particles.The particles show a comparable morphology. In both samples the particles appear agglomerated and the presence of two phases can be noted: the former, likely carbon, is characterized by very small spheres, while the second one PO4 , obtained only from P2 and/or P3 and Li2CO3 precursors, have been prepared and characterized.Furthermore, taking in consideration this electrochemical results, a series of LiMnxFe(1−x)PO4  are shown in The powder X-ray diffraction spectra of LiMn4 to LiMnPO4 the cell size slightly increases, while the crystallites size decreases.Moreover, in 2P) impurities have been detected. Replacement of the bivalent hetero-metal atom does not affect the crystalline structure of pure lithium iron and manganese phosphate. Indeed the crystalline structure (space group) is the same but substitution of iron with manganese (different atomic radius) in effect shifts the peak positions slightly and this is visible also in the reported XRD spectra. What is important here is that for all samples one, unique crystalline phase has been detected while the mechanic mixture clearly shows peak splitting due to the presence of two crystalline phases. On the other hand, when simply mixing together (0.5:0.5) LiMnPO4 and LiFePO4, the observed X-ray diffractogram shows a splitting of the peaks, as reported in 0.5Fe0.5PO4, formation of only one crystalline phase has been observed.In some samples  the presence of iron phosphide  decreases the capacity of LiFePO4 more than the substituition of manganese with iron in LiMnPO4. Indeed the plateaus at 3.5 V for LiMn0.9Fe0.1PO4 and LiMn0.95Fe0.05PO4 are absent, while for LiMn0.5Fe0.5PO4 the plateau at 3.5 V is shorter than the one at 4.15 V. Even if there is just one phase, as it is possible to note by XRD, lithium ion insertion into the structure induces either reduction of Fe+3 to Fe+2 at about 3.5 V or the reduction of Mn+3 to Mn+2 at about 4.1 V, producing in both cases an equilibrium between the oxidized and reduced form , and then determining the stress inside the structure. Such stress, produced by hetero-atom reduction, is even enhanced when its amount is very low, since, in this case, around its position dissimilar atoms are present and the redox reaction could be inhibited. However, the presence of low quantity of manganese can help the lithium insertion into the iron-base structure, but not vice versa.The LiMnAmong the different compounds of the series, only those of lithium iron-phosphate with low manganese content have been taken into account for further investigation, since they have the highest capacities. To evaluate the performances of those samples, the reversibility degree of the insertion-deinsertion process, the pseudo-diffusion lithium coefficient and the charge transfer resistance, were calculate by PSV, PITT, and EIS experiments, respectively.4, LiMn0.05Fe0.95PO4, and LiMn0.1Fe0.9PO4. In this picture, the insertion-deinsertion process can be observed in correspondence of two peaks. The upward peaks correspond to the oxidation process at approximately 3.47 V for all samples (odd sweeps), while the downward peaks represent the reduction process at approximately 3.37, 3.40, and 3.42 V (even sweeps) for LiFePO4, LiMn0.05Fe0.95PO4, and LiMn0.1Fe0.9PO4, respectively. In general terms, the shorter the distance between oxidation and reduction peaks, the higher the reversibility of the process. In the present case it is possible to recognize that manganese content in LiFePO4 increases the process reversibility. The average value of pseudo-diffusion coefficient has been also evaluated determining the Cottrell region for the potential step voltammetry corresponding to the deinsertion process, by using the PITT technique [−4 cm. Indeed, as said before, the insertion of lithium takes place by means of several reaction fronts, and a pseudo-diffusivity coefficient should be more correctly defined, despite McKinnon and Hearing’s assumption [4 and the materials with low manganese content has been reported. Increasing the manganese content, the value enhances very slightly, so that only few changes can be reached with manganese adding.In echnique ,30,31, asumption , who fouImpedance spectroscopy has been also performed on these three materials and in el is the first intercept of the semicircle with the x-axis, while the second intercept minus the electrolytic resistance Rel represents the charge transfer resistance Rct. Therefore, it is possible to state that for all cases Rct is about 15 Ω. It is known by literature that the transfer charge resistance for pristine LiFePO4 without in situ carbon formation is higher than 40–50 Ω [The electrolytic resistance R 40–50 Ω ,34, so txFe(1−x)PO4 already described in the literature and reported in Finally, we compared the synthesis and the electrochemical performances of our materials with analogous LiMnxFe(1−x)PO4 /C  with in situ carbon formation, by using the solvothermal method. They obtained a specific capacity equal to 150, 65, and 50 mAh/g for x equal to 0, 0.5 and 1, respectively, after 20 cycles at C/5. The same material obtained by Yoncheva et al. [xFe(1−x)PO4/C by solid state reaction with x = 0.7, 0.8, and 0.9, obtaining at C/10 a capacity ranging from 110 to 130 mAh/g as x decreases. Xu et al. [Saravanan et al.  produceda et al.  at 500 °a et al.  producedu et al.  synthesixFe(1−x)PO4 by mechano-activation assisted synthesis, reached, the best performance of 125 mAh/g for x equal to 0.2 at C/10.Finally Wang et al. , which aOn the basis of these considerations, our results are consistent and, in some cases, even better than those found in the literature.−1) over the range 15° ≤ 2θ ≤ 80°. The powder diffraction pattern was indexed by using a Rietveld profile analysis [SEM analysis were obtained by the high-resolution microscope FE-SEM Auriga-Zeiss. The apparatus is also equipped with an EDX (Energy Dispersive X-Ray) detector . Room temperature powder X-ray diffraction was performed by using Cu-Kα radiation λ = 0.15418 nm (Philips PW 1830 generator and Seifert XRD-3000 diffractometers). The data were collected with a step size of 0.02° and at count time of 4 s per step  analyses have been performed at liquid nitrogen temperature and using gaseous N2 to evaluate the specific surface of powders. Elemental analysis has been performed by the Servizio di Microanalisi del ISM-CNR, Monterotondo, Rome, Italy. Electrochemical characterization of samples was performed in T-shaped battery cells with lithium metal as counter (anode) and reference electrode. The cathode electrode contains about 10 mg of electroactive material with 10 wt % of Carbon Super S and 5 wt % of Teflon. The electrolyte is constituted by a glass wool separator filled with a 1 M solution of LiPF6 in 1/1 ethylene carbonate/diethyl carbonate. Potential step voltammetry (PSV) was carried out in a three-electrode cell configuration by using the following setting values: potential step: 0.02 V, relaxation time: 10 min, step duration: until I > Io/30 or 10 s if Io < 0.01 mA and in the range 3.2–3.7 V versus lithium. The same configuration was used for the potentiostatic–intermittent titration technique (PITT) experiments. The electrochemical impedance spectroscopy (EIS) has been performed in a frequency range from 105 to 10−2 Hz, with a voltage amplitude of 0.01 V applied on a cell voltage of 3.47 V. A frequency response analyzer (Solartron 1255 HF and Solartron 1286 models by EG and G) and a galvanostat–potentiostat (Mac-Pile II Biologic) were used for these experiments.Thermogravimetric (DSC-TGA) data of the precursor mixtures were obtained in flowing dry nitrogen at a heating rate of 10 °C·min(1−x)MnxPO4 composites. First of all, LiMn0.1Fe0.9PO4 has been prepared following two different synthetic routes, i.e., using as organic precursors either Fe0.9Mn0.1(C6H5PO3)·H2O (P1 precursor) or a mixture of Fe(C6H5PO3)·H2O and Mn(C6H5PO3)·H2O (P2 and P3 precursors). The materials thus obtained show similar behaviour, even if the sample prepared by using a mixture of P2 and P3 precursors presents a slightly higher capacity and seems to be more stable upon cycling. Subsequently, a series of LiMn(1−x)FexPO4  has been produced by using only P2, P3, and Li2CO3 precursors mixtures. Structural and morphological characterizations have been carried out analysing the effect of the reciprocal presence of iron and manganese on the electrochemical performances. Enhancing the manganese content, the capacity decreases remarkably and the only advantage is the presence of a second charge-discharge plateau with higher potential value. Moreover, the reversibility degree of the insertion-deinsertion process increases, the pseudo-diffusion lithium coefficient increases only slightly and the charge transfer resistance almost keeps constant, being in every cases quite lower than the corresponding values reported in literature for pristine LiFePO4. This is due to the presence of carbon produced in situ during the synthesis, which seems to be the only component able to increase substantially the electrochemical performances of this cathode material.Hybrid organic-inorganic precursors based on metal(II) phenylphosphonates have been synthesized, characterized and used for the synthesis of different LiFe


While the cellular machinery involved has been extensively explored, to what degree this important signalling cascade occurs in organised brain tissue has remained unclear. Here we adapted two-photon excitation microscopy and calibrated time-resolved imaging (FLIM), coupled with patch-clamp electrophysiology, to monitor Ca2+ concentration ([Ca2+]) inside individual astrocytes and principal neurons in acute brain slices. Inside the slice tissue local micro-ejection of Aβ in sub-micromolar concentrations triggered prominent [Ca2+] elevations in an adjacent astrocyte translated as an approximately two-fold increase (averaged over ∼5 min) in basal [Ca2+]. This elevation did not spread to neighbouring cells and appeared comparable in amplitude with commonly documented spontaneous [Ca2+] rises in astroglia. Principal nerve cells  also showed Ca2+ sensitivity, albeit to a lesser degree. These observations shed light on the extent and dynamics of the acute physiological effects of Aβ on brain cells in situ, in the context of AD.The pathogenesis of Alzheimer’s disease (AD) is thought to involve acute neurotoxic effects exerted by oligomeric forms of amyloid-β 1-42 (Aβ). Application of Aβ oligomers in physiological concentrations have been shown to transiently elevate internal Ca Although Aβ has been shown to dysregulate synaptic proteins triggering degeneration of dendritic spines , the undresponse . It has response . It apperesponse . Furthereceptors .2+ elevations and regenerative Ca2+ waves to integrate and propagate physiological signals . Intrigin cells , and rec in vivo .2+ elevations induced in cultured astroglia by the acute application of Aβ oligomers have long been documented  was employed to purify HiLyte Fluor 647 Aβ 42 (Cambridge Bioscience LDT), with pH 7.4 SSPE  as the running buffer. Prior to purification, the peptide was kept in ice, flash frozen immediately after purification and stored at −80 °C. In experimental aliquots, purified Aβ 42 was diluted to 500 nM in PBS and left shaking at 37 °C, 200 rpm for 5 h. It was centrifuged at 14,5002.23 25, KCl 2.5, NaH2PO4 1.25, MgCl2 7, CaCl2 0.5, glucose 25 (osmolarity 300–305 mOsM) stored in the slicing solution at 34 °C for 20 min before transferred for 40 min storage in solution containing (in mM) NaCl 119, NaHCO3 25, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3,CaCl2 2.5, and glucose 11 (osmolarity 300–305 mOsM). All solutions were continuously bubbled with 95% O2/5% CO2.Acute hippocampal slices (350 μm) thick were prepared from P21–24 Sprague-Dawley rats, in full compliance with the national guidelines, the European Communities Council Directive of 24 November 1986, and the European Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes. Slices were prepared in an ice-cold slicing solution containing (in mM) NaCl 87, sucrose 75, NaHCO2.3stratum radiatum astrocytes and CA1 pyramidal neurons were performed. During recordings slices were maintained in solution containing (in mM) NaCl 119, NaHCO3 25, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3,CaCl2 2.5, and glucose 11 (osmolarity 300–305 mOsM). Whole-cell recordings were obtained with patch pipettes (3–4 MΩ) with an intracellular solution containing (in mM): KCH3O3S 135, HEPES 10, disodium phosphocreatine 10, MgCl2 4, Na2ATP 4, NaGTP 0.4 . Protoplasmic astrocytes were identified by small soma size, low input resistance (<10 MΩ), linear current-voltage relationship and low rest membrane potential (<–80 mV). Pyramidal neurons were held at −70 mV. The cell impermeable Ca2+ indicator OGB-1 (200 μM) was added to the internal solution. In order visualise gap- junction coupled (GJC) astrocytes at least 20 min was required for dye diffusion through the syncytium. Aβ 42 was applied via puff pipette (3–4 MΩ) next to one of the GJC astrocytes or CA1 neuron and Ca2+ was monitored in this and neighbouring cells.Whole-cell patch-clamp recordings of electrically-passive 2.4Two-photon excitation by femtosecond infrared laser pulses was used to restrict excitation and emission collection to a thin focal excitation plane 50–110 μm deep into the slice. The imaging system was based on the Femto2D microscope equipped with a Becker and Hickl FLIM detector . We ensured that no contaminating fluorescence signal was collected from damaged tissue near the slice surface, and no autofluorescence was detected at these depths (before applying OGB-1). A short-pass 700 nm filter was placed in front of the detector to block out any escaped light from the laser source. A bandpass 500–560 nm filter was also placed in front of the detector to spectrally separate OGB-1 signal from reference fluorescence of SR101 (see below) and Alexa Fluor 594. The two-photon laser source was a Newport-Spectraphysics Ti:Sapphire MaiTai laser pulsing at 80 Mhz, with a pulse width of ∼220 fs and a wavelength of 800 nm optimized for OGB-1 excitation. The laser power was kept below 8 mW under the objective at all times for slice preparations, to minimize phototoxic damage. In fluorescence intensity measurements, experiment-wise baseline trends due to photobleaching, AM dye extrusion or focus drift  were subtracted using linear regression.t,x,y,T) data cube representing an x-y images with distribution of nanosecond delay time (t) of photons at each pixel over the frame duration (T). Average image acquisition times were 120–300 s (maximum laser exposure time of <100 s to minimize phototoxic damage) depending on the total photon count, and the maximum photon count rate was on average <105 s−1 which is well below effect of photon pile-up . In FLIM tests, the fluorescence intensity were routinely recovered from FLIM data by integration of  photon counting data at any given pixel or ROI.In fluorescence lifetime imaging (FLIM) experiments, images were acquired at a laser line scanning rate of 500 lines per second and stored as a 256 × 512 × 512 x n . pH was adjusted using KOH, and the KCl concentration in the intracellular solution was adjusted accordingly, to keep ion constituents in the solution unchanged. The estimated [Ca2+] was finely adjusted using WEBMAXC program at Stanford by Chris Patton (http://www.stanford.edu/∼cpatton/webmaxcS.htm).The calibration protocol was similar to the standard calibration method provided by the Invitrogen Ca earlier . In brie33.12+ monitoring under acute application of Aβ to astroglia in situ, we incubated acute hippocampal slices with a high-affinity Ca2+ indicator Oregon Green BAPTA-1 . In some experiments, we also used sulfarhodamine101 , a widely used empirical marker of astroglia, to confirm positions of astrocyte bodies, as detailed previously  of local slice tissue (2+ concentration ([Ca2+]) monitoring method that takes advantage of the fact that OGB-1 fluorescence lifetime is highly sensitive to free nanomolar Ca2+  induced a prominent Cathe puff B–C. A sie tissue . To avoilar Ca2+ . While pn tissue .3.22+] readout, as detailed previously for an alternative imaging system , for the [Cag system . The nanrvations   cells C. Import2+] in the focal plane of view  over the bodies of GJC astroglia using a false colour table for illustration purposes  showed, if anything, less significant Aβ-induced [Ca2+] elevations  showed no Caeriments C, centreevations C, right.44.12+ sensitivity of astroglia to the physiological concentrations of Aβ oligomers in primary cell culture is relevant to astrocytes in situ. The rationale for this investigation was three-fold.The overall aim of the present study was to ascertain whether the established Cain situ has a sponge-like, space-filling architecture, contributing significantly to the 3D microenvironment of synaptic connections between neurons. A similarly prominent disparity has been reported regarding astroglial physiology, in particular the expression and function of signalling molecules including receptors, transporters and cellular devices such as mitochondria or Ca2+ stores .in vivo models of AD (Secondly, astroglia or neurons in culture do not face the narrow extracellular space which, in addition to providing a natural obstacle to diffusion in organised brain tissue, also contains a milieu of extracellular matrix proteins and other large macromolecules. Thus, our earlier experiments in culture, while being important for establishing the dose-response characteristics of Aβ application , are unlls of AD .2+-dependent fluorescence changes.Finally, cell-permeable indicators, such as OGB-1 AM, could, at least in some conditions, be taken up and hydrolysed inside intracellular organelles, such as ER or mitochondria, rather than being contained entirely within the cell cytoplasm. In that case, it might be difficult to unambiguously the origin of Ca4.22+] is monitored in astroglia that are filled with a soluble Ca2+ indicator through gap junctions using a FLIM technique (2+] readout which was independent of all major concomitants of cell function (2+] elevations in individual astroglia, while having lesser effects in neurons. This confirms our earlier observations in culture (2+] rises do not readily propagate via gap junctions to other astroglia.In the present work, we used a recently developed experimental approach in which intracellular [Caechnique . This mefunction . We thus culture . In our in situ, also providing novel insights into the efficiency and spread of such actions in organised brain tissue. The Ca2+ monitoring imaging method we adapted and tested should help future studies aiming to probe Aβ action directly in brain slices or in vivo. Here we were able to detect an acute, albeit short-lived, effect of Aβ on astrocyte Ca2+in situ. The transient nature of the effect was likely due to the adopted experimental design in which application of Aβ was also transient. To establish the extent and the endogenous dynamics of such effects in vivo, in conditions compatible with AD, is an important question which requires a separate study.The present observations thus improve our understanding of direct physiological actions of Aβ oligomers on astroglia The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.O. Tyurikova carried out experimental studies; K. Zheng designed FLIM calibration experiments and performed FLIM analyses; A. Rings did control DHPG and Aβ tests in astroglia; A. Drews obtained Aβ oligomers; D. Rusakov and D. Klenerman narrated the study; D. Rusakov wrote the manuscript which was subsequently modified by all authors.


This article presented the data related to the research article entitled “Calibration-free concentration analysis for an analyte prone to self-association”  [1]. The data included surface plasmon resonance (SPR) responses of the variants of protein G with different masses under mass transport limitation. The friction factors of the proteins analyzed by an ultracentrifugation were recorded. Calculation of the SPR response of the proteins was also described. Specifications TableValue of the data•The present experimental SPR responses of proteins with different masses under mass transport limitation could be valuable for researchers interested in improvement or progress of a theory for calibration-free concentration analysis.•Calculated data of a theoretical SPR responses help interpretation of experimental data.•The friction factors of the monomer and the linked dimer determined by an analytical ultracentrifugation could be helpful data for researchers interested in diffusion of multimers.1In this data article, the data of the SPR measurements using a system with Protein G and Immunoglobulin G1 (IgG1) as an analyte and a ligand, respectively, are measured. The experimental data and the calculated curve according to equations described in Ref. 22.1Escherichia coli and purified as previously reported −1 (g/L)−1 for PG0919 and 1.628 cm−1 (g/L)−1 for the linked dimer of PG0919.The variant of streptococcal protein G B1 domain (Q32H/D36E/N37H/D40H/E42H/D47P/A48E), PG0919, designed and characterized previously 2.22) of a monoclonal humanized immunoglobulin G1 with a molecular weight of 148 kDa was immobilized on a sensor chip CM5 . HBS-T buffer solution was used for the measurement. The temperature was set at 298 K. The concentrations of PG0919 and the linked dimer of PG0919 were 9.28×10−2 and 7.08×10−2 µg/mL, respectively.SPR measurement was performed with a Biacore T200 . 10 kRU  with rotor speeds of 40 krpm at 293 K. Absorbance at 280 nm was used to monitor the protein concentration in a double sector cell. Prior to the measurements, the proteins were dialyzed overnight against a buffer solution containing 0.01 M sodium phosphate and 0.15 M NaCl (pH 6.9). The initial protein concentrations were set at 0.6 and 0.5 mg/mL for PG0919 and the linked dimer of PG0919, respectively. The sedimentation velocity data were processed with the program SEDFIT using sedimentation coefficient distribution analysis 2.4R, is expressed as:R is given by:RP1/dt = MP1Gkc,P1[P1bulk], dRP2/dt = MP2Gkc,P2[P2bulk], M is the molecular weight of the analyte, G is a factor converting concentration to an R value, and kc is the mass transport coefficient. [P1bulk] and [P2bulk] are the concentration of the monomer and the linked dimer, respectively, in the bulk. The weight concentration of the monomer and the linked dimer are expressed by:The additional amino acid residues introduced into the linked dimer of PG0919 make the molecular weight  2.86 times larger than that of PG0919 (6427 Da), the monomer. Because the ratio between the molecular weights of the monomer and the linked dimer is non-natural number, the formulas for calculating a mass-dependent SPR response described in the reference Eq. bulk]W, is defined by:bulk]W = (1–γ)[Pbulk]W, [P2bulk]W = γ[Pbulk]W, and γ is a constant (0≤γ ≤1). Eq. The total weight concentration of the analytes, W and (dRP2/dt)/[P2bulk]W were experimentally available. dRP1/dt and dRP2/dt were determined by the SPR method. [P1bulk]W and [P2bulk]W were determined by a ultraviolet absorption measurement. The experimental data are shown in kc,P1/kc,P2 of 1.31±0.01. The error represents the uncertainty of the value in the fitting analysis. Eq. q=2.86 gives kc,P1/kc,P2 of 1.26. By using the physicochemical parameters [(f/f0)P1=1.25 and (f/f0)P2=1.56 determined by the present analytical ultracentrifugation, vP1=0.730 and vP2=0.716 cm3/g calculated on the basis of the sequence kc,P1/kc,P2 of 1.46. We also measured the SPR of the solution of the mixture of the monomer and the linked dimer with γ=0.43. With respect to the relation:sP1/sP2=1 and sP1/sP2=1.24, respectively, the latter of which was calculated by use of the physicochemical parameters. The experimental and theoretical values were close to each other.


Nlgn3, a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other’s behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals’ behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers’ behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3, being more influential in males than females.In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of  Nlgn3 gene and demonstrated that, indeed, transgenic mice and their wild-type littermates can modify each other’s behavior. These observations could profoundly affect experiments using mouse models of psychiatric disorders, as they suggest that the use of wild-type littermate mice as controls may lead to misinterpretation of results.Several studies have shown that the behavior and physiology of socially dominant mice differs from that of socially submissive mice. Despite this knowledge, no study has so far addressed the influence of group inequality, and in particular social hierarchy, on behavioral and Physiological measures obtained in mouse models of autism spectrum disorders and their wild-type littermates. In this study, we used a mouse model lacking the In the majority of mammalian species, social groups are not egalitarian but organized in social hierarchies that can influence individuals’ behavior and stress. For example, in despotic hierarchies, the dominant individuals are more anxious than the submissive ones, whereas the contrary is found when the dominance is maintained through intimidation . Inbred Many mouse models for autism spectrum disorders (ASD) show social behavior defects and traits associated with social submission , namely Nlgn3, coding for the postsynaptic adhesion protein Neuroligin-3 exclusively expressed in the brain ] and, at a lower frequency, litters in which males were all of the same genotype, y/+Nlgn3 or y/Nlgn3– mice [referred to as single genotype housing (SGH)]. Importantly, mice from SGH only ever encountered mice of the same genotype. +/–Nlgn3 mice were crossed with y/+ Pvalbcre/creNlgn3 mice to generate litters containing y/+ Pvalbcre/+Nlgn3 and y/–Pvalbcre/+Nlgn3 mice  Act 1986, as amended, and in accordance with the Cardiff University animal care committee’s regulations. Mice containing a stop cassette flanked by loxP sites in the promoter region lacking e/+ mice –6. Nlgn3e in SGH . Nlgn3+/–/– mice . Sires wMice were kept on a 12-h light/dark cycle with free access to food and water. All behavior was assessed during the light cycle. Experiments in adult mice were conducted when mice were 2–4 mo old Figs. 2–, and 7 aBefore experiments, vaginal smears were stained with modified Giemsa solution (fixative and blue/azure dye) to determine the stage of estrus cycle . Test maSocial dominance within cages was assessed using the tube test apparatus (Noldus). This consists of a smooth transparent acrylic tube  with automated doors at both entrances and in the center. All mice were habituated to the tube for at least 10 min/d over at least 4 d. On testing days, mice were placed at opposite ends of the tube and released to enter the tube. When mice reached the center of the tube, the middle door opened and mice were challenged to push their opponent out of the tube. The mouse pushed out of the tube was declared the submissive mouse of that trial. To determine the cage hierarchy, mice from each cage were tested in pairs in a round-robin design, ensuring that all pairings of mice had been tested. The sides of entry for the mice were alternated to avoid bias. The test was repeated three times every 5 d, and the hierarchy on the third testing day was quantified. For testing using unfamiliar mice, we identified submissive C57Bl/6 mice using tube and ultrasonic vocalization tests before testing, and each test mouse encountered three different submissive mice. The percentage of wins, (number of wins)/ × 100, was quantified.Spontaneous activity of mice was recorded in a 40 × 20-cm open field (OF) arena for 20 min in the dark using an infrared video camera. EthoVision XT tracking software (Noldus) was used to measure the distance traveled in the OF (average centimeters traveled per second) and the normalized distance traveled in the center of the arena , expressed as (distance traveled in the center of the OF)/. For spontaneous activity over 30 h, mice were individually housed in clear cages (40 × 24 × 18 cm) with free access to food and water and maintained on their standard light/dark cycle. Three infrared beams passed the bottom part of the cage. Total beam breaks were recorded over 33 h, the first 3 h corresponding to a phase of habituation to the cages and the next 30 h to the testing phase. For the elevated plus maze (EPM), mice were placed for 5 min in a maze composed of four perpendicular 40-cm arms, two opposite arms with high walls, and two other opposite arms without walls. The exploration of the EPM was recorded by a computer-linked video camera located above the arena, and the time spent in the open arms was quantified using EthoVision XT.Social odors originated from two cages of three C57Bl/6 male mice with different parental origins, maintained for 6 d with the same home cage bedding to allow for a concentration of odorants. Before the test, swabs were wiped in a zigzag pattern across the bottom surface of the cage to collect the olfactory cues. Mice were acclimatized for 30 min to the presence of a cotton swab before testing. For the experiment presented in Mice were placed in individual cages (28 × 17 cm) that each contained 20 marbles arranged in rows of four, on top of 4-cm-deep bedding, to allow burying behavior. The room used was dimly lit, with equal light distribution for all mice in the trial. In the days before testing, mice were habituated to the test cage for 30 min. On test days, mice were left for 30 min in the test cage containing the marbles. Marbles were manually counted and defined as buried when >50% of the marble was hidden. For quantification, experimenters were blind to the genotype.Urine was collected from the mice immediately after bladder voiding and frozen instantly on dry ice. The urine was then stored at –20°C until testosterone quantification. Testosterone was quantified using ELISA in accordance with manufacturer instructions (Arbor Assays).T method. Primers used were Cyp2d9 forward, 5′-AGTCTCTGGCTTAATTCCTGAT-3′, Cyp2d9 reverse, 5′-CGCAAGAGTATCGGGAATGC-3′, 18S forward, 5′-GTCTGTGATGCCCTTAGATG-3′, and 18S reverse′ 5′-AGCTTATGACCCGCACTTAC-3′.Total RNA from liver was isolated with TRIzol reagent (Thermo Fisher Scientific) and purified using the RNeasy kit (Qiagen). cDNA was synthesized using Superscript III (Thermo Fisher Scientific). Quantitative real-time PCR analysis was performed using Fast SYBR green Master Mix (Thermo Fisher Scientific) on a real-time PCR System (Thermo Fisher Scientific). Relative expression levels were determined by normalization to 18S rRNA expression using the comparative ΔΔCm Tris-HCl, 1 mm EDTA, 0.1% sodium dodecyl sulfate, 150 mm NaCl, 1% Triton X-100, 10 mm NaF, 1 mm NaVO4, 1 mm dithiothreitol, and complete protease inhibitor (Sigma-Aldrich)]. The amount of protein in each sample was quantified using a bicinchoninic acid protein assay. Samples were then diluted in lithium dodecyl sulfate buffer , and 25 mg of each sample was loaded onto 4–12% Bis-Tris polyacrylamide gels and transferred onto nitrocellulose membranes. Rabbit monoclonal anti–Neuroligin-3  and chicken anti–β-actin  antibodies were used. Signals were detected using horseradish peroxidase and Alexa Fluor 488–conjugated secondary antibodies (Thermo Fisher Scientific). The level of re-expression was quantified as (Neuroligin-3/actin) in y/–PvalbCre/+Nlgn3 mice normalized to (Neuroligin-3/actin) in y/+Nlgn3 mice.Brain regions were dissected from adult mice after death and immediately frozen in liquid nitrogen. A volume of 100 ml lysis buffer was added to 10 mg tissue [50 mn for each figure. Each test included enough animals to reach a power close to or higher than 0.8. Mice were not systematically tested for all tasks. We designed our test groups to have mice from the different groups tested at the same time. We used GraphPad Prism to systematically test for normality using the D’Agostino–Pearson test and for outliers using the regression and outlier removal (ROUT) method with Q = 1 to ensure that no outliers would modify the outcome and power of the statistical tests. No animals were removed from the analyses. For each experiment, at least three independent litters were analyzed. Pairwise comparisons were analyzed by two-tailed Student’s t test for normally distributed datasets or two-tailed Mann–Whitney test for non–normally distributed datasets. Multiple comparisons were performed using one-way ANOVA for normally distributed datasets or Mann–Whitney test for non–normally distributed datasets, followed by Tukey’s or Dunn’s post hoc tests for multiple comparisons when appropriate. All datasets used for two-way nonrepeated and repeated-measure ANOVAs were normally distributed and, when appropriate, followed by post hoc Sidak’s test. All statistical data are presented as mean ± SEM.y/–Nlgn3 mice and their wild-type (y/+Nlgn3) littermates, we analyzed the behavior of mice from litters consisting of both genotypes (MGH) in comparison to litters in which male mice were all of the same y/+Nlgn3 genotype (SGH). As previously reported  = 1.49; y/–Nlgn3 mice from MGH lost more frequently when competing against y/+Nlgn3 mice from MGH  = 9.85; y/+Nlgn3 mice from SGH and MGH and increased compared with y/–Nlgn3 mice from SGH and MGH [y/+Nlgn3 SGH 13.6 ± 2.7 s, y/–Nlgn3 SGH 6.38 ± 3.2 s, y/+Nlgn3 MGH 22.2 ± 3.5 s, and y/–Nlgn3 MGH 9.8 ± 2.8 s; main effect, P = 0.014, F = 4.62; Cyp2d9, a marker of sexual dimorphism in the liver, was not affected by housing  = 57.7; main effect of genotype, P = 0.0006, F = 7.0; interaction odor × genotype, P < 0.0001, F = 9.7; y/+Nlgn3 mice from MGH spent a similar amount of time exploring social odors and control presented simultaneously, showing that their social discrimination was also affected [y/+Nlgn3 MGH: social 7 ± 0.7 s, control 8 ± 1.2 s; y/–Nlgn3 MGH: social 8 ± 0.8 s, control 6 ± 0.6 s; y/+PvalbCre/+Nlgn3: social 18 ± 6.4 s, control 6.3 ± 1.7 s; y/–PvalbCre/+Nlgn3: social 17.4 ± 3.6 s, control 9.1 ± 2.5 s; main effect of odor, P < 0.0001, F(1110) =15.6; main effect of genotype, P < 0.0001, F(3110) = 10.7; interaction Pvalb × odor, P < 0.0001, F(1.114) = 31.9; y/–Nlgn3 mice from SGH and y/–Nlgn3 from MGH spent more time investigating social cues, y/–Nlgn3 mice from SGH spent more time investigating the social odor than y/–Nlgn3 mice from MGH. Note that y/–Nlgn3 mice from MGH showed more interest in social odor than banana -expressing interneurons, known to regulate social behavior in mice , brainstem (∼0.15), striatum (∼0.1), thalamus (∼0.1), and cortex (∼0.04) and failed to detect it in the hippocampus  = 11.4; P < 0.001 in Nlgn3 in Pvalb-expressing cells rescues the interest in social interaction of y/–Nlgn3 and y/+Nlgn3 mice to levels similar to those of mice from SGH. Independently of the housing and re-expression in Pvalb-expressing interneurons, y/–Nlgn3 mice traveled greater total distance in the OF than y/+Nlgn3 mice [y/+Nlgn3 SGH 3977 ± 189 cm, y/–Nlgn3 SGH 4753 ± 202 cm, y/+Nlgn3 MGH 3796 ± 186 cm, y/–Nlgn3 MGH 5299 ± 426 cm, y/–PvalbCre/+Nlgn3 3683 ± 314 cm, y/+PvalbCre/+Nlgn3 4856 ± 363 cm; main effect of genotype P = 0.0018, F = 10.49; y/+Nlgn3 SGH 0.32 ± 0.02 cm, y/–Nlgn3 SGH 0.29 ± 0.01 cm, y/+Nlgn3 MGH 0.29 ± 0.01 cm, y/–Nlgn3 MGH 0.29 ± 0.01 cm, y/–PvalbCre/+Nlgn3 0.25 ± 0.02 cm, y/+PvalbCre/+Nlgn3 0.31 ± 0.02 cm; y/+Nlgn3 SGH 71.3 ± 7.5 s, y/–Nlgn3 SGH 87.26 ± 9.0 s, y/+Nlgn3 MGH 89.6 ± 9.6 s, y/–Nlgn3 MGH 110.5 ± 9.6 s, y/–PvalbCre/+Nlgn3 95.9 ± 12.1 s, y/+PvalbCre/+Nlgn3 100.4 ± 12.2 s; y/–Nlgn3 and y/+Nlgn3 mice from MGH are not more anxious than mice from SGH. Note that there are no significant differences between the behavior of y/+Nlgn3 mice from SGH or MGH and yNlgn3/+PvalbCre/+ mice  spent a similar amount of time in social contact with –/–Nlgn3 mice from SGH , in comparison with litters in which male mice were all of the same genotype, y/+Nlgn3 or y/–Nlgn3 (SGH). y/–Nlgn3 mice are hyperactive and less stressed than their y/+Nlgn3 littermates. Importantly, y/–Nlgn3 mice showed increased activity in the OF, increased exploration of the hole board, and decreased burying behavior but not change in the exploration of the EPM, indicating that only certain aspects of their anxiety were affected  = 8.82; interaction housing × genotype P = 0.017, F = 5.94; y/+Nlgn3 mice, y/–Nlgn3 mice from MGH traveled an increased distance in comparison to y/–Nlgn3 from SGH  of y/+Nlgn3mice [y/+Nlgn3 SGH 0.19 ± 0.01, y/–Nlgn3 SGH 0.37 ± 0.04, y/+Nlgn3 MGH 0.26 ± 0.01, and y/–Nlgn3 MGH 0.28 ± 0.02; main effect of genotype P < 0.0001, F = 22.35; interaction genotype × housing P < 0.0001, F= 17.29; y/+Nlgn3 SGH 12.6 ± 1.2, y/–Nlgn3 SGH 7.6 ± 1.4, y/+Nlgn3 MGH 14.6 ± 3.9, and y/–Nlgn3 MGH 11.4 ± 1.2; main effect of housing P = 0.0407, F = 4.45; main effect of genotype P = 0.0044, F = 9.06; y/–Nlgn3 and, to a lesser extent, y/+Nlgn3 mice. These results also show that y/–Nlgn3 mice are more reactive to novelty when raised in MGH compared with SGH, indicating that anxiety related to novelty is increased in these mice when raised with wild-type littermates.The social submission of affected . Consistfrom SGH A. HoweveNlgn3 would show behavior similar to that of y/–Nlgn3 mice and, in particular, influence the behavior of their littermates. Because Nlgn3 is an X-linked gene, we could not naturally obtain littermate cages containing –/–Nlgn3 and +/+Nlgn3 mice. Therefore, we investigated the effect of –/–Nlgn3 or +/+Nlgn3 mouse behavior on that of +/–Nlgn3littermates. We found that +/–Nlgn3 mice raised with +/+Nlgn3 littermates were indistinguishable from +/+Nlgn3 mice in the time spent in contact with an unfamiliar female [+/+Nlgn3 99.8 ± 8.36 s, +/–Nlgn3WT 114.1 ± 6 s, +/–Nlgn3KO 68 ± 3.8 s, –/–Nlgn3 65.8 ± 6.3 s, P < 0.0001, F = 12.5; +/+Nlgn3 4515 ± 321 cm, +/–Nlgn3WT 4042 ± 206 cm, +/–Nlgn3KO 4398 ± 508 cm, –/–Nlgn3 5870 ± 274 cm, P = 0.0011; +/+Nlgn3 0.23 ± 0.02, +/–Nlgn3WT 0.25 ± 0.01, +/–Nlgn3KO 0.24 ± 0.02, –/–Nlgn3 0.31 ± 0.01, P = 0.0009, F = 6.5; +/+Nlgn3 130.4 ± 7.1 s, +/–Nlgn3WT 109.3 ± 8.8 s, +/–Nlgn3KO 127.6 ± 9.1 s, –/–Nlgn3 124 ± 13.3 s; –/–Nlgn3 mice showed less time spent in contact with an unfamiliar female, decreased thigmotaxis, and similar amount of time in the open arms of the EPM compared with +/+Nlgn3 mice , consistent with an absence of mothering effect. Second, activation of the Pvalb promoter in the brain occurs around P14 (http://developingmouse.brain-map.org); therefore, the interference of behavior between y/+Nlgn3 mice and their y/–Nlgn3 littermates is unlikely to be due to prenatal events. Third, transfer of anxiety or fear (y/+Nlgn3 mice in MGH. However, we did not find any increased anxiety in young animals or adult female mice, showing that the defective social behavior observed in y/+Nlgn3 mice from MGH is not due to social anxiety as a result of MGH. Finally, re-expression of Nlgn3 in parvalbumin-expressing cells restores the social behavior of y/–Nlgn3 mice and that of their y/+Nlgn3 littermates without affecting the hyperactivity of y/–Nlgn3 mice. This result suggests that it is not the hyperactivity of y/–Nlgn3 mice that causes the social phenotype in y/+Nlgn3 mice but rather their social behavior deficits.Using  or fear  could exNlgn3 in parvalbumin cells controls social behavior is consistent with studies showing that mice lacking parvalbumin display deficits in social behavior (y/–Nlgn3 mice is also underlined by a deficit in E/I balance caused by a decrease in inhibitory but not excitatory postsynaptic currents in the striatum (Nlgn3 in parvalbumin-expressing interneurons is therefore likely to be sufficient to restore inhibitory postsynaptic currents and E/I balance in neuronal circuits, rescuing some aspect of the Nlgn3y/– mouse phenotype. Theories on the pathophysiology of ASD have hypothesized that some of the symptoms associated with ASD may be caused by an E/I imbalance in the cortex or the striatum (y/–PvalbCre/+Nlgn3 mice, restoration of the E/I balance is more likely to occur in the striatum, where we detected re-expression of Nlgn3, rather than in the cortex, where we detected low levels of re-expression, close to background. We also detected a high level of re-expression of Nlgn3 in the cerebellum, a brain region associated with the y/–Nlgn3 mouse phenotype (y/–Nlgn3 mice could therefore arise from a restoration of neuronal network activity in the striatum or cerebellum.The finding that behavior . Loss ofbehavior . Resultsstriatum . Re-exprstriatum . In Nlgnhenotype , ASD-asshenotype , and symhenotype . The allNlgn3. Indeed, like the y/–Nlgn3 mice studied here and in –/–Nlgn3 mice show increased activity in the OF and spend similar amounts of time in the open arms of the EPM compared with wild-type mice. Unlike –/–Nlgn3 mice, +/–Nlgn3 mice show behavior similar to that of their +/+Nlgn3 littermates. These results demonstrate that the effect of a loss-of-function mutation in Nlgn3 is dose dependent and, because Nlgn3 is an X-linked gene, less likely to affect females than males. In addition, –/–Nlgn3 mice are likely modifying the social behavior of their littermates, suggesting that, as in males, social hierarchy or at least group inequality may exist in female laboratory mice. In contrast to y/–Nlgn3 mice, young –/–Nlgn3 mice show no sensitivity to the social environment. Importantly, these results indicate that the social environment has a sexually dimorphic effect on the phenotypes associated with ASD, which could in part explain the increased prevalence in boys versus girls in humans.Our results also show similarities in phenotypes of male and female mice lacking y/–Nlgn3 mice but could extend to other models. First, several genetic mouse models of ASD display social submission (y/–Nlgn3 mice and their wild-type littermates. Second, the behavioral phenotype of y/–Nlgn3 is associated with dysfunctions of Pvalb-expressing interneurons [y/–Nlgn3 mice, associated with a common social behavior phenotype; therefore, these transgenic mice could also modify the behavior of their wild-type littermates. Our observation that the phenotype of y/–Nlgn3 mice modifies the social behavior of their y/+Nlgn3 littermates implies that using wild-type littermates as unique controls for experiments with animal models may lead to false interpretations of results. Therefore, these findings could profoundly affect the interpretation of experiments using mouse models of ASD, and we strongly recommend that standard laboratory practice should take into account the potential confounding effect of social heterogeneity by including independent nonlittermate wild-type mice as additional controls.Our observation that the behavior of transgenic mice can affect that of their wild-type littermates, and vice versa, is a crucial parameter to take into account in the interpretation of experiments. Much evidence indicates that such a phenomenon may not be limited to bmission  or courtbmission . Raisingrneurons  and 5 an


Atractylodes macrocephala, has shown various pharmacological activities such as antitumor and antioxidant activities. In this work, we aim to study the protective effect of biatractylolide on glutamate-induced rat adrenal pheochromocytoma cell (PC12) and human bone marrow neuroblastoma cell line (SH-SY5Y) injury and preliminarily explore its mechanism. The results showed that glutamate was cytotoxic with an inhibitory concentration 50% (IC50) of 8.5 mM in PC12 and 10 mM in SH-SY5Y cells. In this work, the preincubation with biatractylolide  observably improved cell viability, inhibited the apoptosis of cells induced by glutamate, and reduced the activity of LDH. AO staining revealed that apoptosis of cells was decreased. Additionally, the results of western blotting manifested that pretreatment with biatractylolide could downregulate GSK3β protein expression and upregulate p-Akt protein expression, thereby protecting PC12 and SH-SY5Y cells from injury. All these findings indicate that biatractylolide has a neuroprotective effect on glutamate-induced injury in PC12 and SH-SY5Y cells through a mechanism of the PI3K-Akt-GSK3β-dependent pathways.Biatractylolide, isolated from the ethyl acetate extract of Alzheimer's disease (AD) is a progressive neurodegenerative disease, used to describe a wide range of conditions, and its appearance was related to advanced age . The mai, which can cause nerve excitability toxicity. Several studies have demonstrated that the main reason for the loss of neurons in the central nervous system is due to the overexcitation of glutamate receptors and high concentration of glutamate induces PC12 and SH-SY5Y cell death [PC12 and SH-SY5Y cell, neural cell lines, has become an ideal cell model for exploring the mechanism of nerve cell injury in vitro and the protective effect of drugs on neuronal cells because of its typical characteristics of neuroendocrine cells , 7. A lall death –13. Therβ1−40 [The roots of Atractylodis Macrocephalae Rhizoma have been widely used in traditional Chinese medicine due to its various pharmacological activities including antioxidation , gastropβ1−40 . Importaβ1−40 . However Atractylodes macrocephala by multistep chromatographic processing [β, phosphor-GSK3β, and β-actin which were purchased from Cell Signaling Technology .Biatractylolide was isolated from the ethyl acetate extract ofocessing   were routinely cultured in DMEM medium in the presence of 10% FBS and 1% double antibody. All cells were cultured at 37°C in a humidified 5% COBy adding different concentrations of glutamate ranging from 0 to 30 mM in the preliminary experiment, we selected the concentrations (7.5 mM and 10 mM) as the most optimal injured concentrations by determining dose-response curves. The experiment was as follows: (1) blank control group; (2) glutamate injury model group; (3) drug treatment group .4 cells were seeded in 96-well culture plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. The medium was washed off with PBS and 20 μL MTT Reagent was added to each well and the plate was incubated at 37°C for 4 h. Finally, the medium was removed from each well and 150 μl of DMSO was added to dissolve formazan crystals. The product was quantified by measuring absorbance at 490 nm using a Dynatech MR5000 plate reader.1 × 105 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in 6-well plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. Then treated cells were digested and washed twice with cold PBS. The cell precipitation was resuspended and added to 5 μL FITC Annexin V and 10 μl PI and then incubated for 15 min at room temperature in dark. After adding 300 μL of binding buffer, labeled cells were counted by flow cytometry within 1 h. All early apoptotic cells and necrotic/late apoptotic cells as well as living cells were detected by FACSCalibur flow cytometer and subsequently analyzed by Cell Quest software (Becton Dickinson).6 × 105 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in 6-well plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. The treated cells were washed with cold PBS twice and fixed with formaldehyde for 30 min according to AO/EB staining assay kit instructions. Finally, they were incubated with AO (6 μg/ml) for 10 min at room temperature in the dark and then observed by fluorescence microscopy.6 × 105 PC12 cells and 6 × 105 SH-SY5Y cells were seeded in 24-well plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. Twenty-four hours later, the supernatant was collected and centrifuged at 3500 rpm for 3 min and the OD value of LDH in the medium was determined by colorimetric method according to LDH assay kit.5 × 105 PC12 cells and 8 × 105 SH-SY5Y cells were seeded in 6-well plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. Next, the treated cells were washed with cold PBS twice and fixed with formaldehyde for 30 min according to ROS assay kit instructions. The intracellular ROS was observed by fluorescence microscopy.6 × 105 PC12 cells and 6 × 105 SH-SY5Y cells were seeded in 24-well plates and cultured overnight. Then the cells were treated with various concentrations of biatractylolide for 30 min before glutamate treatment for 24 hours in incubator. The treated cells were harvested and resuspended in the medium and, finally, incubated with Rhodamine-123 (10 μg/ml) for 10 min at room temperature in the dark and then observed by fluorescence microscopy.5 × 10Western blotting assay was performed using methods described by Su et al. . Primaryt-test and the variance analysis was performed with One-Way ANOVA method and statistical significance was assumed at a value of p < 0.05 or less.All data are presented as mean ± SEM and GraphPad Prism statistical software was used for analysis. Comparison between the two groups using Student's p < 0.001) and 15 mM glutamate-treated SH-SY5Y cells led to 39.12 ± 2.1% decrease of cell viability  on the proliferation of SH-SY5Y cells and PC12 cells using MTT test. The result indicated that the inhibition rate of 7.5 mM glutamate-treated PC12 cells reached 48.2 ± 1.5% .Glutamate-induced cell apoptosis was quantitatively tested by using FITC and PI. As shown in Figures We observed the morphological characteristic of apoptotic cells using the AO/EB staining test. And p < 0.01; Figures μM and 20 μM significantly obstructed LDH release in the PC12 cells, which was decreased from 180.5 ± 0.9% to about 195.3 ± 2.1% as compared to model group  distinctly decreased LDH activity of SH-SY5Y cells, being 174.2 ± 0.4% (p < 0.001), 243.6 ± 0.1% (p < 0.005), and 272.1 ± 2.9% (p < 0.001) as compared to model group   distinctly decreased ROS production of SH-SY5Y cells, being 161.2 ± 1.3% (p < 0.05), 133.6 ± 2.9% (p < 0.01), and 105.9 ± 3.4% (p < 0.01) as compared to model group  . In addiel group .p < 0.001). Pretreatment with biatractylolide  could significantly protect PC12 and SH-SY5Y cells from the glutamate-induced reduction of MMP.As can be seen in β signal transduction pathway on GSK3β is most important in the regulation of apoptosis. Studies have revealed that activation of PI3K-Akt-GSK3β pathway can have neuroprotective effects [β was observably increased after glutamate treatment compared with control group (p < 0.05) and pretreatment with biatractylolide led to marked decreases in level of GSK3β of compared to the model group. In comparison, treatment of cells with glutamate induced a significant decrease in the level of p-Akt compared to control group (p < 0.01) and pretreatment with biatractylolide led to significant increases in level of p-Akt compared to the model group.PI3K-Akt signaling pathway is essential for the survival of neurons and the effect of PI3K-Akt-GSK3 effects . To valiNatural drug has been widely used in the development and research of neuroprotective drugs because of its low side effects, multiple targets, and high efficiency. Some studies have reported that biatractylolide could slow down the isolated guinea pig right atrium heart rate and reduce shrinkage force . FurtherGlutamate-induced neurotoxicity may be caused by glutamate and NMDA receptors, which could produce oxidative damage, thus leading to apoptosis . AcridinOxidative stress refers to the imbalance of generation and clearance of oxygen free radicals in the body, leading to oxidative damage caused by accumulation of active oxygen and active nitrogen in the body. Numerous studies have shown that there are increases in the level of ROS in neurodegenerative diseases , 29, whiRh123 , 31 is aβ signal transduction pathway plays important roles in the neuroprotective process [β increased significantly compared with the control group while the level of t-Akt was almost unchanged. After treating with biatractylolide, the levels of p-Akt increased and GSK3β decreased in a concentration-dependent manner, suggesting that these changes were abolished by pretreatment with biatractylolide and biatractylolide may play a neuroprotective role by activating PI3K-Akt-GSK3β pathway.The process of glutamate-induced nerve cell damage is related to multiple signal transduction pathways. Numerous studies show that PI3K-Akt-GSK3 process , 33. Ourβ pathway.As we mentioned above, biatractylolide effectively protects PC12 and SH-SY5Y cells from the glutamate-induced neuronal damage and it is suggested that the mechanism of protective effect of biatractylolide may be mainly related to PI3K-Akt-GSK3