Using RT-qPCR, the expression of IFN-β and IFN-λ was quantified following stimulation along with the expression of specific ISGs. Luciferase based assays evaluated the combined expression of ISRE-promoter driven ISGs under IFN-β stimulation. Cellular LD content did not alter the entry of fluorescently labelled viral mimics into cells, but significantly decreased the ability of both Huh-7 and HeLa cells to produce type I and III IFN, as well as downstream ISG expression, indicative of an impeded innate immune response. This observation was also seen during Sendai virus infection of HeLa cells, where both control and LD reduced cells replicated the virus to the same level, but a significantly impaired type I and III IFN response was observed in the LD reduced cells. In addition to altered IFN production, cells with reduced LD content exhibited decreased expression of specific antiviral ISGs: Viperin, IFIT-1 and OAS-1 under IFN-β stimulation; However the overall induction of the ISRE-promoter was not effected. This study implicates a role for LDs in an efficient early innate host response to viral infection and future work will endeavour to determine the precise role these important organelles play in induction of an antiviral response.The cellular localisation of many innate signalling events following viral infection has yet to be elucidated, however there has been a few cases in which membranes of certain cellular organelles have acted as platforms to these events. Of these, lipid droplets (LDs) have recently been identified as signalling platforms for innate TLR7 and 9 signalling. Despite their wide range of similar roles in various metabolic pathways, LDs have been overlooked as potential platforms for antiviral innate signalling events. This study established an  The innate immune response constitutes the first line of host defence to invading viruses; as such, viral infection of a mammalian cell triggers the activation of a number of pattern recognition receptors (PRRs), with subsequent pathway activation resulting in the production of interferon (IFN). IFNs are secreted cytokines, released into the extracellular milieu where they act in both an autocrine and a paracrine manner, binding to specific receptors on the surface of infected and uninfected cells . The actThe localisation of many of the key adaptor molecules within the PRR or JAK/STAT signalling pathways remains elusive, although a defining feature of eukaryotic cells is the use of membrane-bound organelles to compartmentalize activities and serve as scaffolds for signal transduction . SignallThe role of LDs as a signalling platform in the early innate immune response is relatively unexplored. Lipid droplets (LDs) consist of a neutral lipid core, predominantly triglycerides and sterol esters, surrounded by a monolayer of phospholipids and a variety of proteins , and thInterestingly, the role of the LDs in anti-viral signalling events remains relatively unexplored, therefore this work sought to describe the innate response to viral stimulation in cells with a decreased LD content, to assess the value of these organelles in the transduction of an anti-viral innate immune response.The human hepatoma cell line Huh-7 and Human epithelial cell line HeLa were originally obtained from the ATCC and were maintained as previously described . Sendai 2 flasks for 48 hours prior to plating at the required cell density. Cell culture media on all experiments was changed 30 minutes prior to the beginning of the experiment, with all transfections and experiments being performed in 10% FCS.Cells were either given low serum media containing 2% FCS, or control serum media containing 10% FCS and were incubated in T75cm4/well, 24 hours prior to transfection/infection, and performed at least in triplicate. Total RNA was extracted from cells using TriSure reagent (Bioline). First strand cDNA was synthesized from total RNA and reverse transcribed using a Tetro cDNA synthesis kit (Bioline). Quantitative real-time PCR was performed in a CFX Connect Real-Time Detection System (Bioline) to quantitate the relative levels of IFN and ISG mRNA in comparison to the house keeping gene RPLPO. Primers can be seen in All experiments involving real-time PCR were performed in 12 well plates with Huh-7 and HeLa cells seeded at 7 × 10http://imagej.nih.gov/ij/), provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). All bands were normalized to β-actin and graphed as LC3I/II.Huh-7 cells were given low serum media for up to 72 h before cells were lysed and lysates subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed for LC3-b ) with detection of complexes with goat-anti-rabbit-HRP conjugate (Sigma) and chemiluminescence. Protein loading was normalised by re-probing filters for β-actin ). Membranes were scanned using an Amersham 600 chemiluminescent imager. Densitometry to quantify western blot bands was performed using ImageJ software  with 1μg pISRE-firefly luciferase in combination with 10 ng of the constitutively Renilla expressing plasmid pRL-TK. Twenty-four hours following transfection, cells were stimulated with IFN-β (1000 U/ml) for 5 hours. Input renilla luciferase and ISRE driven firefly luciferase were measured at 5 hrs post IFN-β stimulation using the Stop and Glo dual-luciferase kit (Promega) on a CLARIOstar plate reader. All experiments were performed in at least triplicate.Luciferase experiments were performed essentially as previously described . Huh-7 aCells were infected at 50 HA units/ml of Sendai virus, in a volume of 300 μl per well of a 12 well culture plate. Cells were then washed with PBS three times before being re-incubated in their respective culture media. At the indicated time points post infection (pi), cells were harvested for RNA as described earlier, and real-time PCR was performed utilising primers outlined in 2O. Slides were rinsed with 60% isopropanol prior to staining with a filtered 5% Oil Red O solution for 30 minutes, and subsequent rinsing with 60% isopropanol. Bodipy staining was performed as previously [i-E inverted microscope ; Quantification of mean fluorescence intensity was performed with defined regions of interest (ROI), using NIS Elements AR v.3.22, and exported to excel for further analysis. Data was normalised by background signal subtraction, and user defined ROI’s accounted for cell counts.Oil red O staining was performed on cells that were fixed in 10% formalin for 5 minutes, followed by a 1 minute wash in ddHeviously , with nuResults are expressed as mean ± S.E. Student’s t test was used for statistical analysis. p < 0.05 was considered to be significant. All statistical analysis was performed using Prism 6 (GraphPad Software). All experiments were performed in at least triplicate.Reducing the FCS in cell culture media is a widely used method in the literature to decrease the LD mass in cells –24. ThisIn order to reduce the lipid content in cells we utilised a final FCS concentration of 2% for both Huh-7 and HeLa cell lines, and determined the reduction in relative LD abundance over time. Cells were initially plated in reduced FCS media for 24, 48 and 72 hours, and as can be seen in The ability for cells to regenerate lipids after starvation is well documented in the literature –34. AlthWe next wanted to assess whether a reduction in LD mass affected the ability of cells to respond to type I IFN, the predominant cytokine produced in response to viral infection of a host cell. Huh-7 and HeLa cells were plated in either low serum (2% FCS) or control serum (10% FCS) media, for 48 hours. All cells were placed into control serum media 30 minutes prior to transfection of an interferon stimulated response element (ISRE) promoter linked to the firefly luciferase gene, and stimulated 24 hrs post transfection with IFN-β for 5 hrs. As can be seen in Prior to analysis of the host PRR signalling pathways in reduced LD cells, we first wanted to assess whether a lowered LD content would impact the delivery of viral mimics into cells. Utilising Huh-7 cells as a model cell line, we treated the cells as described above with lowered serum or control serum conditions for 48 hrs, prior to returning them to standard media. Cells were then transfected with a rhodamine conjugated poly I:C, and allowed to settle for 24 hours before imaging. As can be seen in Viral DNA in the cytosol of host infected cells is detected via a number of interferon induced cytosolic receptors, which all activate the common adaptor protein STING (stimulator of interferon genes), to ultimately induce the production of IFN . To inveIn order to determine if the reduction of IFN expression resulted in a decline in ISG expression we assessed the mRNA levels of the ISG viperin, which is known to be regulated via both type I and type III IFN . As can RNA viruses are detected in the cytosol and during endosomal mediated entry through the RIG-I like and TLR3/7 pattern recognition receptor families of mammalian host cells (As reviewed in ). To invp<0.05), with a much larger 70-fold decrease at 24 hrs post stimulation in the low serum cells compared to the control (p<0.001), which is converse to what was observed for dsDNA stimulation. This may indicate that LDs may have a larger role to play at early time points in the context of dsDNA stimulation of the ISG viperin, in contrast to later time points for induction of viperin via dsRNA . The inin vitro model of reduced LD mass. Functional biogenesis LD genes such as Arf79F in Aedes aegypti and mammalian Perilipin 1 and 2 have been knocked down effectively using RNAi technology, and demonstrate the inhibition of LD biogenesis [in vitro. This method was found to be highly reproducible when cells were placed in reduced serum media for 48 hrs prior to experimentation, with a complete complementation of LDs returning within 32 hrs, with no harmful effects such as autophagy induced.To examine the broader role of LD’s in the context of induction of an IFN response to a viral infection, we sought to firstly establish an ogenesis , 45. Howogenesis . Consequin vitro, by examining the activation of the ISRE promoter. However, we saw no changes in the response of either cell line to IFN in the presence of a lowered LD content, perhaps suggesting that LDs are not a generally integral organelle of the JAK/STAT pathway. To look at this in a more focused fashion, we examined the upregulation of individual ISGs known to be anti-viral effectors, in the presence of a reduction in LDs. Interestingly, a significant decrease in the ability of type I IFN to drive expression of these genes was observed in Huh-7 cells, but not in Hela cells in vitro, indicating that LDs may play a role in the IFN induction of ISGs in a cell type specific manner. Although the production of IFN is induced in essentially all cell types, the induction of some ISGs has been shown to be more cell-type specific [We initially examined the ability of a lowered LD content to impact the cellular response to IFN specific . The twospecific , in contspecific . This diin vitro. The magnitude of the IFN response to dsDNA and dsRNA was most notable at 8 hrs, with a significantly impeded upregulation of both IFN-β and IFN-λ in both cell lines. Interestingly, in the Hela cells there was little to no difference in the production of type I and III IFN in response to either nucleic acid at 24 hrs, with the levels of each representing a near steady state between 8 and 24 hrs in the LD reduced cells, and a reduction in expression levels in the control cells; indicating that in the Hela cells, a reduction in LD content mainly impacts the magnitude of the IFN response to RNA and DNA viral mimics. This effect was also seen in the context of the RNA virus, SeV, where infection of Hela cells resulted in a significant reduction of both type I and III IFNs in the cells with a lowered LD content, despite viral load being the same , and a In this study, we have demonstrated a reduced activation of both the dsDNA and dsRNA signalling pathways under reduced LD conditions. This work underpins the hypothesis that the LD is an important organelle in the context of certain innate immune signalling pathways by adding further data to the observation that the LD is vital in TLR7 and 9 signalling. Clearly more work is required to fully elucidate the full role of the LD in the context of the early innate immune response to viral infection, however recent work has indicated that the LD may also be another key organelle in the context of antiviral signalling cascades.


In line with previous studies, we found that local and transported emissions as well as thermal differences in sensor systems drive variability, particularly for high-time resolution data. While this level of variability is unlikely to affect data on larger averaging scales, this variability could impact analysis if the user is interested in high-time resolution or examining local sources. However, with thoughtful placement and thorough documentation, high-time resolution data at the neighborhood level has the potential to provide us with entirely new information on local air quality trends and emissions.The increased use of low-cost air quality sensor systems, particularly by communities, calls for the further development of best-practices to ensure these systems collect usable data. One area identified as requiring more attention is that of deployment logistics, that is, how to select deployment sites and how to strategically place sensors at these sites. Given that sensors are often placed at homes and businesses, ideal placement is not always possible. Considerations such as convenience, access, aesthetics, and safety are also important. To explore this issue, we placed multiple sensor systems at an existing field site allowing us to examine both neighborhood-level and building-level variability during a concurrent period for CO As research into and the use of low-cost air quality sensor systems continues to expand there is great potential for this technology to support community-level investigations. Furthermore, given the nature of these sensor systems, such investigations provide data with increased resolution on both temporal and spatial scales. Ideally, such sensor systems offer greater insight into personal exposure , small-sPrevious studies have demonstrated that pollutant variability can exist on small spatial scales utilizing six-minute or hourly averaged data. Variability has been observed across a street or within a few hundred meters, especially in more complex urban environments ,9. There3 sensors and the impact of siting conditions/type on the overall dataset [3 monitors set up by citizen scientists can supplement existing reference networks and provide new information, as limited variability was introduced due to siting choices and this variability was minimal over typical reporting scales  [A recent study in New Zealand thoroughly examined specific aspects of this question by determining the intra-site variability of paired O dataset . Miskell dataset . The teaed data) .3 and the impacts of siting choices in this context. However, it is possible that communities may wish to use sensors for the collection of high-time resolution data on smaller spatial scales rather than the larger averaging times and regional scales studied by Miskell and colleagues. To explore the impact of siting choices in this alternate context we undertook a small case study during a larger deployment of sensor systems in Los Angeles, CA, USA. We added four additional sensor systems to one sampling site to observe the variability across several sensors on one building. We compared this building-level variability to the neighborhood-level variability. This analysis includes data from both metal oxide O3 sensors and non-dispersive infrared CO2 sensors–providing the opportunity to examine a primary and a secondary pollutant. The differing spatial scales  and higher temporal resolution (utilizing primarily minute-median data) as well as the addition of CO2 data offers a small, complementary dataset providing additional information to inform recommendations for siting practices.This study by Miskell and colleagues provided a comprehensive example of how sensor systems can support existing monitoring networks for OFurthermore, while there currently exist several valuable resources contributing best practices and supporting community-based investigations using low-costs sensors, such as the US EPA’s Air Sensor Guidebook , South C3 sensors (model MiCS-2611) and ELT non-dispersive infrared CO2 sensors (model S-300) as well as data from environmental sensors . These sensor systems, or similar ones  operating the same sensors, have been used in prior sensor quantification and spatial variability studies [The sensor systems utilized for this study, called Y-Pods , contain several gas-phase and environmental sensors. This analysis utilizes data from the SGX  metal oxide semiconductor O studies ,16,17,18As previously mentioned, this study was integrated into a larger field deployment in Los Angeles allowing us to leverage one of the existing study sites and ongoing sensor calibration efforts. The study area is primarily high density residential with schools and some businesses nearby. In addition to local traffic and businesses (such as restaurants) other emission sources include two major highways to the North and East of the sampling area. The diagram in This study relies on comparing co-located sensor data with spatially deployed sensor data, therefore we limited the data utilized to match the lengths of our co-located datasets meaning approximately three weeks of data were included in the analysis. 2 and O3 sensors, voltage values were recorded to the SD card as ADC values. These voltages were used as is for the CO2 sensor, but for the O3 sensor they were converted to a normalized resistance prior to analysis [3 data from Y-Pod N3, on which the O3 sensor appears to have malfunctioned. Thus, this data has been excluded from the analysis.Sensor signals were saved to a text file on a micro-SD card on the Y-Pod every 6–25 s, depending on the programming. As some of the metal oxide sensors used here require a warm-up period, the first half hour of data after a pod has been powered off for half an hour or more was removed. Minute-medians were computed; using medians instead of averages removes any single extreme points likely the result of electronic noise. For both the COanalysis ,19. NoteSensor signals were converted to concentrations using field calibration, which involves: (1) co-location with high-quality reference instruments; 2) the development of a calibration model using the air quality sensor signals, environmental sensor signals, and trusted reference data as well as a technique such as multiple linear regression; and (3) the evaluation of that model and its application to testing or validation data. Ideally; the sensors are co-located before and after the field deployment to better facilitate corrections for drift. It is common to incorporate environmental parameters into these calibration models as low-cost sensors are often cross-sensitive to temperature, humidity, and sometimes other pollutants . This me the deve2 sensor quantification, the Y-Pods were twice co-located with a LI-840A  placed at a regulatory monitoring site near downtown Los Angeles. The Licor LI-840A has an expected uncertainty of <1% of the reading as stated by the manufacturer, and the instrument is calibrated using a zero and two-point span calibration with gas standards. The Licor used in this study was calibrated prior to a deployment during the previous summer and was stored between these deployments. As a result of the time lag, we expect drift to have impacted the CO2 reference data. However, as we are interested in sensor to sensor comparisons and the sensor data is baseline shifted (as described below), this drift is of minimal concern. These two co-locations with the Licor were 8 weeks apart and included 17 days total, 12 of which were used for calibration model training and 5 of which were used for model testing. In this instance more of the co-location data was designated for training in order to increase the robustness of the model and expand the environmental conditions for which the model was trained. The model used, Equation (1), included predictors for temperature (Temp), absolute humidity (AH), time (t), and the sensor signal or voltage (V) and solves for the CO2 concentration (C):For CO2 calibration model is extrapolating in time, additional drift was expected. For this reason, the CO2 data was converted using the calibration model and then this data was baseline corrected (to remove drift), and finally the 10th percentile value from each Y-Pod was normalized to 400 ppm. We selected 400 ppm as it is the approximate atmospheric background concentration of CO2 [Due to logistics and a lack of available reference data, both calibration co-locations occurred prior to the building-scale variability study . For thin of CO2 . In ligh3 sensor quantification, the Y-Pods were co-located with API/Teledyne 400 instruments  at two different regulatory monitoring sites. The first site was in Los Angeles in a mixed-use area with some nearby housing and industry. The second site was outside of Los Angles in Shafter, a rural Californian community. These two co-locations occurred prior to and following the building-scale field deployment and therefore no additional signal processing was necessary. The model, Equation (2), used included predictors for temperature (Temp), absolute humidity (AH), time (t), the normalized sensor resistance (R/R0), as well as an interaction term between temperature and concentration, and solves for the O3 concentration (C). The interaction term is intended to address not only changes in baseline driven by temperature but changes in the magnitude of sensor response driven by temperature. This model has been demonstrated as well performing for this sensor in previous studies [For OEquation , used in studies :C =  to Week 2 (deployed to field sites), there is increased variability in both the CO3, spatial variability across field sites was much more apparent. Although there was little change in the correlation coefficient, there was an increase in the spread in both the scatterplot and the boxplot  and O3 a secondary pollutant , it is also worth noting that the CO2 sensor has a lower signal/noise than the O3 sensor in this application.For O boxplot . For Wee boxplot  confirme2, there was a decrease in the correlations on the same scale as occurred across some of the neighborhood sites  and Week 2 (deployed). For COod sites . For O3,n spread , similarn spread  showed t3 data [2 data.Hourly-averaged data was added to both 3 data . However2 and decreasing spikes for O3 that are smaller in magnitude. The range of responses observed in the sensors, along with this contextual information affirms that multiple pollutant sources were impacting the building in an uneven manner.For both pollutants, the most dramatic differences were between sites B1 and B4/B5, the two sites at the back of the building. Speaking with community partners from the project we determined that the building has both a natural gas hot water heater and natural gas dryers toward the back of the building where there are also pipes that appear to be venting these emissions. Sources on the building would seem to explain the large magnitude of the observed variability. By comparison, for the sites B2 and B3, which were on the front of the building above the road, there were occasional increasing spikes for CO2 (in blue) and O3 (in red). There are periods where the differences between CO2 and O3 were well-correlated indicating a shared source. Following this period were instances where the differences were primarily visible in one pollutant or the other. This lack of correlation likely indicates two separate sources, one with relatively more CO2 and another with more NO. Furthermore, there were many instances where these differences between the two building sites were well above the RMSE values. In 2 and decreases in O3 at the B5 site. The correlation between differences in CO2 and O3 occur primarily in the evening hours, while the uncorrelated periods result in enhancements during early morning and daytime hours. These temporal patterns also point to separate sources influencing the sensor data.Providing further evidence for multiple sources, In addition to nearby emission events, Miskell and colleagues observed that direct sunlight causes thermal variations in the instruments causing variability . We compSiting choices and additional shading for the sensor systems could reduce this variability. Although some of the variability between building-sites can be attributed to thermal differences, it is important to recall that this variability is displayed as a bias rather than the larger spread associated with the variability driven by nearby emissions. Therefore, this variability would be unlikely to affect any conclusions about spatial differences due to sources in the same way the short-term enhancements would when examining high temporal resolution data.3 concentrations on typical reporting scales (hourly or eight-hour averages for example). However, the same is not necessarily true for CO2 suggesting it may be valuable to further investigate this aspect of variability for primary pollutants. The spatial variability observed here becomes especially important for communities interested in high-time resolution data, which may be used to assess exposure and/or understand the impact of local emission sources within a neighborhood. When high temporal and spatial resolution is of interest, incorrect placement could result in the inappropriate attribution of sensor responses or failing to record emissions that are present. In agreement with the findings of Miskell and colleagues, we have observed that local emissions or plumes can drive intra-site variability as well as temperature differences caused by exposure to direct sunlight . Also, a3 datasets and only a few instances in the hourly CO2 data where the mean of the B5 dataset differs beyond the standard deviation of the B1 dataset. However, examining the minute-median data for either pollutant, one might draw different conclusions regarding the neighborhood variability depending on which building site was selected. For example, one might anticipate more variability with B5 selected, or fewer local sources capable of scavenging O3 with B1 selected. If examining the maximum daily CO2 concentrations, the results for several days would differ. Regardless of which building site is selected, the diurnal trends are consistent potentially providing an indication of regional trends. Also, for the minute data, this difference between the datasets is more extreme for the CO2 data possibly due to CO2 being a primary pollutant and less well-mixed in the atmosphere.To further explore the impact of the building-scale variations on the community-scale spatial differences, Compare Sensors: Co-locating sensors in the field will support a better understanding of inter-sensor variability prior to their deployment, which will aid in attributing new variability introduced by the deployment of sensor systems to separate sites. These relative comparisons can also be valuable if there are problems with the calibration.Placement and Distribution: To study a particular emission source, place sensors upwind and downwind of the site of interest, at varying distances. Some of the sensors should have a line of sight to the emission source. Consider factors such as typical wind directions and potential obstructions, which may impact the transport of emissions. These placements should also minimize added variability when possible. For example, shading all sensor systems, placing them on the same sides of buildings, or placing them exclusively on rooftops could reduce the variability and biases that result from occasional direct sunlight.Document Deployment: Document your deployment in writing and with photos (take photos of the sensor systems from different angles and photos from the sensors of what they “see”). Learning about nearby activities could provide contextual information that can aid in data interpretation and reduce the misinterpretation of sensor data.There are a few aspects of this study that limit generalizability: we used short periods of data, we only examined the variability around one building in Los Angeles (variability might look different around a different structure or in a different city), and the two sensors types we used rely on different operating principles. Given these limitations, there are still recommendations based on this analysis that can be made. As the following recommendations are intended for individuals or groups interested in conducting sensor studies, more general “best practice” recommendations have been included as well. While some of these are more general, specifically the first and fourth ones, the results of the study nonetheless affirm their value. Furthermore, these suggestions complement the US EPA’s existing recommendations for planning a study and siting sensors . These r2 and O3, measured using low-cost sensors, across a single sampling site can be comparable to the variability across several sites in a neighborhood. However, this spatial variability occurs primarily in high-time resolution (<1 h) data as it seems to be driven by nearby emission plumes and occasional thermal differences. As Miskell and colleagues reported these differences do not persist at typical reporting scales [This deployment demonstrated how the variability in COg scales , but if While minute-level data is not currently utilized for regulatory purposes, this level of data can provide powerful preliminary and


We report a systematical study on the liquid assisted femtosecond laser machining of quartz plate in water and under different etching solutions. The ablation features in liquid showed a better structuring quality and improved resolution with 1/3~1/2 smaller features as compared with those made in air. It has been demonstrated that laser induced periodic structures are present to a lesser extent when laser processed in water solutions. The redistribution of oxygen revealed a strong surface modification, which is related to the etching selectivity of laser irradiated regions. Laser ablation in KOH and HF solution showed very different morphology, which relates to the evolution of laser induced plasma on the formation of micro/nano-features in liquid. This work extends laser precision fabrication of hard materials. The mechanism of strong absorption in the regions with permittivity (epsilon) near zero is discussed. Femtosecond (fs) laser has proved to be an efficient tool for micro/nanomachining  in microIn this paper, we report a systematical study of liquid assisted fs-laser machining of quartz plates in water, KOH, and HF solutions as explored in LIBWE geometry. The obtained micro-holes in liquid have a better morphology and a smaller diameter (1/3~1/2) compared with those fabricated in air. It has been found that the formation of laser induced periodic structures was decreased in solutions. Due to the redistribution of oxygen at the laser ablation site, chemical reactions affected formation of the induced periodic structures and provided better control over surface modification.The schematics of the employed experimental laser microfabrication system are shown in After laser fabrication, the sample was cleaned in an ultrasonic bath with KOH solution  at room temperature, which removed all the debris from the surface thoroughly. After cleaning, the morphology of the ablated region was obtained with a scanning electron microscope . At each machining parameter, a 5 × 5 hole-array was fabricated and all the results were the average of these 25 dots.N = 1 to 100. Apparently, it indicates that the size of the holes ablated in deionized water was much smaller than those produced in air ambient (in LIBWE geometry). The interior of holes made in deionized water were relatively cleaner. In air, an unstructured ablation hole was observed at one pulse regardless of the pulse energy. Grating-like ripples with an orientation perpendicular to the laser polarization  [At first, the morphology of the holes fabricated with different laser pulse energies and pulse numbers were investigated after fabrication in air and deionized water. As shown in ripples)  were fouIn water, the ablated products may interact and undergo chemical modifications in a short time after the laser pulse, especially inside the cavitation bubble . CompareD2) on the irradiation pulse energy. By fitting the data according to the equation (assuming Gaussian intensity profile) [ω0 is the beam waist (radius) at the focal plane and thE is the threshold energy. The ln(thE) could be obtained as asymptotic value when D2→0. The threshold fluences, thϕ, could be calculated for the peak amplitudeThe diameter of the ablated area with the pulse number in air (a) and in water (b) is shown in profile) (1)D2=2ω2 and 5.3 J/cm2, respectively.For a single shot, the calculated threshold pulse energies were about 46.4 nJ and 35.7 nJ in air and in water, respectively, which corresponded to the fluences of 2.3 J/cmLaser induced periodic structures were found depressed in water as shown in From the cross-section, it can be seen that the depth of periodic structures extended a micrometer into silica. Interestingly, the structures become squeezed with smaller periods at larger depths, expandable by the effective medium theory, i.e., a larger effective refractive index more far from the ablated interface. The redistribution of the oxygen revealed lower concentration in the center but higher at the ablation border by energy disperse spectrum (EDS) measurement, as shown in The laser ablation process caused elemental redistribution, which is the most probable reason for selective etching of the ablated area and non-ablated area . For exaThe morphologies of laser ablation in the etching solutions were very different for the HF and KOH: the surface becomes cleaner without laser induced ripples. Nanovoids of elliptical shape similar with those water but with a nanogaps extending perpendicular with the laser polarization were observed for KOH and HF. This can be attributed to the electronic heat conductivity enhancement along the electrical component of the oscillating light E-field which facilitates chemical reaction and thermal diffusion . E-field2SiO3 is less dissolvable compared to SiF4.Femtosecond laser machining is discussed as ‘cold processing’  due to hN = 5) of pulses  in air, water, KOH, and HF solutions in LIBWE geometry. Smaller holes and smooth surface were obtained in water and water solutions. The threshold in water had a similar decreasing trend with pulse accumulation. In addition, laser induced periodic structures were less pronounced in water. Etching enhancement along the E-field of a linearly polarized laser beam was observed in the laser ablation in solution, which is attributed to the thermal effect enhanced chemical reaction and E-field enhanced electronic conductivity. Our systematical investigation opens up prospects for a better controlled high precision nano-/micro-scale fabrication of hard and chemically inert materials. The mechanism of energy deposition into the ENZ regions is discussed.


Intrinsic neuronal excitability has been reported to change during normal aging. The bed nucleus of the stria terminalis (BNST), a limbic forebrain structure, is involved in fear, stress and anxiety; behavioral features that exhibit age-dependent properties. To examine the effect of aging on intrinsic neuronal properties in BNST we compared patch clamp recordings from cohorts of female mice at two ages, 3–4 months (Young) and 29–30 months (Aged) focusing on 2 types of BNST neurons. Aged Type I neurons exhibited a hyperpolarized resting membrane potential (RMP) of circa -80 mV compared to circa -70 mV in the Young. A key finding in this study is a hyper-excitability of Type II neurons with age reflected in an increase in firing frequency in response to depolarizing current injections; activation of Type II neurons is believed to dampen anxiety like responses. Such age-related changes in intrinsic neurophysiological function are likely to modulate how the limbic system, acting via BNST, shapes function in the HPA-axis. Chronic stress, anxiety and related mood disorders have a substantial effect on the well-being of all global societies. In the developed world, conditions within this spectrum of disorders have become a major economic burden, in terms of both direct healthcare costs and lost economic productivity .Although seen at most ages from childhood onward, the development and nature of chronic stress exhibits a complex age-related nature  the basiAlthough the mammalian limbic system unquestionably has pivotal roles in eliciting and modulating stress signaling, it is not extensively connected to key hypothalamic areas involved in stress responses, including the paraventricular hypothalamic nucleus (PVN). The PVN is pivotal in this regard as it releases corticotrophin-releasing factor (CRF) into the median eminence to drive pituitary adrenocorticotropic hormone (ACTH) release into the systemic circulation and ultimately glucocorticoid release from the adrenal cortex. The PVN also exerts substantial influences on autonomic activity via its connections to various brainstem/spinal cord networks .Due to the above mentioned lack of direct connectivity, limbic system influences on hypothalamic and brain stem-dependent outcomes are necessarily relayed through intermediate brain areas. One area of particular interest in this regard is the bed nucleus of the stria terminalis (BNST). In the mammalian brain the BNST is located in the caudal pallidum, adjacent to the caudate putamen, and either side of the anterior commissure. It receives afferent projects from many areas of the limbic system including the amygdala, the ventral subiculum, the hippocampus and the prefrontal cortex. Recent brain imaging-based analysis of BNST connectivity in humans indicates a very similar connectivity to that described in rodents, indicating that functional studies in rodents are likely to reflect largely human neural physiology. Furthermore, multiple functional magnetic resonance imaging (fMRI) studies in man have demonstrated the role of BNST in the human central nervous systems (CNS) response to fear and anxious anticipation .ALG)  .ALG which can be characterized based on their electrophysiological properties  were compared to animals aged 29–30 months (Aged). Experimental days employing brain slices obtained from the two different ages of mouse were interleaved throughout the duration of the study.All experiments were carried out in accordance with the animals (scientific procedures) act 1986. All tissues for this study were harvested from female C57-Bl6 mice purchased from Charles River. For their entire lifespan animals had D-Glucose, 26 NaHCO3, 3 KCl, 5 MgSO4, 0.1 CaCl2, 1.25 NaH2PO4. A Leica VT1200 vibratome was then used to cut serial 300 μm thick coronal sections. Following their preparation slices were allowed to recover at room temperature for at least 60 min in our standard artificial cerebrospinal fluid (aCSF). This was composed of (in mM):124 NaCl, 3 KCl, 24 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4, 10 D-Glucose, and was continuously gassed with carbogen Animals were killed by cervical dislocation in accordance with schedule 1 of the UK Animals (Scientific Procedures) Act 1986. The skull was opened and the brain was rapidly removed and placed immediately in an ice-cold slicing medium consisting of (in mM): 189 Sucrose, 10 ALG came from approximately Bregma -0.1 to +0.3, and were identified with the aid of the Paxinos and Franklin mouse brain atlas using the anterior commissure as a key landmark. Recordings were carried out in the dorsal portion of the BNSTALG. Typically one or two suitable BNST-containing coronal sections per animal could be used and by bisecting these along the dorsal-ventral midline we were able to obtain two to four usable tissue sections per mouse.Slices containing the BNSTAll recordings were made using the whole cell patch clamp technique. The BNST containing brain slice was transferred into a submerged recording chamber which was perfused with gassed aCSF and maintained at a temperature of ∼34.5∘C. The recording chamber was mounted on the stage of an upright microscope (Olympus BX51).K-Gluconate-based internal solution used for all recordings. This was composed of (in mM): 130 K-Gluconate, 20 KCl, 10 HEPES free acid, 0.2 EGTA, 0.3 GTP-Na salt, ATP-Mg salt, pH adjusted to 7.3 with KOH. The 15 mV junction potential error produced by pairing this pipette solution with our aCSF was corrected for during analysis.A Flaming Browning P-97 micropipette puller was used to produce the microelectrodes used in this study. These had a resistance of 3–5 MΩ when filled with the Cells within the BNST were visually identified using the microscope’s infrared differential interference contrast optics and a coupled IR-sensitive CMOS camera (Thor Labs). All recordings were made with a Multiclamp 700B amplifier (Molecular Devices) interfaced to a Digidata 1440A (Molecular Devices). Experiments were controlled and data collected using the Clampex program within the pClamp 10.4 software suite. All data were stored directly onto a personal computer (Hewlett-Packard) and backed-up to a network drive.A sequential series of protocols were carried out in both voltage clamp and current clamp mode to assess the intrinsic and synaptic properties of BNST neurons. Initially cells were voltage clamped at a holding potential of -70 mV for 60 s. Here intermittent spontaneous postsynaptic currents could be observed. We recorded the average holding current, and following their detection, the mean frequency and amplitude of the spontaneous inward-going synaptic events were determined. Following the initial 60 s period in voltage clamp, the amplifier was switched to current clamp mode for the remainder of the recording allowing cellular voltage responses to be studied. Firstly, we recorded a period of activity in the absence of any injected current  this allowed us to assess both the resting potential and also the proportion of cells exhibiting any spontaneous action potential firing.Next by the application of a suitable level of bias current cells were set at a prestimulus membrane potential of -70 or -80 mV. Once the prestimulus membrane potential was set to the desired level a series of nine, 500 ms duration, current injections ranging from -40 to +80 pA in 15 pA increments was applied to each cell; the time between the stimuli was 10 s. The hyperpolarization caused by the first, i.e., -40 pA, current injection was used to determine passive, subthreshold membrane properties. Input resistance was calculated from the difference between the pre-stimulus voltage and average voltage during the final 100 ms of the hyperpolarizing current stimulus. The series of depolarizing pulses were used to characterize the firing properties of the cells. The action potential properties of each cell were characterized from the first action potential generated in response to the depolarizing steps. For each recording we measured action potential threshold, maximum rate of rise, zenith  and width. Another way to examine excitability is to look at rheobase . This was achieved by applying current injections lasting 300 ms that were increased in amplitude by 2 pA per sweep until a spike was observed, the time between stimuli was 1 s. Cells were sorted into 3 different populations based on the criteria outlined by t-test was carried out, if data were not normally distributed a Mann–Whitney U was carried out. Part of the data was examined from two prestimulus potentials; if the data was normally distributed at both prestimulus membrane potentials a repeated measure two-way ANOVA was carried out with age as the between subject effect and prestimulus membrane potential as the within subject effect. If the data were not normally distributed a Mann–Whitney U test was performed at each prestimulus membrane potential. If the data were normally distributed at only one prestimulus membrane potential an unpaired t-test was carried out on this set and a Mann–Whitney U on the non-normally distributed set. Firing frequencies in response to each depolarizing current injection were analyzed via repeated measure three-way ANOVA. All statistics were carried out in SPSS. Proportion of cells firing was determined by chi squared, these tests were carried out in Excel. Figures were prepared with Origin 2016.Data were analyzed using a range of custom written MATLAB scripts and pClamp 10.4 software. Determination of appropriate statistical test was based on assessment of normal distribution using the Shapiro–Wilk normality test. A number of properties were examined from only one membrane potential, i.e., firing frequency at rest; for these parameters if data were normally distributed an unpaired This study was exempt from approval by an ethics committee; animals used in this study were killed by cervical dislocation, this procedure is regulated by the schedule 1 of the animals (scientific procedures) act of 1986 of the United Kingdom.ALG. The aged neurons consisted of 8 Type I cells, 16 Type II cells and 4 Type III cells, the young neurons consisted of 18 Type I cells, 18 Type II cells and 7 Type III neurons. Due to the low numbers of Type III cells recorded we did not make cross-age comparisons and they are not included in this report.A total of 28 cells from the Aged cohort and 43 cells from the Young cohort were recorded in the BNSTn = 18) was over 10 mV more depolarized than that of the Aged population , . The proportion of cells exhibiting any spontaneous action potential firing was 12 % in the Aged slices (1 of 8 cells) while 50% were firing in the Young group . Of the cells which did not fire spontaneously there was no difference in rheobase .Cells were placed into current clamp to examine properties at their resting membrane potential; the mean resting membrane potential of the young cohort  and -80 mV  was dependent on age , reflecting the age-dependence of resting membrane potential.Holding cells at a set membrane potential was achieved by application of suitable level of “steady state” bias current. The amount of current required to hold cells at -70 mV  which was >30% faster than those from the Aged population . The membrane time constant from a prestimulus potential -80 mV averaged 29 ± 3 ms in the Young cohort (n = 19) and 35 ± 4 ms in the Aged cohort (n = 8) which was not significantly different . There was no statistically significant effect of age on input resistance, capacitance or hyperpolarization activated “sag” (Table 1), although the direction of the difference in input resistance was in line with the observed membrane time constants.Once cells were held at a set prestimulus membrane potential, a series of depolarizing and hyperpolarizing current steps were injected. Injection of -40 pA of current was used to assess passive membrane properties of the cells; the averages of these traces are shown in n = 14) than the Young group  . Changes in holding voltages are often determined by two key factors, resting membrane potential and input resistance, as the input resistance of this population did not differ with age the changes in resting membrane potential are most likely to be causing the observed change. Imposed upon the baseline current of both groups was a considerable barrage of spontaneous inward-going synaptic events. The frequency of these events varied considerably from cell to cell but did not differ with age . The average amplitude of these events was -16 pA  and was not age-dependent.The spontaneous synaptic events were examined in voltage clamp mode while holding the voltage at -70 mV. The mean holding current required to maintain cells at -70 mV in voltage clamp mode was higher in the aged cohort  related to stimulus amplitude, this did not differ with age . Examples of current injections from both age cohorts are shown in Figure 3. The relationship between current injected and proportion of cells firing one or more spike is shown in Figure 3B for prestimulus potentials of -70 and -80 mV. As expected less current was required to generate spiking from the more depolarized prestimulus membrane potential. At one of the weaker effective current stimulus levels of 20 pA a higher proportion of cells fired in the aged cohort; from a prestimulus potential of -70 mV where 6/18 of the young cells generated action potentials while 8/8 aged cells fired  and from a prestimulus potential of -80 mV where 3/18 young cells fired and 3/8 aged cells fired (chi-squared p = 0.2), however, this failed to reach statistical significance .n = 16) than cells in the Aged cohort  . From a prestimulus membrane potential of -70 mV the mean current required to elicit a spike was more than double in the Young cohort  than the Aged neurons  but failed to reach statistical significance . Finally we looked at the latency to the first action potential generated following the injection of 80 pA; no age-dependent differences were observed .The rheobase protocol in which the increment between successive current stimuli was only 2 pA was used to examine excitability. When cells were at -80 mV significantly more current was required to produce the first spike in the Young neurons . Spikes were noticeably broader in Aged animals, thus the action potential width measured at the threshold was a mean of >0.4 ms longer in the Aged cohort in comparison to the Young cohort .The action potential properties were measured from the first action potential generated in response to the series of depolarizing current injections. Average APs from the Young and Aged groups are presented in n = 16, Aged: 7 ± 2 Hz, n = 14, Mann–Whitney U, p = 0.3, Z = -1.2), however, the average amplitude of these events was a mean of ∼20% larger in the Young cohort  in comparison to the Aged . The holding current required to hold Type II cells at -70 mV in voltage clamp mode did not differ with age .Similar to Type I neurons type II cells exhibited an ongoing barrage of spontaneous inward–going synaptic events. The frequency of these events did not differ with age . The proportion of cells spontaneously firing, however, was far greater in the Aged cohort with 10/16 (69%) of cells firing while only 5/18 (28%) of cells fired in the Young cohort (Chi squared p = 0.04). The somewhat more depolarized mean resting potential is likely to be a contributing factor to the more prevalent spontaneous firing in the Aged cohort; but other factors may also contribute such as action potential threshold or input resistance. Of the cells which were spontaneously firing in the two groups there was no significant effect of age on the firing frequency ; equally for the cells lacking spontaneous firing the rheobase was independent of age at our sample size .When type II cells were switched into current clamp mode there was no difference in the resting membrane potential (Table 3). Capacitance was a mean of over 10 pF higher in the young cohort, however, this was not statistically significant with this sample size .As for type I cells the 500 ms -40 pA step of the current injection series was used to determine input resistance, membrane time constant, sag and capacitance. Input resistance, membrane time constant and sag were not age-dependent . The averages for these action potentials are presented in Figure 6; no differences were observed in action potential zenith or the maximum rate of rise of the action potential; as the threshold differed in this population action potential width was measured from a set potential of -20 mV, this was also not dependent upon age (Table 4).The first action potential generated in response to the depolarizing 500 ms current injections was used to assess action potential properties. Threshold was more depolarized in the young cells .The shift in action potential threshold appeared to have no effect on the amount of current required to produce at least one spike in the “rheobase” protocol both set prestimulus membrane potentials . From a prestimulus membrane potential of -70 mV the first step (5 pA) caused a higher proportion of Aged cells to fire one or more action potentials with 11/16 (69%) Aged cells firing while only 2/19 (11%) cell from Young animals produce a spike (chi-squared p = 0.0004). There was no difference in the likelihood of firing in any other steps from this prestimulus membrane potential. This is most likely due to the fact that at the 20 pA injection >78% of cells in both populations generated at least 1 spike, and at 50 pA step all cells in both cohorts were firing. From -80 mV the likelihood of firing did not differ for any of the current steps. Another measure of excitability is the average frequency of action potentials during each of the current steps. There were significantly higher rates of firing in the Aged cohort in comparison to the Young . Furthermore, following the injection of 80 pA of depolarizing current the Aged neurons generated spikes ∼33% earlier their Younger counterparts .Given the more hyperpolarized threshold of type II neurons in aged animals one might expect to see an increase in excitability in these cells. Excitability was examined using a series of incremental 500 ms depolarizing current injections. Sample traces containing components of these protocols can be seen in The data presented here describe differences in the intrinsic properties of two classes of BNST neurons observed when comparisons were made between patch clamp recordings performed in the anterolateral area of the BNST in brain slices obtained either from post-pubescent Young (3–4 months) or very Aged (29–30 months) female C57-BL6 mice. In work by others, neurons in this area have been classified into three types . Due to In type I neurons the mean resting membrane potential of the Aged neurons was >10 mV more hyperpolarized. This would place the cells away from action potential threshold, which was similar at the two ages, making them less likely to fire, however, there was no significant difference in the proportion of Type I cell firing at rest. The differences in prestimulus membrane current for both the voltage clamp recordings and the current clamp recordings performed at set potentials can be accounted for by these differences in resting membrane potential, in the face of relatively similar input resistances. In Type II neurons a significantly higher proportion of the Aged cohort were firing at rest.in vivo. This commonly made assumption is one we have certainly questioned previously in the light of how intrinsic properties can be readily and persistently modified by “neurophysiological experiences”  that might be expected to occur during the process of preparing acute brain slices ], their afferent axons are for the most part detached from their cell-bodies, and thus are highly likely to be silent. Consequently, one must also consider the level of spontaneous neural activity in the light of this functional denervation. For example, the preparation of slices and the use of a high chloride intracellular solution could have disrupted some form of strong inhibitory synaptic tone that in vivo holds BNST cells more negative reducing electrogenesis of spikes. Having said this, some of the very first neurophysiological studies of the BNST, in vivo recordings in anesthetized female rats, revealed that the majority of BNST neurons that exhibited any activity fired at >1 Hz, and this activity may have to some degree been affected by estrogen status  or resist (low threshold K+ channels) AP genesis. Furthermore, quite subtle changes in combinations of these factors could synergize to promote enhanced excitability. In the aged cohort a significant hyperpolarized shift in action potential threshold of Type II neurons were seen. Using macropatch recordings from hippocampal neurons we have previously shown how aging-associated changes in action potential threshold were paralleled by equivalent shifts in the voltage-dependence of Na+ channel gating (+ channels underlying spike initiation. The differences in threshold of Type II neurons could account for the higher proportion of cells spontaneously firing at rest.When the membrane potential was pre-set to fixed levels of either -80 or -70 mV prior to application of current stimuli, the depolarization-evoked action-potential frequency was higher in type II Aged animals. Theoretically such age-associated increase in excitability could result from a number of underlying sources. These include, but are not limited to, a negative shift in AP threshold (akin to that we reported in hippocampal neurons, l gating , althougThe majority of neurons in the BNST are GABAergic; BNST originating GABAergic projections innervate multiple areas of the CNS, for example those involved in reward, feeding and stress . One keyAn interesting observation seen in Type I neurons was a ∼25% widening of action potentials with age. If this outcome, seen in our recordings at the cell body, translated to similar changes in spike width at the presynaptic terminals formed by BNST neurons, a substantial increase in release probability might be expected, and thus a corresponding increase in drive to down-stream synaptic targets of BNST neurons. This might be expected to increase the inhibitory influence of BNST on the PVN and thus reduce overall HPA activity by limiting CRF output from PVN neurons .This increase in excitability in the type II neurons could also contribute to a decrease in anxiety with age. This study did not address the anxiety levels of the mice prior to culling, however, in humans a decrease in the prevalence of anxiety disorders with age has been reported . As thisAlthough it can be proposed that neurophysiological changes in BNST neurons develop as a consequence of what might be regarded as autonomous aging-related processes within the BNST itself, another possibility is that the aging-associated neurophysiological changes that arise in type I and type II cells represents an adaptive change in response to an altered afferent synaptic drive from, for example, other limbic structures. Certainly other components of the limbic system with well-established BNST connectivity have been previously shown to exhibit significant neurophysiological changes associated with aging .The BNST is well known for its sexually dimorphic nature; due to their availability for this first study of BNST neurophysiology in Aged animals we focused entirely on females. An interesting extension to this work would be to compare how aging alters the neurophysiological properties of male mice. It would also be of interest to investigate how neurophysiological profiles in sub-regions of the BNST respond to aging, although such work would perhaps be better performed in rats. This is because subregions have been better defined in that species and also the greater physical size of the rat brain would make such studies more experimentally tractable. Notably our data provide no indication of when the neurophysiological changes we report develop within the mouse lifespan. A more detailed longitudinal study would be required for this, preferably sampling multiple age points from a large cohort of littermate animals raised throughout life in the same facility.Overall this study identified a hyper-excitability of Type II neurons and a hyperpolarized resting membrane potential of Type I in aged female mice. Due to the central role of the anterolateral BNST in the limbic influences over the HPA axis, the changes observed in this study could shape changes of HPA axis function with age.HS planned and carried out the experiments, she was also responsible for the analysis of the data and contributed significantly to the writing of the manuscript. JT was involved in the securing of the initial grant and also proved expertise in stress pathways. JB was involved in the analysis of the data, provided all the Matlab scripts used during analysis and contributed to the statistics used. AR was involved in the securing of funding, the analysis of the data and the preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Klebsiella pneumoniae, containing highly-resistant antimicrobial profiles. Using 78 patient isolate test-plates, we demonstrated that our mobile-reader meets the FDA-defined AST criteria, with a well-turbidity detection accuracy of 98.21%, minimum-inhibitory-concentration accuracy of 95.12%, and a drug-susceptibility interpretation accuracy of 99.23%, with no very major errors. This mobile-reader could eliminate the need for trained diagnosticians to perform AST, reduce the cost-barrier for routine testing, and assist in spatio-temporal tracking of bacterial resistance.Routine antimicrobial susceptibility testing (AST) can prevent deaths due to bacteria and reduce the spread of multi-drug-resistance, but cannot be regularly performed in resource-limited-settings due to technological challenges, high-costs, and lack of trained professionals. We demonstrate an automated and cost-effective cellphone-based 96-well microtiter-plate (MTP) reader, capable of performing AST without the need for trained diagnosticians. Our system includes a 3D-printed smartphone attachment that holds and illuminates the MTP using a light-emitting-diode array. An inexpensive optical fiber-array enables the capture of the transmitted light of each well through the smartphone camera. A custom-designed application sends the captured image to a server to automatically determine well-turbidity, with results returned to the smartphone in ~1 minute. We tested this mobile-reader using MTPs prepared with 17 antibiotics targeting Gram-negative bacteria on clinical isolates of  The increasing prevalence of antimicrobial resistance represents a severe threat to global health16789quantitative result that allows tracking of resistance. The MIC value must be interpreted using a table of values that relate to the proven clinical efficacy of each antibiotic for various bacterial species. An interpretive criterion  is assigned to each bacteria/drug combination in order to guide the physician in treatment decisions. These interpretive criteria have been established by both the U.S. Food and Drug Administration (FDA) and the Clinical Laboratory Standards Institute (CLSI) using data from animal studies, microbiological studies, and clinical efficacy data. A susceptible result indicates that the patient’s organism should respond to therapy, while a resistant organism will not be inhibited by the concentrations of antibiotic achieved with normal dosages used for that drug.The gold standard for antimicrobial susceptibility testing is the broth microdilution methodAn important part of the global challenge for this gold standard testing is that a high level of clinical microbiology expertise and tedious examination of the well plate is required to read the turbidity from the MTP and to establish the interpretive criteria necessary for treatment. An additional global challenge is that the data collected in clinical microbiology laboratories are not easily available for epidemiological studies, and are not available at all in regions of the world where antimicrobial susceptibility testing is not regularly performed.Over the past several years, smartphones and other portable consumer electronics devices have demonstrated their capability for serving as a multiplexed platform for building biomedical imaging, sensing and diagnostic systems121314151617181920212223242526272829303132333435363738394041Klebsiella pneumoniae. This species of bacteria can exhibit highly resistant antimicrobial profiles and contain members of the Carbapenem resistant Enterobacteriaceae (CRE), with a very high mortality rate in multiple disease states including sepsis and pneumonia43444546474849We demonstrated this mobile system’s ability to automatically determine drug-specific MIC and corresponding drug resistance through a comprehensive clinical evaluation performed at the UCLA Clinical Microbiology Laboratory using plates containing 17 different antibiotics targeted for Gram-negative bacteria and tested on patient isolates of Our AST system  is comprOn receiving a new request, the server (implemented in Python using the Twisted framework) adds the request to the job queue and saves the images and job details to local storage. Each DNG image is converted into 16-bit tagged image file format (TIFF) and the pixels corresponding to the blue channel are extracted from the raw Bayer image. Subsequent processing of the blue channel images is performed using MATLAB. The server also has pre-recorded information of the mapping between the fiber-optic cables of our AST device and the individual wells of a 96-well plate. This 2D mapping does not change from image to image, and only needs to be determined once for a given device design. To find this mapping function, a bright-field image  of a plate is taken with deionized (DI) water with no turbidity in the wells and the centre of each fiber-optic cable is digitally calculated. To map successive images of the AST plates to this control image, corner wells are found using a threshold-based approach followed by morphological operations to separate adjacent wells. The corner wells are then used to remove scaling and alignment issues that might be caused by the camera’s auto-focus feature and potential misalignments between the camera and fiber-optic array, where the known physical distances among the fiber-optic cables are exploited to find the centre of each well. A circular mask with a radius of 25 pixels is then applied to reduce the interference from nearby fiber-optic cables before extracting the average intensity of each well’s signal.After extracting each well’s pixel intensity, the average intensities per well for the dim, moderate, and bright images are each normalized with respect to the average intensities of control images at each exposure time to scale the intensities from 0 to 1, with 1 representing complete transmittance of light through the well relative to the DI control. To maximize our dynamic range, the normalized transmittance values for each well from the dim, moderate, and bright images are first scaled respective to their integration times and then averaged to obtain a single higher dynamic range value for each well.first well in each drug-specific set of wells that contains turbidity. Depending on the selected microbe of interest, drug susceptibility interpretations are automatically made based on the MIC for each drug (see Klebsiella pneumoniae).To determine whether a well contains turbidity or not, we use a threshold-based approach to determine the cut-off transmittance value for each well. To choose an accurate threshold value for each well, we extract the normalized transmittance values of wells with no turbidity from a training set of patient plates and blank reference plates . As the turbidity increases, the light transmittance through the well decreases. Thus, from this set of normalized transmittance values from wells with no turbidity, for each well we decided to use a threshold at two times the standard deviation below the mean to determine whether a given well is statically likely to have microbial growth represented by turbidity. For a given plate type , the MIC for each drug is determined by finding the After the turbidity decisions of the wells, the MIC determination, and the drug susceptibility interpretations are all automatically made, the results are stored in a database on the server, and are also sent back to the originating smartphone within 1 minute. On the smartphone application, the user can review the results via the history page see , which lKlebsiella pneumonia isolates from patient samples collected by the UCLA hospital system and prepared and tested at the UCLA Clinical Microbiology Laboratory. Antimicrobial agents are tested using two-fold serial dilutions and the concentration range varies with the drug, the organism tested, and the site of the infection. For the microdilution method, the antimicrobial dilutions are in 0.1 mL volumes that are contained in wells of a 96 well microdilution tray. The drug panels are then stored frozen until they are inoculated. Briefly, a suspension of the tested organism is prepared in sterile saline to a 0.5 McFarland standard using isolated colonies. 1.5 mL of the suspension is transferred to an inoculating tray containing 40 mL of sterile distilled water. The inoculating tray has prongs that allow for transfer of bacteria into each well of the 96 well drug plate. The plate is then incubated for 24 hours at 37 °C. The panels are quality controlled with the appropriate ATCC (American Type Culture Collection) organisms. The bacterial pathogen identification was performed after the culture of the organism by MALDI-TOF (matrix assisted laser desorption/ionization - time of flight) identification method. For each plate, an expert diagnostician inspected the plate and recorded the presence or absence of turbidity in each well, which was used as our gold standard. Each plate was then imaged using our smartphone-based platform. All experiments were conducted at the UCLA Clinical Microbiology Laboratory by a medical personnel trained on how to use the mobile platform. This study was found to be exempt from IRB  review by the UCLA Office of the Human Research Protection Program.We tested our platform using Klebsiella pneumoniae were determined using a chart provided by the UCLA Clinical Microbiology Laboratory . Optical analysis by the digital reader may potentially reveal early turbidity and allow for a more rapid turn-around time of the AST results to the physician.How to cite this article: Feng, S. et al. High-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader. Sci. Rep.6, 39203; doi: 10.1038/srep39203 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Cardiovascular safety has to be proven for antidiabetic therapy for type 2 diabetes according to the guidance from regulatory bodies. Recently, the results of the respective study for the sodium-glucose transporter-2 (SGLT-2) inhibitor empagliflozin were published. Unlike similar studies for other antidiabetic agents that proved cardiovascular safety by non-inferiority compared with standard treatment with regard to a combined cardiovascular primary endpoint, empagliflozin showed superiority with a significantly lower incidence of cardiovascular events. SGLTs are cell membrane proteins that facilitate active glucose transport through the cell membrane. SGLT-2, a kidney specific isoform, is expressed in the renal tubule and is responsible for the physiological reabsorption of glucose from the proximal tubule after glomerular filtration. SGLT-2 inhibition therefore leads to removal of excess glucose via the urine in type 2 diabetes and improves glycaemia without an increased hypoglycaemia risk. SGLT-2 inhibitors have additional actions that may be favourable in type 2 diabetes: due to the glucosuria, a caloric loss occurs and in clinical studies a moderate loss of body weight between 2–3 kg is observed with SGLT-2 inhibitor treatment. Furthermore, blood pressure is reduced by approximately 2–5 mmHg together with slight volume and sodium depletion.6 Almost 50% of the patients enrolled in this study, BI 10773 (Empagliflozin) Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG OUTCOME) trial, had a previous history of myocardial infarction, in almost 75% coronary artery disease had been diagnosed and up to 25% had suffered from a stroke or had signs of peripheral vascular disease. The diabetes duration in the study cohort was also long, with a majority of patients being diabetic for more than 10 years. Likewise, signs of renal complications were also prevalent . The majority of patients were on a combination therapy for their type 2 diabetes with antihyperglycaemic medications. Concomitant medications for hypertension and dyslipidaemia were widely used with >90% of patients receiving antihypertensive agents and 80% receiving statins or other lipid-lowering drugs.A cardiovascular safety study according to the FDA guidance with the SGLT-2 inhibitor empagliflozin has now been published that not only proved cardiovascular safety but also reported a reduced risk of total mortality and a decreased risk of cardiovascular death in patients with type 2 diabetes at specifically high risk of cardiovascular events.6Empagliflozin was added to standard treatment at two doses of either 10 or 25 mg once daily in the intervention group. In the comparator arm, standard care was intensified to reach comparable glycaemic outcomes in all study arms. Both empagliflozin doses resulted in a significant reduction of total mortality and the three-point major adverse cardiac events (MACE) rate, mainly driven by a reduction of cardiovascular death. The number needed to treat (NNT) was 39 over a time period of 3 years. Empagliflozin was well tolerated with an increased rate of genital infection as an adverse event (6.4% versus 1.8%) but no increase in other adverse events.The reported risk reduction was most likely multifactorial and not related only to glycaemic effects, since the difference in event rates occurred early during the study and glycaemic control was comparable in all study groups. SGLT-2 inhibitors may elicit vascular effects and may change renal as well as cardiac regulatory functions in water, electrolyte and blood pressure regulation. Albuminuria and uric acid as established surrogate parameters for cardiovascular risk were also reduced in the empagliflozin-treated patients. Other still unknown potentially beneficial effects of SGLT-2 inhibitors may also contribute to the study results.7Do these findings now make it necessary to change diabetes treatment and guidelines immediately? Certainly, studies showing a significant beneficial effect on cardiovascular outcomes with antidiabetic agents are scarce and comparable studies with dipeptidyl-peptidase-4 inhibitors (DPP-4 inhibitors) have only shown non-inferiority versus standard care, but not superiority as the EMPA-REG OUTCOME trial. The results of the EMPA-REG OUTCOME study were achieved in a cohort with long-standing diabetes and cardiovascular disease and may not be automatically generalised for patients with new onset type 2 diabetes before having developed concomitant vascular complications. Here, metformin is still standard and has proved to be advantageous. The long-term effects of SGLT-2 inhibitors and empagliflozin over >10 years are still unknown and have to be established in comparison to classic antidiabetic drugs. Adverse events and safety characteristics are of utmost importance in medications for chronic diseases. In this respect, the rare reports on ketoacidotic metabolic disturbances that have been associated with the use of SGLT-2 inhibitors have to be taken into account as well. The respective cardiovascular safety study results with the other available SGLT-2 inhibitors canagliflozin and dapagliflozin have also to be completed in order to confirm these data and in order to have a broader picture of the action of the drug class of SGLT-2 inhibitors. At present, there is an indication to treat patients with type 2 diabetes and a similar cardiovascular risk profile as seen in the study population of the EMPA-REG Outcome trial with empagliflozin. In addition, there are now stimulating and positive data to explore the action of SGLT-2 inhibitors further in order to explain the findings of the EMPA-REG Outcome study in more detail and in mechanistic pathophysiological models. This may help to evolve the drug class further and may help to identify patient groups that have a specifically high benefit from such a therapy.


Mice were tested immediately after exposure in the juvenile period (7 weeks of age) and again as adults (12 weeks of age). Measures included electroencephalography (EEG) in response to auditory stimulation, the social choice test, and a 6-arm radial water maze task. Outcome measures include low-frequency EEG responses, event-related potential (ERP) amplitudes, indices of social behavior and indices of spatial working memory. Juvenile exposure to ketamine was associated with electrophysiological abnormalities in adulthood, particularly in induced theta power and the P80 ERP. The social choice test revealed that ketamine-exposed mice failed to exhibit the same age-related decrease in social interaction time as controls. Ketamine-exposed mice outperformed control mice as juveniles on the radial water maze task, but did not show the same age-related improvement as adult controls. These data support the hypothesis that juvenile exposure to ketamine produces long-lasting changes in brain function that are characterized by a failure to progress along normal developmental trajectories.Frequent ketamine abuse in adulthood correlates with increased risk of psychosis, as well as cognitive deficits, including disruption of higher-order executive function and memory formation. Although the primary abusers of ketamine are adolescents and young adults, few studies have evaluated its effects on juvenile cognition. Therefore, the current study analyzes the effect of adolescent ketamine exposure on cognitive development. Juvenile mice (4 weeks of age) were exposed to chronic ketamine (20 mg kg Studies suggest that drug abuse in the adolescent period leaves users with increased vulnerability to cognitive performance deficits, psychosis and neuroanatomical changes that can affect the rest of their lives.35 Social interaction testing, specifically the social choice or social approach test,38 allows controlled measurement of social interactions initiated by the test mouse, and is a replicable measure of sociability in mice.38 EEG measures are known to reflect neurodevelopmental changes during the adolescent period,41 and changes following ketamine treatment in adult mice are well-characterized.43 Furthermore, EEG phenomena, which are in part regulated by the GABA and glutamate systems, are altered in disorders due to abnormal NMDA receptor-mediated glutamate signaling, including ERPs and ER spectral perturbation (ERSP) theta frequencies.46 Alterations in power within the theta frequency range following auditory stimuli reflect performance on cognitive tasks including attention, working and long-term memory.51 There is also evidence that modulations in theta oscillations are reflective of hippocampal spatial memory function.48 As attention and working spatial memory are important aspects of our behavioral testing, theta frequencies are used in this study to shed light on ketamine-induced changes across the lifespan. Furthermore, in a related study, it was discovered that theta power was selectively altered in adult animals following chronic ketamine exposure 13Our study analyzes the effect of adolescent ketamine exposure on cognitive development in mice. Following the chronic exposure of adolescent mice to ketamine or saline vehicle, subjects underwent two stages of assessment, including 1 week following cessation of exposure as juveniles, and again at 5 weeks following cessation as adults. Spatial memory was assessed using a radial arm water maze, social preference was assessed using a social interaction task and EEG indices of brain function were also evaluated, including event-related potential (ERP) and baseline- and event-related power. The radial arm water maze task requires mice to utilize spatial memory to find a platform based on cues in the testing room, and has been used successfully with rats and mice.Delayed emergence of abnormalities in social behavior.Delayed emergence of abnormalities in working memory.Delayed onset of abnormal patterns of EEG.Delayed onset of abnormalities in social, cognitive and EEG measures will reflect a failure to progress rather than a deterioration.The current study tests the hypotheses that exposure to ketamine during adolescence will cause:ad libitum over the duration of the study. On conclusion of the last day of testing, mice were killed and brain samples were removed for future studies. All procedures adhered to the guideline of the NIH Guide for the Care and Use of Laboratory Animals and the local Institutional Laboratory Animal Care and Use Committee.Juvenile C57/Bl6J male mice from Jackson Laboratories  were used. Mice arrived at 21 days of age to our facility and were allowed 1 week for acclimation prior to the start of injections. Mice received 14 days of injections starting at 28 days of age. Electrodes were implanted at 42 days of age, with a week-long recovery period before testing. Mice were tested for all measures at 7–8 weeks of age and again at 11–12 weeks of age. Testing included 1 day of EEG recording, at 49 days and 77 days of age; 1 day of social interaction testing at 50 days and 78 days of age; and 3 days of water maze testing beginning at 51 and 79 days of age. Mice were kept on a 12:12 h light/dark cycle, with chow and water available n=14) or 20 mg kg−1 i.p. ketamine (n=14), injected daily over a 14-day period. This dose was chosen based on previous studies53Mice received either 0.9% saline 13 and dental cement  were used to secure the electrodes to the skull.56 One week of rest was given to allow for recovery before EEG recording took place.Electrode implantation took place under 1% isoflurane anesthesia. A series of three small holes were drilled into the skull at −1.8, −0.8 and +0.2 mm Anterior-Posterior to bregma; and 2.65 mm lateral. A positive electrode was placed into right hippocampal region at 1.8 mm posterior, 2.65 mm lateral and 2.75 mm dorsal to bregma. A reference electrode was lowered onto the surface of the ipsilateral cortex at 0.2 mm anterior, 2.75 mm lateral and 0.75 mm dorsal.The EEG testing apparatus consisted of eight standard mouse cages, modified to allow placement of a speaker on the top of the cage for delivery of auditory stimuli, located within a Faraday cage. Auditory stimuli were generated by Micro1401 hardware and Spike2, version 6.0 . EEG was recorded during auditory click presentations  of a white noise tone . Mice received a total of 300 clicks. To minimize the effects of stress, animals were given 15 min to acclimate to the ERP apparatus prior to each ERP recording session. ERPs were analyzed using Spike2 software.EEG data were processed using EEGLAB. Several mice were excluded from the EEG testing due to poor signals or broken electrodes.57 ERPs are systematic voltage deflections in the averages of EEG recorded during a response to repeated events, as described above; as average value of EEG activity during a repeated stimulus, ERP is believed to reflect bursts of neural activity that are ‘time locked' to these stimulus events.57 The amplitude of ERP waveform components were quantified as the maximum or minimum amplitude changes relative to the previous points of inflection. Latency was calculated as the time point at which the maximum or minimum value occurred during the specified time interval. P20 amplitude was calculated as the maximum value between 10 and 30 ms; N40 amplitude is defined as the minimum value between 25 and 60 ms; and P80 amplitude is defined as the maximum value between 50 and 200 ms.ERP is often used to translate sensory and cognitive changes in schizophrenia models to the patient.54 Baseline measurements were gathered from data prior to the start of auditory stimuli, when raw EEG was recorded for a 60-s period. Inter-trial coherence (ITC) measures the degree of cross-trial entrainment of the oscillation at a particular time-point for a particular frequency range. ITC is calculated by measuring the phase angle of the oscillation, with a value of 1 being complete overlap and 0 being no overlap between cross-trials. ITC was measured in the theta frequency range.ERSP is a reflection of the degree of power at a particular frequency range; in this case, we explored the ERSP data for theta oscillations. Theta frequency ERSP was quantified as the average EEG power between 4 and 12 Hz between 0 and 200 ms post stimulus. Power reflects the oscillation amplitude.The radial arm water maze consists of a circular pool, 90- cm wide by 78-cm high, with six stainless-steel arms of length 25.4 cm and width 20 cm, creating wide paths and an open center area. The pool was filled to 65 cm, submerging a platform placed at the end of one arm by about 3 cm of water. Non-toxic white paint was used to hide any distinguishing features of the maze beneath the waterline, including the platform. Lights were dimmed, and numerous visual cues were placed on walls throughout the room to act as visual cues to help the mice to navigate the maze .Testing was performed for 3 days, with 5 trials per day. At the start of each trial, mice were placed in one of the five empty arms. Start locations were randomized so that no arm was repeated, no mouse started from adjacent arms in consecutive trials and no sequence of arms was repeated. Each day, every mouse swam a full course of five arms, and was allowed 60 s to find the platform on each trial. If mice failed to find the platform in this time, they were guided to the platform before being removed from the maze. Mice were removed from the platform after 10 s, dried and placed in a warming chamber for 10 min before undergoing subsequent trials with different start locations. For juvenile testing, the platform was always placed in arm 5; for adult testing, the platform was always placed in arm 3, to prevent mice from using any long-term spatial memory to complete the task.The performance of mice on each trial was recorded by a single researcher who stood in the same place in the room for each trial. Latency and order of entry into maze arms during each trial were measured for every mouse by the same researcher. The number of errors (accuracy) and latency (the time taken to complete the maze) was analyzed. Efficiency was measured by analyzing the time spent per arm entry.58 The test is performed in a three chambered box (27 × 53 × 23 cm3), as shown in The social preference of subject mice was tested using the controlled social approach  test.P=0.05. A two-way repeated measures analysis of variance was used for both the social interaction and the EEG analyses. Fisher least significant difference post hoc analyses were used.Statistical analyses were performed using STATISTICA . For the water maze data, statistical analysis was conducted using a 2 × 3 × 5 way repeated measures analysis of variance with an alpha threshold of 1,16=29.133, P=0.00006, 1,16=15.740, P=0.00111, post hoc revealing an age-related change only in vehicle-treated mice  in saline-treated mice, 1,16=6.8882, P=0.01840, post hoc: P<0.05). However, there was no change across development in ketamine-treated mice , but there was no difference between groups in adulthood. A significant improvement in accuracy occurred across both developmental testing points, on all days and among all treatment groups by trial 3, which remained consistent through trials 4 and 5 . In human subjects, acute ketamine administration results in significant deficits in understanding facial cues for sadness, as well as a trend toward difficulty in identifying negative emotions.60 Similarly, schizophrenia patients show significant deficits in emotion recognition tasks, particularly with emotions such as fear and disgust.63 These studies may indicate that our mice exhibit a translationally valid effect of delayed emergence of deficits in understanding of social cues in schizophrenia.The current study first tested the hypotheses that exposure to ketamine during adolescence would cause a delayed emergence of abnormalities in social behavior. Data revealed that saline-treated mice showed an age-related decrease in time-per-social interaction that was not evident in ketamine-treated mice. All other measures including number of social interactions and total time spent on social interaction showed significant age-related decreases across both groups. This selective effect of ketamine on the length of individual interactions suggests that ketamine-treated mice may fail to respond to cues related to the termination of social contact. Adult male mice are known to be less social overall when compared with juvenile mice, which may reflect progression to a mature post-pubertal pattern of conspecific male interaction.65 In addition, the observation that ketamine-treated mice did significantly better than controls as juveniles suggests that the effects of ketamine in adult populations may not translate to the adolescent period. Such data would suggest that there may be ways to parse the amnestic and cognitive-impairing effects of ketamine from its properties on other domains. As ketamine is emerging as a potential therapeutic agent for depression, this ability to determine which factors mediate its beneficial vs detrimental effects will be critical. For example, future studies could determine which factors in normal brain development are sensitive or conversely insensitive to ketamine-induced alterations in cognitive capacity. It is important to note that several measures indicate that the test itself was a valid assessment of spatial working memory: mice showed significant improvement in performance over sequential testing days and trials, indicating that procedural learning took place.Our study also tested the hypothesis that chronic ketamine administration would result in the delayed emergence of abnormalities in working memory. We observed that control mice showed improvement on the spatial memory task in adulthood with a reduction in latency to find targets and resulting efficiency scores. Ketamine-treated mice did not change their performance over time. However, this observation may be in part related to the finding that ketamine-treated mice performed better than their control counterparts as juveniles. Furthermore, juvenile ketamine mice performed at a level that was similar to a control adult level. Therefore, data do not support the hypothesis that ketamine abuse in adolescence causes delayed emergence of abnormalities in working memory, though it is interesting to note that it does alter the normal trajectory for spatial working memory development. The failure to progress to more successful outcomes in ketamine-treated mice may reflect a ceiling effect, in which mice cannot perform significantly better than the level already achieved by them as juveniles. Previous studies demonstrate that mice typically achieve error rates of no fewer than three mistakes at their most successful, suggesting that our ketamine-treated mice could not have improved from their juvenile rates.69 Our data provide potential insight to ketamine-related changes in brain function as manifested by theta oscillations that are thought to reflect regional connectivity. Specifically, ketamine-treated mice showed a significant failure to progress normally with age for theta coherence, as well as induced and ERSP measures. Control mice showed a reduction on these measures on reaching adulthood, but ketamine-treated mice did not. Induced activity reflects activity in the brain that is not temporally tethered to the activity of the sensory system.70 As such, it is thought to reflect orienting, and selectively attentional response to the occurrence of an event, rather than the sensory registration associated with that event.71 Thus, the ketamine-treated mice express a juvenile-like pattern of ‘over reacting' to a non-threatening stimulus. A previous study showed that MK801 disrupts normal novel object recognition ability in rodents under acute exposure, and that its administration is known to cause attentional problems.72 Our data is not consistent with lasting attentional deficits shown in this study, but attentional changes are observed in the increased induced theta activity, indicating that attention pathways may be modified by NMDAR antagonists. Similarly, our group previously demonstrated that acute ketamine administration in adult mice yields a reduction of theta activity, indicating that the drug has a consistent influence on generators of theta activity, even though the drug's effects are different when exposure takes place in adulthood.46Finally, we investigated the hypothesis that a delayed onset of abnormal patterns of EEG would emerge as a result of chronic ketamine abuse in the juvenile period. Murine ERP data in the current study are consistent with findings in clinical studies of schizophrenia patients.74 Although the direction of change is not the same as described here in mice, the developmental time course for that change is similar. These data support the idea that although the effects of juvenile ketamine exposure may not appear in adolescence, they do emerge in adulthood. Furthermore, this P80 ERP change with age may be related to progressive deficits in the ability of subjects to withdraw attentional resources from stimuli, such as the auditory stimuli presented during EEG testing.75 The ketamine-related P80 changes are similar to the pattern of changes in the water maze. Ketamine-treated mice have a slightly lower P80 as juveniles compared with controls, and in adulthood both control and experimental mice show similar results. This similarity across experiments further supports the conclusion that ketamine abuse in adolescence causes a failure of age-related changes to occur in mice.Another ‘failure to progress' phenotype was seen in the P80 ERP measure, in that ketamine mice did not exhibit the same age-related reduction as controls. Changes in P80 component amplitude with age have been observed in human ERP studies.76 In our model, we restricted the NMDAR hypofunction only to the late adolescent period. In this scenario, although there were no manifestations of cognitive deficits in mice as juveniles, inappropriate hyper-social activity and a failure of attentional systems to develop normally in adulthood was observed. These data suggest that alterations in NMDAR activity that have been proposed to begin during the schizophrenia prodrome are consistent with a post-adolescent emergence of deficits. Alternatively, the lasting decline in cognition and theta power that occurs following chronic ketamine in adulthood,42 was not observed when chronic ketamine was restricted to the juvenile period. These data suggest that any alteration in NMDAR activity that may begin during the prodromal period in schizophrenia likely persists into adulthood. As such, the current model of adolescent ketamine abuse may not accurately reflect the chronic and persistent nature of clinical schizophrenia in humans. Similarly, the administration of NMDAR antagonists at different life stages can have drastically different effects. Data suggest that ketamine does not have the same level or kind of lasting effects on the juvenile brain as it does on the adult brain, perhaps due to increased plasticity of the juvenile brain. However, there may be a different critical period for the effects of ketamine than the period of development used in this study, which may explain the lower impact changes observed in the cognitive and social function of our subjects.Based on cross-sectional similarities between acute manifestation of NMDAR antagonists and prodromal symptoms, previous investigators have proposed that changes in NMDAR activity may occur before symptoms appear.77 Future studies on the effects of ketamine in the juvenile period might focus on the molecular changes in brain at different stages of development, adjust the timing of drug administration in the juvenile period or use different testing batteries that reveal effects of the drug on long-term memory and cognition.The aim of our study was to evaluate the effects of juvenile ketamine abuse on social behavior, working memory and EEG during and after adolescence in mice. We hypothesized that the administration of chronic ketamine in the juvenile period would result in a delayed incidence of prominent cognitive changes in the post-pubertal period. We found several interesting changes: hypersociality in adulthood and a failure to progress to normal theta and P80 activity as reflected in EEG measures. However, our results did not show the dramatic differences we expected to observe between control and ketamine-treated mice. Our findings indicate that ketamine abuse in the juvenile period does not produce the same kind of cognitive deficits as observed in post-pubertal abuse, perhaps due to the increased plasticity of the developing brain. Interestingly, we recently found that NMDA receptor function was decreased in mice treated with ketamine in adulthood, while it was increased in those treated in juvenile period.