We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem’s influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers. In drug delivery the utilization of nanocarrier systems provides increased bioavailability as well as the generation of specific targeted effects and for this reason is highly in focus of current research . During Alonso et al. advanced a method to obtain colloidal nanocapsules based on solvent displacement (or spontaneous emulsification) . In furtex vivo as well as in animal models [We have developed a nanocapsule drug delivery system based on the biopolymer chitosan which is known to increase paracellular permeability through epithelial barriers. Chitosan, a family of cationic natural aminopolysaccharides, is known for its various interactions with biological barriers, like mucoadhesive properties , the abil models . Severall models  and morel models  convene l models . These el models . Afterwal models . Recentll models . The infl models . Chitosal models ,17,31–35In this work, we utilized chitosan as a coating for capsaicin-loaded nanocapsules which was used as model for lipophilic drugs. Capsaicin is a natural compound occurring in hot chili peppers that is well-known for its pungency . We chosin vitro model for an epithelial barrier, with encapsulated and free capsaicin [In a previous study, we incubated Madin Darby canine kidney (MDCK)-C7 cells, an apsaicin . We founThe aim of this study is to investigate the influence of nanoencapsulated and free capsaicin on cell migration compared to its free form which, to the best of our knowledge, has not been addressed before, and to analyze its impact on cellular morphology.To this end, we developed a novel microscopy assay based on digital holographic microscopy (DHM) –51, an i2 flasks. The cultures were kept in an incubator set to 5% of CO2 and 37°C . For all experiments, the passages 41 to 47 were used. After forming a confluent monolayer, the cells were rinsed with 10 ml of Phosphate buffered saline (PBS) and trypsinized with 10 ml of trypsin buffer (0.05% trypsin EDTA). The trypsin buffer was diluted after detachment with 10 ml of cell culture medium. The cells were centrifuged at 300 rcf for 5 min in a falcon tube . Supernatant was removed, and the cell pellet was resuspended in 1 ml of cell culture medium. For counting, 10 μl of cell suspension was diluted with 90 μl of trypan blue and transferred to an improved Neubauer chamber. Afterwards, the cells were used for experiments. For subculture, the cells were split in the ratio of 1:10. All experiments were performed independently in triplicates on three different days.Madin Darby canine kidney (MDCK)-C7 cells were cultured using MEM supplemented with 10% of fetal bovine serum, 1% of L-glutamine (200 mM) and 1% of penicillin-streptomycin  in 75 cmin vitro experiments, 400 μl of a 100 mg/ml ethanolic lecithin solution  was mixed with 530 μl of the capsaicin stock solution (24 mg/ml). This was supplemented with 125 μl Miglyol 812 N  and 9.5 ml ethanol. The organic solution was immediately poured into 20 ml chitosan in the aqueous phase (0.5 mg/ml in 5% stoichiometric excess of HCl). The milky mixture was concentrated in a rotavapor  at 50°C until 3.5–4.0 ml remained and the volume was topped up to 4.0 ml with milliQ water if necessary to yield a final capsaicin concentration of ~10 mM. The nanoemulsions were prepared using the same procedure but without including chitosan. Unloaded nanocapsules and nanoemulsions were prepared by replacing the capsaicin solution with ethanol. The physical characteristics of the nanocapsules have been documented in a previous study [The chitosan-coated nanocapsules were prepared as previously described with modifications ,19. Brieus study . Briefly5 cells/dish and were allowed to attach overnight. The following day the medium was replaced with MEM lacking supplements but containing 50 μM of capsaicin or the nanoformulations with the same capsaicin concentration  in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer. Quantitative label-free DHM imaging of MDCK cells was carried out using an inverted microscope  with an attached DHM module [2-enriched atmosphere) showed a cytotoxic effect of capsaicin starting at a concentration of ~200 μM . From the resulting migration trajectories, velocity and directionality (Eq 1) were calculated and visualized as plots with the freely available Chemotaxis and Migration Tool .For each time lapse measurement, ten cells were tracked manually in the resulting series of DHM quantitative phase images by using the Tracking Plugin of the free software ImageJ  ,61 and re medium ,59 and tncell, nmedium and λ to be constant during the experiment, the average phase contrast Fig 1) after treatment with capsaicin and the nanoformulations. To visualize and quantify such cellular morphology changes, the histograms of quantitative phase contrast images of confluent cell layers were plotted, and the average phase contrast S1 Movie were corrected for background fluctuations using the "subtract background" option of ImageJ. Then, the background corrected phase images were analyzed at certain time points. MDCK-C7 cells migrated very fast within the cell mono layer during the observation periods in the observed field of view (for illustration see video S1 Movie in the Supporting Information). Thus, to compensate for this effect, 4 time-lapse phase images prior and 5 phase images after each time point were considered using the “stack” plugin of ImageJ. The resulting averaged histograms of the phase images around each time point were plotted using Prism v6.0c . Note that the averaging process of the phase data during the histogram generation also reduces the influence of the temporally randomly fluctuating disturbances in the quantitative DHM phase images as visible in video S1 Movie which are caused by the coherence properties of the applied laser light and fluctuating small particles in the cell culture medium. In addition, for each chosen time point the averaged phase contrast was calculated:For a confluent cell layer and assuming the parameters nx and ny denote number of image pixels in x and y direction and Δφi,j denotes the phase values of the image pixels.In Eq , the par2, 1 mM MgCl2, 5 mM KCl, 140 mM NaCl) to remove the excess dye. Finally, 200 μl of HEPES buffer was filled in each well, and the slide was immediately used for the experiment. Imaging was performed using structured illumination fluorescence microscopy (SIFM) based on an inverted fluorescence microscope  equipped with a structured illumination module . Fluorescence images were acquired at a rate of 0.88 Hz. After recording 20 frames, 100 μl of a sample containing 375 μM of the nanoformulation was added with a pipette to reach an overall concentration of 125 μM and the time-lapse experiment was continued for further 5 minutes. All images series were acquired with the same adjustments for excitation light intensity and camera exposure times. The resulting fluorescence images were analyzed using ImageJ. First, the background intensity was subtracted using the “rolling ball algorithm” . Then the brightness of all series was normalized, and the brightness value prior the addition of the treatments was defined as zero. The temporal development of the average image brightness was plotted using Prism version 6.0c .For calcium influx imaging, cells were seeded to form a confluent monolayer in an eight-well microscopy slide  and were allowed to attach to the surface overnight. After a single washing step with supplement free medium in each well the cells were incubated for 20 min 100 μl of cell culture medium containing Fluo-4 (1 μl of 5 mM stock solution in 1 ml). Then cells were rinsed with HEPES-buffered Ringer’s solution . Cells were treated with nanocapsules (1:40) or capsaicin (125μM) for 6 h and fixed with methanol for 20 minutes at -20°C.For analysis of the actin distribution cell membranes were permeabilized with 0.2% Triton-X-100 in PBS (Sigma) for 5 min and incubated with 130 nM Alexa Fluor 488 labeled Phalloidin (Invitrogen) for 45 minutes. Nuclei were stained with 0.5 μM 4’,6-Diamidino-2-phenylindole dihydrochloride  for 10s. Cover slips with fluorescence labeled cells were embedded in Aqua Poly/Mount (Polysciences) and imaged with an Olympus IX-81 inverted fluorescence microscope via a 60x UPLSAPO oil objective (Olympus) and a CCD camera .2, 1 mM MgCl2, 5 mM KCl, 140 mM NaCl) and blocked with 2% BSA in incubation buffer . The primary antibody directed against zonula occludens-1  was diluted in incubation buffer (1:150) and co-incubated with cells for 4 h at room temperature. The secondary FITC-labeled antibody, diluted in incubation buffer (1:200) (BD Pharmingen. 554020), was applied for one hour at room temperature. Cell nuclei were stained with DAPI. Finally, glass cover slips were mounted on a glass object slide using DABCO-Mowiol. Samples were analyzed with an inverted fluorescence microscope  and the ZEN software .For fluorescence imaging of ZO-1, cells were washed with HEPES-buffered Ringer solution . All experiments were statistically analyzed using non-parametric tests. For all experiments, the Kruskal-Wallis test in combination with Dunn's multiple comparison test was applied.S1 Movie  of the obtained quantitative phase images illustrates the swarm-like migration behavior of the cells and shows representative measurement results for each treatment. The control cells migrated slowly in a random movement pattern while the cell contours were not clearly visible. Cells treated with free capsaicin moved very similar but, in contrast, the cell boundaries were visible during the whole experiment and after ~8 h, formation of gaps between the cells was observed. For the cells treated with unloaded and capsaicin-loaded nanocapsules the swarm-like migration behavior altered to highly directed movements. Moreover, the migration speed of the cells treated with unloaded nanocapsules significantly accelerated in comparison to the control, while the cell outlines were more clearly distinct. For capsaicin-loaded nanocapsules, in analogy to the treatment with free capsaicin, after ~13 h, a formation of gaps between the cells was observed.To investigate the migration behavior of the MDCK-C7 cells after incubation with sub-cytotoxic concentrations of capsaicin-loaded nanocapsules, unloaded nanoformulation, free capsaicin and control treatments, we carried out time-lapse measurements using label-free digital holographic microscopy (DHM). To this end, confluent cell monolayers were observed for 18 h by taking digital holograms every ten minutes. Video Fig 2summarizes the results from the quantitative evaluation of the DHM phase contrast images by single-cell tracking. In Fig 2A representative 2D single-cell tracks are plotted. All tracks are arranged by adjusting their origin in the same starting point which is marked with a red dot at the respective x-axis. The lines represent the migration trajectories of each analyzed cell while the black dots indicate the final cell position at the end of each track. Due to their high migration velocities, the cells treated with the nanocapsules already left the field of view much earlier than the cells stimulated with the other treatments. Thus, the time durations of each tracking experiment, given above each plot, are not identical for the different treatments. The trajectories of the control cells show, in correspondence with video S1 Movie (Supporting Information), a mainly non-directed random pattern with an average maximum migration distance of ~100 μm from their initial position. The addition of 50 μM free capsaicin to the cell culture medium changed the initial random movements of the cells to a more straight migration. The tracks from the cells treated with unloaded chitosan nanocapsules show a further increase of directed movements. Treatment with capsaicin-loaded nanocapsules led even to almost linear cell migration trajectories and average maximum migration distances of about ~300 μm from the origin, which represents about the 3-fold distance compared to the control. Due to the high migration velocity some of the cells left the field of view prior the end of the time-lapse observation experiment. Thus, the further quantitative evaluation of the migration trajectories was limited to the first 7 hours of the experimental period. Fig 2B summarizes the retrieved average migration velocities from three independent experiments for each cell treatment and control cells. A statistically significant velocity increase was found for all treatments compared to the control . Fig 2C shows the averaged directionality derived from the migration trajectories. For all treatments, the directionality is found to be higher than for the control. This is in agreement with the appearance of the migration tracks shown in Fig 2A. The two nanoformulations induced a significant increase of the directionality values compared to the control  while for free capsaicin, however, the observed increase was non-significant . Fig 2D depicts the temporal dependency of the averaged accumulated migration distance. Note the almost linear trends of all curves. Fig 2E shows the corresponding data for the Euclidean distance, between the starting point and the end point of the single-cell migration trajectories. In agreement with the results obtained for the accumulated distance for all treatments, highly linear trends are also observed for the Euclidean distance. Notice that the curves of the cells treated with capsaicin-loaded nanocapsules show the highest gradient. In contrast, only a slight difference was found between the data from unloaded nanocaspules and free capsaicin whereas the curves of the control cells showed the smallest slope.Fig 3summarizes the results. In Fig 3A the histograms of quantitative phase images for a representative measurement at t = 0 h, 8 h and 14 h are plotted. The parameter Δφ detects the spatially dependent optical path length delays that are related to the thickness changes of individual cells in the confluent monolayers. Thus, the histograms of the phase values quantify global alterations of the cell layer surface roughness during the time course of the experiments . Fig 4A depicts the images for the controls and the treatment with free capsaicin. For the negative control, in which only the buffer was used, no fluorescence emission was detected. In contrast, in the positive control a sudden strong fluorescence signal was evident. In analogy to the negative control, the stimulation with 100 μM capsaicin induced no fluorescence signal. Fig 4B presents the results after the addition of unloaded and capsaicin-loaded nanocapsules. For both treatments fluorescence was detected. The time course for the measured fluorescence intensities of all treatments is plotted in Fig 4C. The positive control produces the strongest fluorescence signal immediately after stimulation. This response reaches a maximum after ~2 s and persists until the end of the observation period of ~24 s. Also, for the nanoformulations, an overall increase of the fluorescence intensity is detected, ~ 35% lower than for the positive control. In contrast to ionomycin, the observed effect is reversible . In the case of untreated cells and cells treated with free capsaicin, no fluorescence signal occurred during the experiment.Intracellular calcium fluxes are known to influence cell migration  and can S1 File). While the expression of TRPV4 was confirmed, the presence of TRPV1 could not be detected.To gain further insight into the calcium influx results the presence of TRPV1 and TRPV4 receptor ionic channels in the cells was probed by polymerase chain reaction (PCR)  showed continuous sharp lines along the cell-cell contacts for the control cells and the unloaded nanosystems. Notice that for the treatments with capsaicin and capsaicin-loaded nanocapsules, ZO-1 rearranges to discontinuous diffuse and disrupted structures with increased amounts of dislocated proteins in the cytosol and probably also in the nucleus. Note that a stronger effect is observed for free capsaicin than for the capsaicin-loaded nanoformulations which only cause few disturbances of the ZO-1 pattern. The distribution of the actin skeleton was further imaged for the loaded nanoformulation after 24 hours . No changes between the control and the treated sample were observed.To probe the influence of the chitosan nanocapsules on the cytoskeleton and the tight junctions we investigated changes in the actin filament structure and zonula occludens 1 (ZO-1) which is connected to many tight junction proteins and thus is a target to image tight junction protein rearrangements. In our previous studies  we have Fig 2D and 2E). Error bars of similar magnitude support the reliability of the retrieved data. These results matched well with the averaged endpoint measurements  where a higher migration speed corresponds to an increased cumulative migrated distance. The found increased directness of cell migration is also in agreement with increased values for the Euclidean distance. These findings also fit with the evaluation results of the DHM assay were global morphology changes of the observed monolayers  are correlated with rearrangements of the cytoskeleton and the distribution of tight junction proteins . In short, we found that changes of the migration behavior are accompanied by alterations of the global cell morphology in a time-dependent manner and rearrangements of actin and tight junction proteins as further discussed below.In this study, we have analyzed the migration behavior of MDCK-C7 cells after treatment with both, chitosan-based unloaded and capsaicin-loaded nanocapsules, in comparison to free capsaicin, and to untreated control cells. To this end, we recorded time-lapse micrographs by minimally-invasive observation with label-free DHM and analyzed the resulting quantitative phase images by cell tracking. This newly developed image analysis procedure quantifies global cell layer morphology changes from the interferometrically detected optical path length changes. Furthermore, we carried out investigations with fluorescence microscopy to gain insight into changes in the cytosolic calcium concentrations and to analyze rearrangements of the cytoskeleton and tight junction proteins. We found capsaicin and nanocapsules induced profound changes in the migration behavior of the cells. In general, all treatments induced increased cellular motility and speed and changed the migration from a mainly random pattern observed in the control, to a more directed, straight movements. The results from single-cell tracking reveal for all treatments that these effects were present over the entire time course of the experiments, as evidenced by the linear trends of the migration behaviors . Nevertheless, in general, the homeostasis of ions within the cell is also known to have an influence on cell motility [S1 File), in agreement with the lack of intracellular influx of calcium in the cells treated with free capsaicin. This result also suggests the observed effects of capsaicin on cell motility , might be governed by a different mechanism independent of calcium. Also, the cell morphology seemed to be affected  after capsaicin and nanocapsules addition. The fact that the cell contours became clearly visible is an indicator for an increase of the height in the cell center which resulted in a more heterogeneous surface roughness . This implies the nanoformulation itself must have had an impact on the cell motility which may stem from several possible reasons. One explanation could be the generation of a mechanical stimulus on the cell. It is known mechanical stimulation can lead to influx of calcium ions mediated via the TRPV4 receptor, which was proven to be expressed in the investigated cell line (supporting information S1 File). For endothelial cells, it has been shown mechanical stimulation had an impact on cell motility [Fig 4). Even though there was no change in calcium level visible directly after the initial influx it remains to be elucidated if there is a long-term effect on calcium influx related to this finding. The newly developed method to characterize global cell morphology changes using histograms and average phase values of quantitative DHM phase images concurs with the results from the motility analysis. Indeed, the Y-axis intercept of the average DHM phase contrast increased, which is consistent with an instant change of cell morphology. Also, the slope of the average DHM phase images indicated an increasing change in surface roughness of the cell layer after treatment with the nanocapsules . In the time lapse video S1 Movie the cell contours are clearly visible which also indicates a change in surface roughness. The highest values for migration speed and directness were observed for the loaded nanocapsules  which is probably the result of the combined effect of capsaicin and the nanosystem. The DHM morphology assay showed here the highest increase in surface roughness concurring with the highest Y-axis intercept and slope . In the corresponding time lapse video S1 Movie, it was also visible that tight junctions opening was more pronounced compared to the unloaded system. The observed weakening of the entire cellular network mediated by TJs and the actin skeleton could be an explanation for the overall increase in speed of displacement . Indeed, a recent study has revealed interference with a connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and directed cell migration [The increased phase values of the Y-intercepts of the DHM analysis of global morphology changes in us study . This efmotility ,66. Thisigration . Antibodigration . At leasigration , and havigration . Moreoveigration , which min vivo. The developed nanosystem is a potential new drug carrier for hydrophobic drugs due to the tight junction opening abilities which could also be used for functional food applications as it is only comprised of materials of natural origin. Ongoing studies in our groups investigate the feasibility of the system as a gene transfection agent with promising results.In summary, the treatment of MDCK-C7 cells with capsaicin encapsulated by a chitosan-coated nanosystem and in its free form resulted in a change of the cellular morphology as well as velocity and directness of migration compared to control cells while also the unloaded system demonstrated a change in these properties in a very similar way. Calcium influx occurred for the nanoformulations, but not for free capsaicin, which suggests an alternative mechanism could be at play as the TRPV1 receptor was not expressed in the investigated MDCK-C7 cell line. A newly developed DHM assay, that quantifies cell layer surface roughness changes by interferometric detection of cell induced optical path length changes, afforded complementary quantitative information about the global morphological alteration of the cells that are accompanied with the findings from cell tracking and calcium influx measurements. Protein redistributions within the cells  revealed by fluorescence microscopy further support the presence of cell morphology changes. Additional details of the underlying mechanisms at molecular level governing the stimulation of epithelial cells with nanoencapsulated and free capsaicin that induce their motility and change in morphology remain to be elucidated. “Why do the stimulated cells move in an orchestrated manner and the same direction?” is a question that needs to be answered. Chemotaxis induced by local gradients of concentration of nanocapsules as well as other unknown effects, cannot be ruled out, whereas the disruption of ZO-1 function and the associated restructuring of the actin cytoskeleton may provide some clues. It also remains to be discovered what is the significance of these phenomena for single cells, groups of cells and S1 MovieMovie of quantitative phase images resulting from time-lapse observations of MDCK-C7 cells with digital holographic microscopy after treatment with free capsaicin, unloaded chitosan nanocapsules and capsaicin-loaded chitosan nanocapsules in comparison to untreated control cells.(WMV)Click here for additional data file.S2 MovieMovie of temporal development of calcium flux utilizing the dye Fluo-4 as supporting information for (WMV)Click here for additional data file.S1 FileSupporting information about cell viability of MDCK-C7 cells after treatment with free capsaicin for DHM time-lapse observation conditions, ribonucleic acid (RNA) isolation and polymerase chain reaction (PCR) for probing the presents of TRPV1 and TRPV4, and fluorescence microscopy of actin in MDCK-C7 cells 24 hours after treatment with capsaicin loaded chitosan nanocapsules. S1_File.pdf).(PDF)Click here for additional data file.


These spoil-piles are generally found in multiple rows, several kilometers in length and heights of up to 50 or 60 m above the original landscape. They are highly dispersive and erodible. Legislation demands that these spoil piles be rehabilitated to minimize on-site and off-site discharges of sediment and salt into the surrounding environment. To achieve this, the steep slopes must be reduced, stabilized against erosion, covered with topsoil and re-vegetated. Key design criteria  are required for the construction of post-mining landscapes that will result in acceptable erosion rates.Open-cut coal mining in Queensland results in the formation of extensive saline overburden spoil-piles with steep slopes at the angle of repose (approximately 75% or 37A novel user-friendly hillslope computer model MINErosion 3.4 was developed that can accurately predict potential erosion rates from field scale hillslopes using parameters measured with a 3m laboratory tilting flume-rainfall simulator or using routinely measured soil physical and chemical properties. This model links MINErosion 2 with a novel consolidation and above ground vegetation cover factors, to the RUSLE and MUSLE equations to predict the mean annual and storm event erosion rates. The RUSLE-based prediction of the mean annual erosion rates allow minesites to derive the key design criteria of slope length, slope gradient and vegetation cover that would lead to acceptable erosion rates. The MUSLE-based prediction of storm event erosion rates will be useful as input into risk analysis of potential damage from erosion.MINErosion 3.4 was validated against erosion measured on 20 m field erosion plots established on post-mining landscapes at the Oakey Creek and Curragh coalmines, as well as on 120 and 70 m erosion plots on postmining landscapes at Kidston Gold Mine. The nature of open-cut mining is that much larger surface area is disturbed compared to underground mining. In Australia, the deep solid overburden above the mineral or coal seam is blasted using explosives and then removed mechanically using trucks & shovels or draglines. The latter is the most common method used in Central Queensland open-cut coal mines. The high speed of operation using draglines results in landscapes that consist of long parallel tertiary overburden spoil-piles that are generally highly saline, dispersive and highly erodible as shown in -1. These storms will result in severe erosion unless the slope length and steepness are reduced and the disturbed land rehabilitated and revegetated satisfactorily.In Central Queensland, where most of these open-cut mines are located, annual rainfall may range from 200 mm to 1400 mm, with a long-term median of around 600 mm and dominated by several intense tropical rainstorms annually which may reach intensities of over 100 mm.hrIn Queensland alone, the total area of land disturbed by open-cut coal mining and land already rehabilitated exceeds 146,000 and 44,000 hectares, respectively . It is tThe most expensive component of the rehabilitation process is the re-shaping and preparation of the overburden dumps to create a suitable landscape for vegetation growth. Landscape reconstruction typically involves extensive and costly earthworks to produce a post-mining landscape that is resistant to geo-technical failure and surface erosion processes by rainfall and runoff. In the case of high risk overburden material  the DEHP Financial Assurance Calculator provided estimates of default third party cost for reshaping and capping as $136,000 per ha .Severe erosion can occur on post-mining landscapes if slopes are too steep and may result in rehabilitation failures, excessive sediment and salt discharges onto surrounding areas and waterways as well as the mining operational areas which could result in additional costly interruption of mine operations. As the marginal cost per additional degree of slope reduction increase progressively as the final slope gradient is reduced (associated with greater earthworks per degree of slope reduction), and as soil and overburden materials exhibit a very wide range of erodibilities , the extA methodology that assist mine-sites to efficiently and economically design post-mining landscapes prior to reconstruction, and that will result in acceptable erosion rates  would be useful. As these landscapes are particularly vulnerable to erosion during the first few years following rehabilitation and prior to the establishment of adequate vegetation cover, this methodology should also allow the prediction of the probability of damaging erosion events from rainstorms.Existing erosion models such as the Universal Soil Loss Equation (USLE) , and itsIn contrast, in the management of post-mining landscapes, changing slope gradients and slope lengths are the essential first steps necessary to stabilize the landscape. Overburdens are often high in rock content which provides protection from water erosion, while top-soils are often limited in quantity, of poor fertility and may consist of coarse grained materials such as fine gravel. The surface soil or overburden are vegetated, left undisturbed and allowed to consolidate (increase in bulk density and strength) with time. Vegetation cover fluctuates seasonally and may disappear temporarily due to the bushfire which is a natural part of the Australian ecology. In the latter situation, the soil has been consolidated by the processes of wetting and drying and the presence of roots. Therefore, separating the effect of above ground vegetation and the consolidation of the soil including the roots would allow the estimation of erosion rates from bare soils following bushfires. Existing agriculture based erosion models were not intended to design new landscapes for erosion control on post-mining landscapes and a new approach was needed for that purpose.-1 rainstorm for 30 minutes  at 20% slope, followed by slopes of 5, 10, 15 and 30% for 15 minutes each. At these rainfall intensities, steady state was quickly established. The data from these measurements and the derived parameters were used to develop the MINErosion 2 model, which successfully estimates field scale erosion rates on bare unconsolidated simple linear hillslopes with various combinations of slope gradients and lengths. MINErosion 2 can also be used effectively to simulate multiple field plot experiments on a computer, based on a few measurements made on a tilting flume-rainfall simulator facility in the laboratory. This is a highly efficient experimental procedure.The MINErosion 2 model was developed to overcome the problem of scaling across different length of hillslopes. It combines the separate laboratory measured rill and inter-rill erodibility parameters into a combined erodibility and the prediction of erosion from unconsolidated (freshly applied) material at the hillslope scale of Central Queensland  The concThe model was developed as an Excel spreadsheet, with a user interface written in MS Visual Basic . ErodibiThe main output from MINErosion 2 includes rill, inter-rill and total soil loss from different combinations of slope and slope length on hillslopes with bare unconsolidated soil or overburden . HoweverMine-sites require assessments of the potential annual erosion rates from rehabilitated areas for their environmental reporting and completion criteria reporting to the DEHP (Department of Environment and Heritage Protection), the authority responsible for rehabilitation monitoring and compliance. In addition, mine-sites are also concerned about the risk of erosion damage to the rehabilitated landscapes during the early years prior to the establishment of vegetation cover. In the transition period from bare unconsolidated to vegetated and consolidated soil/spoil, appropriate algorithms and models for the effect of above-ground vegetation cover and consolidation had to be developed.As the RUSLE mean annual erosion rates are required by DEHP as a measure of erosion rates, MINErosion 2 was then linked to the RUSLE equations by incorporating the above ground vegetation cover and consolidation factors, to predict mean annual erosion rates, and to the Modified USLE or MUSLE  equationTherefore, the objectives of this paper are:To develop the above-ground vegetation cover (C) and consolidation (Ccons) factors that are necessary to apply RUSLE and MUSLE to rehabilitated mine-sites;To develop MINErosion 3 as a tool for the prediction of event-based and annual soil erosion rates from soil erodibility measured with a tilting flume-rainfall simulator facility in the laboratory or from routinely measured soil physical and chemical properties;To validate the MINErosion 3 model with field data collected using erosion plots in the field.vegetation cover (C) function was developed from measurements on the tilting flume-rainfall simulator facility shown in The Chloris guyana, and a stoloniferous Sabi grass, Urochloa mosambicensis) and 2 replications. Plants were watered regularly and maintained at the field capacity. After reaching adequate growth with more than 50% cover, they were trimmed successively to have 50%, 25%, 10% and 0% standing vegetative cover. Trimming was conducted immediately following the completion of a series of measurements at each level of cover and each series is completed within one or two days. Visual and photographic assessments were made of the vegetative cover. At each stage, 30 minutes’ rainfall simulations  were conducted at 10, 15, 20 and 30% slope. Samples of run-off were collected for 10 seconds each at 3, 5, 8, 12, 16, 20 and 25 minutes, to determine run-off rates and sediment concentrations. As the steady state is achieved within 15 mins, the average of the last 3 measurements were used as the steady state run-off and sediment concentrations. The results were statistically analysed by computing the standard errors of the mean values, and an analysis of variance conducted at the p = 0.05 to determine the effect of soil, slope and vegetation cover on steady state run-off, steady state sediment concentration, soil loss and relative soil loss rates. Details of this procedure are given in The tilting flume was equipped with 8 inserts (3 m long x 0.8 m wide x 0.25m deep), which can be lifted into the flume for a simulation run and lifted out and placed in the glasshouse. The inserts were packed with 2 soil types, a heavy black clay  and a sandy loam ), planted with 2 types of grasses  were calculated for each level of vegetative cover to develop the vegetation cover function independent of soil properties.The reduction of the above-ground vegetation cover was also intended to simulate the effect of frequent loss of cover by fire  which leaves roots intact within the soil. The effect of roots in the soil was included as part of the soil consolidation factor (see below). The separation of roots from the above ground component is different from the USLE models  where veconsolidation function (Ccons) was derived from field measurements of soil erodibilities on two mine-sites and described in details in MUSLE associated with the age of rehabilitation. At the Blair Athol Mine, Central Queensland  measurements were taken on a sandy loam  and clay soils  using a 4-m long field rainfall simulator similar to that shown in The o 43’ S, 139o 29’ E) on a sandy loam  with some rocks and gravel [Similar simulations were also conducted at the Mount Isa Mines, Queensland  to measure rill erodibilities. Run-off water and sediment were measured at different intervals during the simulations. Total sediment delivery, steady state run-off and sediment delivery rates were calculated from the data, and used to calculate rill and inter-rill erodibilities of the sites. Relative KMUSLE (Consolidated/Unconsolidated erodibilities) were calculated and plotted against the age of rehabilitation to develop the consolidation function. As roots were left in the soil and considered as part of the soil, its effect was included in the consolidation factor.All above ground vegetation was removed and rainfall simulation was conducted over a plot of 4 m x 1.5 m using 100 mm.hrFor each site, bulk soil samples were collected with a backhoe and transported to the laboratory for a standard simulation on the tilting flume to determine the slope adjustment factors for each soil material. For each flume, three 200 l drums of soil are required and are sieved to <50 mm before use.The starting point for MINErosion 3 is the MINErosion 2 model, and details of the equations involved have been published previously , 11, and-1 and on slopes that have been vegetated and left to consolidate with time, MINErosion 2 needs to be developed further and linked to the RUSLE and MUSLE equations.The output from MINErosion 2 includes the rill, inter-rill and total erosion rates per 1m plot width for any selected slope and slope length combinations of bare unconsolidated soil/spoil on simple uniform slopes . For prep) into a a single-valued erodibility using the MUSLE equation. This was done by simulating erosion for a standard plot  on bare unconsolidated soil/spoil to derive the erodibility KMUSLE as defined by the MUSLE equation [In MINErosion 3, the rill and inter-rill erodibilities from MINErosion 2 are combined with estimates of the total runoff volume (Q) and peak runoff rate  and the peak runoff rate (mm.hr-1), respectively. At the steady state, they are estimated for the simulated conditions from the following equationsp is the peak run-off rate(mm.hr-1), I is the rainfall intensity (mm.hr-1), Ir is the steady state infiltration rate (mm.hr-1) and D is the rainfall event duration in hr.Q and qMUSLE).LS is the USLE slope length and gradient factor for the standard plot, and C and P are the above-ground vegetation cover factor described above and soil protection factors , storm duration and its recurrence interval can be estimated using the IFD (intensity-frequency-duration) curve for the location [For the estimation of event-based erosion rates, a relationship between the recurrence interval (probability of occurrence) of rainstorm intensity and the rate of erosion is generated. For any given rainfall intensity above 60 mm.hrlocation , 22. Sto30 is computed using the long-term average rainfall pattern for the selected location . The default calculation is for a bare, unconsolidated soil/spoil. Computation is repeated for soil/spoil that has been consolidated for various periods and degrees of vegetation cover for selected combinations of slope lengths and slope gradients. Mean annual soil loss rates are then plotted against slope length for selected slope gradients. As the slope gradient, slope length and vegetation cover are the three landscape factors relevant for design and construction of post-mining landscapes that will result in acceptable  erosion rates, these graphs can readily be used to derive suitable landscape design factors.The mean annual soil loss was computed using the RUSLE, and a relationship between the average annual soil loss rate and slope length is generated for various slope gradients. The mean annual EIThe flow chart and the processing steps of the MINErosion 2 and 3 models are presented in o 30’ S, 148o 50’ E) and Oaky Creek mines  near Emerald, Queensland in 1992. These field plots were 20 meters long (up and down the slope) and 5 meters wide across the slope. The slope varied from 10, 20 and 30% with different percentage of vegetation cover of trees and grasses and time of consolidation [Field erosion plots were initiated on the Curragh  on the soil plots by cutting and drying.The site was instrumented with 2 recording rain-gauges at the top of the 10% and 30% plots. Each plot had a double bedload sediment trough at the bottom to collect the coarse sediment (bedload). The total run-off volume and peak run-off rate were recorded with a tipping bucket and the number of tip per minute was recorded on a data-logger. Total tips were also recorded on a mechanical counter as a backup. To determine suspended sediment concentration in the run-off, a small sample (0.1% of flow) was collected using a 10 cm copper tube with a 1 mm slit and accumulated in a 20 l plastic drum. A sub-sample was taken to determine suspended solid concentration, pH and EC. Soil loss was measured as the sum of bedload and suspended sediment load. Bedload was collected after each event and weighed. Vegetative cover was quantified from 3 photographs taken at 5 m intervals and covered an area of 30 mo 6’ S, 143o 26’ E) where in 1996 erosion plots of 120 m long and 20 m wide were setup on 20o (44%) slopes, and 70 m long plots x 20 m wide on 37o (75%) slopes [o slopes, and two treatments  x 2 replications on the 37o slopes. There were also two older plots initiated in 1991 on 17o and 37o consisting of spoil + vegetation treatments. Soil capping consists of 20–30 cm soil material. Run-off and soil loss were measured in a similar fashion as at the other two mine-sites. Because of the size of the plots, a 3-trough system was used to collect and measure the bedload. This comprises a modified triple Gerlach troughs (20m x 0.5m x 0.4m each), to collect soil and water and direct the water either through simple manifolds to tipping buckets, which measure total flows and thence to automatic samplers for collection of samples for sediment analysis or through modified Parshall flumes, which measure flow rates and total water volume and thence to automatic samplers. Five automatic samplers are being used on plots where high runoff of water and sediment is expected on the 20o slopes and five tipping buckets in lower runoff situations on the 37o slopes.The location of the three sites is shown in ) slopes . There wDetails of the procedures used for validation of annual and individual rainstorm event erosion rates are given in The Analysis of Variance (AOV) shows there were no differences in the steady state (SS) run-off (RO) rates between the combinations of soil, slope and vegetative cover, but the SS sediment concentration and hence SS erosion rate increase as veg cover decrease and slope increase. However, when erosion rate is expressed as a ratio in comparison with that from bare soil there were no differences due to soil and slope and the ratios were averaged across different soils and slopes. There was, however, a significant effect of vegetation type see . These rThe data in MUSLE (Kconsolidated/Kunconsolidated) is plotted against time (years) of consolidation in consolidated/Kunconsolidated and X time (years) since rehabilitation. Hence it is reasonable to assume that this relationship should also apply to most other soils as well. The relative KAt the Curragh and Oakey Creek mines, 6 years of data were available to estimate the average annual soil loss from 20 m long bare plots. At Curragh the first 3 years were below the average rainfall followed by 3 years of above average rainfall. The long term mean EI30 was used for MINErosion 3 prediction.rd year of the field trial. They were derived from 120 m bare plots(20o slope); 3 years rehabilitated and vegetated plots on 20o (120 m long) and 37o (70 m long) slopes, and 9 years of rehabilitated and vegetated plots on 17o and 37o slopes. With one year of data, the measured EI30 for that year was used which amounted to 79% of the mean annual EI30 At Kidston Goldmine, there was only one year of complete set of data available from the 32 of 0.86** and the Nash–Sutcliffe model efficiency coefficient value is 0.755 indicating good agreement between predicted and measured values of the annual soil loss. Although data were limited and of a short term nature, it covered a wide range of conditions and this shows that MINErosion 3 can be used to predict the annual soil loss from field plots with reasonable confidence using data derived from laboratory flumes under simulated rainfall.The comparison is plotted in -1 rainfall intensity, the observed storm events were replaced with equivalent 100 mmhr-1 rainstorms with a duration that would result in the same EI30 as the observed storm events. Only uninterrupted single peak rainstorms with peak intensities greater than 60 mm.hr-1 were selected for validation of the model. Eight major single peak storm events for Curragh from 1996 to 1999 were considered suitable for validation. Storms with multiple peaks were not included at this stage. The rainfall data collected by the data logger on the Curragh mine plot areas include the total amount of rainfall, date, duration, the total energy and EI30 values.As MINErosion 3 assumes a steady state condition with a constant 100 mm.hrThe occurrence of the 8 storms on the various treatments (bare and vegetated plots) resulted in thirteen (13) erosion events from plots covered with soil  and twen2 of 0.649**. A Nash and Suttcliffe coefficient of efficiency [2 of 0.625** and a Nash Sutcliffe coefficient of efficiency of 0.612 [ficiency  was calcMINErosion 3 was developed as a model to predict potential event based and annual erosion rates from steep rehabilitated post-mining landscapes under intense tropical rainstorms, based on measurements conducted in the laboratory under simulated rainfall. As laboratory measurements can be conducted prior to landscape reconstruction or even prior to commencement of mining, it provides mine-sites with the ability to estimate the rate of annual and event based erosion from any proposed post-mining landscapes, thus providing various options for post mining rehabilitation planning. Results from this work shows that the prediction of erosion rates from defined post-mining landscapes are accurate and reliable.-1 based on analysis of rill density data by Gilley et al. [The assumptions that underpin MINErosion 2 and 3 are that run-off and soil loss processes following intense tropical rainstorms on uniform, unconsolidated, bare soil/spoil on slopes of low surface roughness, can be modelled as a time-invariant steady-state process . Under ty et al.  and our -1 with a duration that would result in the same EI30 value as that of the observed storm. Run-off properties were estimated from this design storm and the soils infiltration rates, while the vegetation cover used was measured on the field plots and consolidation estimated from the age of rehabilitation. Similarly for the annual soil loss, the mean cumulative annual EI30 was used. The agreement between predicted and measured event and annual erosion rates were excellent, confirming that the assumptions used in this model are reasonable, in particular that erosion from intense tropical rainstorms can be modelled as steady state processes.As raindrop kinetic energy is a major driving force for erosion, in the validation of the model for event-based erosion, the predicted erosion rate was calculated for a design storm of 100 mm.hrAs the main target users of this model are busy environmental officers with limited available time, MINErosion 3 was designed as a user friendly model to derive appropriate landscape design parameters. Detailed data of 35 soil and spoil materials from Central Queensland mines are embedded in the model. They can be readily viewed and are available to the user to select from. For soil or spoil from areas other than Central Queensland, the user can select a soil/spoil from the database with properties that is closest to the soil/spoil of interest. Alternatively they can use their soil/spoil physical and chemical properties to estimate the interrill and rill erodibilities, or they can make their own measurements (detailed procedures embedded in the model) and processed the data through the MINErosion 3 model. Detailed explanatory notes were embedded in the model to enable users to familiarize themselves quickly or to refresh on the details of the model. This includes an introduction to erosion and erosion modelling, procedures used for conducting laboratory and field rainfall simulation, and procedures for soil physical and chemical analysis used to derive the parameters used in the model. Other target users are students and the embedded features of the model would also be useful as an educational tool. Outputs as graphs and/or tables can readily be obtained and printed. -1.year-1.The question of acceptable erosion rates is probably best defined as the average erosion rates of the surrounding natural unmined areas. These can be estimated by using MINErosion 3 with consolidation factor of 10 years or more  with an Another possible use of MINErosion 3 is to estimate erosion onto the mine working area. On one side of the working pit of an open-cut coal mine is the newly formed overburden spoil pile at the angle of repose (75%) and could be well over 50 m high, and is therefore vulnerable to erosion from intense rainstorms. This could affect the working pit conditions. The probability of these severe erosion event from the recurring rainstorms is shown in A unique feature of this model is an embedded database of soil physical and chemical properties and erodibilities of 35 materials i.e. 17 soils and 17 overburdens from open-cut coalmines and 1 soil from an open-cut goldmine in Central Queensland (CQ), which can readily be selected from a pull-down menu. This covers a wide range of soils and spoils from Central Queensland (CQ) and enables environmental officers to readily make erosion predictions for most CQ mine-sites. For Australian soils or overburdens that are not covered by this database, additional measurements can be made with a tilting flume-rainfall simulator facility similar to that available at Griffith University, to determine the rill and inter-rill erodibilities of the new material, and they can be entered into the database. Alternatively, they can be estimated (as a first approximation) from routine soil physical and chemical properties  and these options are built into the model. As the climatic data in the current model is limited to the Australian continent, for locations outside the Australian continent, the annual EI30 can be used to estimate the average annual soil loss, but local climatic data (frequency intensity duration of rainstorms) is required to determine storm event erosion with specific recurrence intervals.h.so@griffith.edu.au; ashraf.khalifa@gmail.com) or downloaded from the following link: https://www.dropbox.com/s/oq4487w7btd7r1u/setup.exe?dl=0.The current version is the MINErosion 3.4 and a copy of the installation file is available for download from the authors (The model has not been adequately tested for use on very low slopes and cropping land (< 6%). Further work will be required to check the validity of the model for these conditions. As MINErosion 3.4 is a hillslope erosion model, it cannot be used to estimate the total erosion from a catchment or the overall post-mining landscape with multiple catchments. For that, MINErosion 3.4 needs to be combined with a GIS system (ArcGIS) into a landscape erosion model (MINErosion 4) and this will be presented and discussed in a following paper.The model’s vegetation cover is limited to grass vegetation cover at this stage. No data are available for shrubs or tree cover and further measurements and work is required to develop appropriate cover functions for such vegetation.In conclusion, this paper has shown that the hillslope erosion model MINErosion 3.4 can be used to estimate the expected mean annual erosion rate and event erosion rates from steep post-mining landscapes using parameters measured on laboratory tilting flumes under simulated rainfall. As MINErosion 3.4 is based on steady state processes, erosion on steep landscapes under intense tropical rainstorms are adequately approximated and modelled by steady state processes. Finally, MINErosion 3.4 is a user friendly application that allows mine-sites to readily derive key landscape design parameters  to construct post-mining landscapes that will result in acceptable erosion rates. MINErosion 3.4 prediction was validated against measured annual erosion rates and event erosion rates from field erosion plots on rehabilitated post-mining landscapes at two open-cut coalmines and one open-cut goldmine in Central Queensland with excellent agreement. The software MINErosion 3.4 allows mine-sites to estimate mean annual erosion rates from proposed or constructed post-mining landscapes, as well as erosion rates from rainstorms with selected average recurrence intervals and use this as input into their risk analysis for potential erosion damage assessment.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


Overdiagnosis and overtreatment are rarely discussed in the context of NTDs despite their relevance for patients under the care of health services with limited resources where the risks of therapy induced complications are often disproportionate to the benefit. The advantages of cyst staging-based management of patients with cystic echinococcosis (CE) are not yet fully explored. Questions are: Do inactive cysts (CE 4 and CE 5) need treatment and is there a difference between cysts which reach CE4 and CE5 naturally or by benzimidazole therapy?Analysis of long-term follow-up data from a prospective CE patient cohort of 223 patients of a national clinical center for echinococcosis. The event of interest “relapse” was defined as the reversal of a cyst from an inactive stage  back to an active stage. The watch &wait (ww) group included 30 patients with 46 inactive cysts who never received medical treatment. The benzimidazole-treated (med) group included 15 patients with 17 cysts. There was no relapse in the ww-group whereas 8/17 cysts showed relapse within 18 months after treatment in the med-group. Loss to follow-up was 15.5%.Data from the watch & wait group impressively show how stable naturally inactivated cysts are in contrast to cysts which reach inactivity through treatment with benzimidazoles. A substantial proportion of patients can be spared from treatment through cyst staging. Cysts which inactivated through a natural course do not relapse with very high likelihood. We recommend follow up of 5 years to confirm the stability of the inactive stage. Cysts driven into inactivity through benzimidazole therapy instead need careful monitoring to identify those which reactivate (around 50% within 18 months). 5 years follow-up appears safe to make a final decision on the need for further monitoring. Cystic echinococcosis (CE) is a zoonosis of worldwide distribution causing cystic lesions in the liver and lung. The prevalence of disease is highest in countries with limited resources. Overdiagnosis and overtreatment are increasingly discussed in medical practice not surprisingly also for patients with cystic echinococcosis. In neglected tropical diseases (NTD) this is particularly relevant since most patients are under the care of health services with limited resources where the risk of therapy induced complications are often disproportionate to the benefit of interventions. Our study aims at answering the following Questions: Do inactive cysts (CE 4 and CE 5) need treatment and is there a difference between cysts which reach CE4 and CE5 naturally or by benzimidazole therapy? Results of our study impressively show that a substantial proportion of patients can be spared from any treatment through cyst staging. Cysts which inactivated through a natural course of involution do not relapse with very high likelihood. To rule out unlikely events, we recommend follow up of 5 years to confirm the stability of the inactive stage. Cysts driven into inactivity instead need careful monitoring to identify those which reactivate (around 50% within 18 months). 5 years follow-up appears safe to make a final decision. Overdiagnosis and overtreatment are increasingly discussed in medical practice not surprisingly also for patients with cystic echinococcosis (CE). In neglected tropical diseases (NTD) this is particularly relevant since most patients are under the care of health services with limited resources where the risk of therapy induced complications are often disproportionate to the benefit of interventions. In high-tech environments conditions which would never have surfaced and bothered a person regularly get unintentionally diagnosed and unnecessarily treated .CE cysts are staged on the basis of the WHO-IWGE ultrasound (US)-based cyst classification  into “acThe objective of this study is to test these two hypotheses in a prospectively followed-up well documented clinical cohort.The Section of Clinical Tropical Medicine at Heidelberg University Hospital runs an interdisciplinary clinic for echinococcosis in cooperation with the Department of Diagnostic and Interventional Radiology with weekly radiological conferences, the Department of Surgery, the Department of Thoracic Surgery, the Interdisciplinary Endoscopy Center, and the Department of Parasitology since 1999. Our unit is a national clinical center for echinococcosis. Patient data is collected prospectively, at every visit of CE patients a defined set of cyst and treatment data are entered into the CE database. The CE patient database contained 223 consecutive patients in February 2014. The database was screened for patients with w&w management or medical treatment and all follow-up data of patients eligible for the study were updated in June 2016.Of 223 patients entered into the database, 114 patients were male and 109 female, the mean age was 46.14 (2.62–90.79). Further details of the patient cohort are shown in Patients with single or multiple (maximum 8) inactive CE4 or CE5 cysts who never received medical treatment for CE.Patients with single or multiple (maximum 8) active CE cysts which were treated with benzimidazoles for CE and became inactive.To determine the time to relapse with sufficient accuracy, relapse from CE4 and CE5 to an active cyst stage was only included into the analysis if the time between the visit when the cyst was still inactive and the visit when it relapsed was ≤ 18 months. Patients were followed-up by ultrasound or magnetic resonance imaging. A patient was considered lost to follow-up if he did not complete a minimum follow-up of 5 years or the last follow-up visit was > 24 months ago. Patients with disseminated CE were excluded.The Ethical Board of the University of Heidelberg (S126/2010) approved the analysis of patient data form the CE patient database without additional patient consent. All patient medical data analyzed were anonymized.The event of interest “relapse” was defined as the reversal of a cyst from an inactive stage  back to an active stage.Proportion of cyst relapse in the med-group was calculated for each follow-up year since reaching the inactive stage. The numerator consists of the number of cysts with relapse during the respective time window, the denominator is the number of cysts still under observation which are at risk for relapse. A binomial exact 95% confidence interval was calculated for each follow-up year.In case medical treatment was still on going at the time of the visit, the time at risk started at the end of therapy. If this date did not coincide with a visit to the clinic, the cyst stage from the last visit was considered (last observation carried forward).In order to account for censoring we performed a time-to-event analysis for the med group. The start of the time at risk for each cyst was defined as the date of the visit when the inactive stage was diagnosed. In case medical treatment was still ongoing at the time of the visit, the time at risk started at the end of therapy. If this date did not coincide with a visit to the clinic, the cyst stage from the last visit was considered (last observation carried forward).We analyzed time to relapse using the Kaplan-Meier method. Cumulative incidence of relapse was calculated as one minus the Kaplan-Meier estimate.All analyses were performed using Stata Version 13.1  and R software version 3.3.1 .Descriptive results for continuous variables are given as mean  if not stated otherwise.The selection of study patients of the Heidelberg CE patient cohort is displayed in Forty-five patients of the Heidelberg CE patient cohort fulfilled the inclusion criteria. Thirty patients with 46 cysts were included in the ww-group, and 15 patients with 17 cysts into the med-group. The mean age of ww-patients was 41.5 (8–69), 18 patients were male, 12 patients female, mean follow-up for ww-patients was 5.4 (0.5–10.9) years. 6 patients were lost to follow-up in the ww-group. The mean age of med-patients was 35.8 (13–65), 4 patients were male, 11 patients female, mean follow-up of med-patients was 7.3 (1.5–11.8) years. One patient was lost to follow-up in the med-group. Patients were from the following countries: Turkey: 16, countries of the former Yugoslavia: 10, Romania: 4, Commonwealth of Independent States: 7, Italy: 3, Germany: 1, Hungary: 1, Northern Africa: 2, Iran: 1.Forty-six cysts have been included in the analysis. All cysts were CE4 or CE5 at entry into the cohort. Forty-three cysts were localized in the liver; there was 1 splenic and 1 peritoneal cyst. Seventeen cysts were included in the med-group, 8 of the 17 analyzed cysts showed relapse. Regardless of cyst stage before treatment all cysts except one relapsed to stage CE3b. All cysts without relapse reached cyst stage CE4 after treatment with benzimidazoles. Cyst stage, localization and diameter at the beginning and end of medical treatment and at relapse are displayed in Tables Kaplan-Meier-Analysis of time to relapse in the med-group shows a cumulative incidence of relapse of around 50% within 18 months .The benefits of cyst staging-based management of patients with cystic echinococcosis are not yet fully explored. Very obvious questions are: Do inactive cysts (CE 4 and CE 5) need treatment and is there a difference between cysts which reach CE4 and CE5 naturally or by benzimidazole therapy? So far only two CE clinical centers systematically explored the watch and wait approach, the Centre at the Pavia University Hospital ,8 and ouIn the ww-group data impressively show how stable naturally inactivated cysts are. In the first 3–5 years of observation confidence intervals predicting relapse are narrow. The later increase is due to the decreasing number of cysts under observation and must be judged by biological plausibility. Over time inactive cysts increasingly consolidate and calcify making relapse less and less likely. Our data strongly support the watch and wait approach in patients with uncomplicated asymptomatic cysts in anatomical sites where they do not compromise vital structures such as vessels. Based on our data we suggest that a minimum follow-up of 3–5 years is sufficient in patients with naturally inactivated cysts.In contrast cysts which reach inactivity through treatment with benzimidazoles behave differently. The relapse rate of 50% shown in the Kaplan-Meier analysis  correspoThe small sample size is a limitation of the study; however, the results relapse after w&w only and relapse after medical treatment are very robust.In conclusion, a substantial proportion of patients can be spared from any treatment through cyst staging. Cysts which inactivated through a natural course of involution do not relapse with very high likelihood. We recommend follow up of 5 years to confirm the stability of the inactive stage. Cysts driven into inactivity instead need careful monitoring to identify those which reactivate (around 50% within 18 months). 5 years follow-up after reaching an inactive stage appears safe to make a final decision on the need for further monitoring.S1 Checklist(DOCX)Click here for additional data file.


Escherichia coli is reconstituted into liposomes and activated to its different sub-open states by titrating lysophosphatidylcholine (LPC) into the lipid bilayer. Activated channels are closed back by the removal of LPC out of the membrane by bovine serum albumin (BSA). Electron paramagnetic resonance spectra showed the LPC-dose-dependent gradual opening of the channel pore in the form of incrementally increasing spin label mobility and decreasing spin-spin interaction. A method to reversibly open and close mechanosensitive channels to distinct sub-open conformations during their journey from the closed to the fully open state enables detailed structural studies to follow the conformational changes during channel functioning. The ability of BSA to revert the action of LPC opens new perspectives for the functional studies of other membrane proteins that are known to be activated by LPC.Understanding the functioning of ion channels, as well as utilizing their properties for biochemical applications requires control over channel activity. Herein we report a reversible control over the functioning of a mechanosensitive ion channel by interfering with its interaction with the lipid bilayer. The mechanosensitive channel of large conductance from  Elucidating the mechanisms by which ion channels function remains an ongoing challenge in physiology. Even though invaluable structural information is obtained from crystal structures, it is not always possible to derive the conformational transitions necessary for function from the static structures. Therefore, complementary approaches that give information about the dynamic conformers are needed.Escherichia coli (E. coli). In nature, MSC's sense the changes in membrane tension and are involved in hearing, the sense of equilibrium, touch, pain, as well as renal and cardiovascular regulation  protects cells from a sudden hypoosmotic shock in the external environment by opening a non-selective, temporary pore and releasing small solutes out of the cell  from One of the challenges of studying a mechanosensitive ion channel by spectroscopy is to design a tool to control the channel opening and closing. Here we show an approach to achieve this goal. In patch clamp electrophysiology, the membrane can be stretched by applying suction via the recording pipette in a controlled manner. However, this level of control over the channel activity in a spectroscopy setting is missing. To this end, pioneering works by Perozo et al. ,b explorin situ by LPC and BSA  methanethiosulfonate (MTSSL) and Methoxypoly(ethyleneglycol)5000 Amidopropionyl Methanethio-sulfonate (MTS-PEG5000) were purchased from Toronto Research Chemicals Inc. (TRC), Toronto, Canada. Azolectin was from Avanti Polar Lipids. Polycarbonate membrane was from Avestin Europe, GmbH. Biobeads (SM-2 Absorbents) were from Bio-Rad Laboratories, Inc., CA, USA.G22C mutant of MscL channels was produced and isolated as described before  was calculated as explained before :Csamp is the concentration of MTSSL in the labeled protein, Nsamp and Nref are the evaluated integrals of the absorption spectra of the sample and the reference, respectively, and Cref is the reference concentration. Typically, 100 mM MTSSL dissolved in DMSO was used as a reference. Then, Csamp was used to calculate the SLE by Equation (2):Where, Cprot is the concentration of the labeled protein determined by the standard Bradford assay.Where, The protein was reconstituted into liposomes as previously described . Next, a 2 μL of proteoliposomes were added to a cuvette filled with 2100 μL efflux buffer. Fluorescence emission was monitored at 515 ± 2 nm (excitation at 495 ± 2 nm) in a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.). Channel activation at different LPC concentrations was achieved by the addition of LPC to the sample and followed for approximately 3 min. Channel deactivation was achieved by adding BSA to already LPC-containing sample and followed for 1 min. Six different final LPC concentrations were tested: 1, 1.5, 2.0, 2.5, 3.0, and 3.5 μM, whereas 25 mol% BSA with respect to LPC concentration was added for channel deactivation. At the end of each experiment, proteoliposomes were lysed by the addition of Triton X100, and the resulting fluorescence value was taken as 100 %.The release of calcein was calculated as a percentage of the total calcein amount present in the proteoliposomes:100 is the fluorescence intensity upon complete lysis of the proteoliposomes, which was elicited by adding 100 μL Triton X-100 from a 10% stock.Where, I is the measured fluorescence intensity at a given time, Io is the initial background fluorescence intensity, and IContinuous wave (cw) EPR measurements were performed using a MiniScope benchtop X-band EPR spectrometer  with a rectangular TE102 resonator. The cavity was fluxed with gaseous nitrogen to keep the temperature stable. The microwave power and the B-field modulation amplitude were set to 10 mW and 0.20 mT, respectively (see supplementary information for determination of overmodulation in this particular instrument). EPR glass capillaries with 0.9 mm inner diameter were filled with a sample volume of 10 μL (for LPC) and 14 μL (for BSA) at a final MscL concentration of 170–200 μM.The microwave frequency was 9.41 GHz; the modulation frequency was 100 kHz. Each spectrum of maximally labeled protein corresponds to the accumulation of 16 scans while spectra of minimally labeled protein correspond to the accumulation of 25 scans.EPR experiments at increasing LPC concentrations were carried out from a single stock of spin labeled MscL (MscL-SL) reconstituted in liposomes. The stock was divided into six aliquots . A desired LPC to azolectin ratio was obtained by adding the required amount of LPC to a constant final volume (10 μL) always 1 h before starting the EPR experiment. Channel closure was achieved by adding BSA to LPC-activated channels at 25 mol% with respect to LPC concentration (LPC: BSA molar ratio) to 14 μL final volume.−1 and the second moment <ΔB2> were used. After base-line correction, using the MS400–Analysis software , the first and second moments of the EPR spectra were calculated numerically using the UNISPC program .To determine spin-label mobility the inverse peak-to-peak central linewidth ΔH2> was calculated according to the following equation  and non-broadened (non-interacting spins) EPR spectra, respectively; SI(B) is the EPR absorption spectrum in which the dipolar spin-spin interaction is present, whereas SN(B) is the spectrum without spin-spin interaction; BFI and BFN are the first spectral moments of the respective spectra, and B is the static magnetic field.Where < ΔBblack line). If, on the other hand, BSA (25 mol% with respect to LPC concentration) was added to a duplicate of the first sample before the release reaches the plateau, the release stopped immediately , indicating that LPC and BSA can be used to open and close the channel, respectively. We showed that the cycle of opening and closing of the channel could be repeated multiple times, as shown with the red line in Figure First, to demonstrate the action of LPC and BSA on MscL, we performed a fluorescence dequenching assay using MscL reconstituted into liposomes , the channels were reconstituted into liposomes and used for EPR measurements. The black line in the middle and bottom panel of Figure middle panel, red line). Increase of the LPC concentration above 40 mol% did not change the EPR spectrum anymore. The addition of 25 mol% BSA (relative to LPC concentration), on the other hand, generated a signal that completely reverses the changes seen in the middle panel and results in a spectrum that is very similar to that of the closed MscL channel .After showing that LPC has no effect on detergent solubilized MscL  revealed a narrowing of the spectral lines with increasing LPC concentration. In addition, the hallmarks of the slow rotation in nitroxide EPR spectra, as marked by arrows in Figure The EPR spectra of maximally and minimally labeled MscL samples  and second moment (<ΔB2>), were calculated from the EPR spectra of the minimally labeled sample. The inverse central line width increases as the mobility of the spin label increases, since less mobility causes broadening of the EPR lines. The second moment  with respect to the minimally labeled  EPR spectra LPC concentrations up to 30% of LPC. This is most obvious in the spectrum with 20 mol% LPC at field values around 333 mT .The second trend manifests itself in a broadening of the EPR spectra of the maximally labeled Figure , black wd Figure , red EPR2>, the values of which are given in Table Line broadening in the maximally labeled with respect to the minimally labeled sample suggests that there is a spin-spin interaction in the maximally labeled sample when the channel is in its closed state . However, as the LPC concentration increases, the spin-spin interaction between spin labels attached to different subunits of MscL reduces, suggesting pore opening. A measure for the amount of broadening of the EPR spectra is the difference in spectral second moments (Equation 4), Δ <ΔB2> values show a sharp decrease between 20 and 30% LPC concentration suggestive of channel opening . The difference in second moment values at and above 30 mol% LPC are so small, that spin-spin interaction is considered to be negligible.Although overall small, the Δ <ΔBin situ was a reversible light switch, which was covalently linked to the pore region of MscL , which is part of the Netherlands Organisation for Scientific Research (NWO) (to MH) and by European Research Council (Starting Grant 208814 to AK) is gratefully acknowledged.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.


Escherichia coli to assess the global transcriptional consequences of interference with lipoprotein transport. Exposure of E. coli to the LolCDE inhibitor at concentrations leading to minimal and significant growth inhibition, followed by transcriptome sequencing, identified a small group of genes whose transcript levels were decreased and a larger group whose mRNA levels increased 10- to 100-fold compared to those of untreated cells. The majority of the genes whose mRNA concentrations were reduced were part of the flagellar assembly pathway, which contains an essential lipoprotein component. Most of the genes whose transcript levels were elevated encode proteins involved in selected cell stress pathways. Many of these genes are involved with envelope stress responses induced by the mislocalization of outer membrane lipoproteins. Although several of the genes whose RNAs were induced have previously been shown to be associated with the general perturbation of the cell envelope by antibiotics, a small subset was affected only by LolCDE inhibition. Findings from this work suggest that the efficiency of the Lol system function may be coupled to a specific monitoring system, which could be exploited in the development of reporter constructs suitable for use for screening for additional inhibitors of lipoprotein trafficking.In Gram-negative bacteria, a dedicated machinery consisting of LolABCDE components targets lipoproteins to the outer membrane. We used a previously identified small-molecule inhibitor of the LolCDE complex of IMPORTANCE Inhibition of the lipoprotein transport pathway leads to E. coli death and subsequent lysis. Early significant changes in the levels of RNA for a subset of genes identified to be associated with some periplasmic and envelope stress responses were observed. Together these findings suggest that disruption of this key pathway can have a severe impact on balanced outer membrane synthesis sufficient to affect viability. Enterobacteriaceae (CRE), it is clear that new sources of efficacious compounds to address infections caused by Gram-negative bacteria are a necessity (–Multiply drug-resistant (MDR) bacterial pathogens pose a serious challenge in clinical medicine. Currently, the options for the treatment of serious infections caused by Gram-negative organisms are narrowing. With the emergence of carbapenem-resistant ecessity , 2. The ecessity –5. Wherecessity –, 6. Varicessity –. Becausecessity –, 8. An acessity –.The unique components of the outer membrane of Gram-negative bacteria are assembled during cell elongation and division. Three outer membrane assembly pathways with components located in each of the membranes and in the periplasm are known to exist in these bacteria: Bam (β-barrel assembly machine), Lpt (lipopolysaccharide transport proteins), and Lol (lipoprotein transport) –12. Each–Escherichia coli there are more than 90 different lipoproteins, with the majority residing in the inner leaflet of the outer membrane , it is conceivable that the compound recognizes a structurally related fold interfering with the function or assembly of the LolCDE complex.Until recently, the only known small molecule capable of interfering with lipoprotein transport was globomycin, an inhibitor of the type II signal peptidase Lsp . EmployiE. coli . This moE. coli . It exhiE. coli. The impact of the compound on bacterial viability and cell integrity over time was assessed, and the early transcriptional changes brought about by exposure to the compound are described. In addition, we compared the transcriptional levels of several key genes with altered expression levels resulting from inhibition of the Lol pathway with the transcriptional levels in the presence of several antibiotics with different mechanisms of action. Our findings demonstrate that pharmacological inhibition of the Lol pathway results in increased transcription in several envelope stress pathways.We exploited the activity of the small-molecule inhibitor of the Lol pathway to investigate additional physiological effects of interference with lipoprotein transport in Escherichia coli BW25113 [rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1] with the ΔacrB deletion (CGSC JW0451-2 8609) was employed , for which the MIC in LB is 0.6 μg/ml. All experiments were performed in LB broth cultures at 37°C with shaking at 300 rpm. For experiments in which compounds or antibiotics were added, the MIC values determined in LB broth microtiter plates with low concentrations of bacterial cells (5 × 105 CFU/ml) (600), and the concentrations that produced reductions in the growth rate were employed for the experiments .For all experiments, employed . Use of  CFU/ml)  were useacrB were selected on LB agar plates with 5 μg/ml (8× MIC) of compound 2. After 20 h of incubation, selected colonies were transferred onto fresh LB plates with 5 μg/ml of compound 2 and grown overnight at 37°C. The growth from these plates was stored in 10% glycerol in LB at −80°C. Genomic DNA was isolated from several clones, and LolC and LolE were amplified. A mutant with a previously reported  lysis mix-acid phenol solution. Lysis mix consisted of 320 mM sodium acetate, 8% SDS, and 16 mM EDTA  in nuclease-free water. One hundred microliters of the above-described lysis mix was combined with 700 μl of acid phenol-chloroform (Ambion) in 2-ml tubes. The cells and lysis mix-acid phenol were rapidly mixed on a vortex mixer and kept at 65°C with vortexing for 5 to 10 s every minute for 10 min. After centrifugation for 5 min at 12,000 × g, the upper phase was carefully removed, transferred into 700 μl of phenol-chloroform-isoamyl alcohol (Ambion), and vortexed to mix. This extraction procedure was repeated twice, and centrifugation at 12,000 × g for 5 min was used to separate the phases, with the upper phase being taken each time. Finally, the upper phase was transferred to 600 μl  of chloroform-isoamyl alcohol and again centrifuged as described above. The upper phase was removed, and RNA was precipitated by treatment with 2 volumes of 100% ethanol overnight at 80°C. On the following morning, the mixture was centrifuged at 18,000 × g at 4°C for 10 min. The pellets were washed once with 70% ethanol in DNase- and RNase-free water (Invitrogen). The pellets were dried under vacuum in a Savant SpeedVac system for 5 to 10 min. The dried pellets were resuspended in 30 μl of diethyl pyrocarbonate-treated water (Invitrogen). RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Fisher). Ribosome integrity numbers (RINs) were determined with an Agilent Bioanalyzer 2100 instrument and an Agilent RNA 6000 Nano kit . The initial RIN for all six RNA samples was 10. DNase treatment was carried out with a Turbo DNA-free kit (Ambion). The RINs were again checked after DNase treatment, and the values ranged from 9.4 to 9.9. The rRNA was depleted using a Ribo Zero rRNA removal kit for Gram-negative bacteria (Illumina). RNA libraries were prepared with an NEBNext ultradirectional RNA library preparation kit for the Illumina system using NEBNext multiplex oligonucleotides for Illumina index primer set 1 (New England BioLabs). The size distribution of the library was tested with an Agilent 2200 TapeStation high-sensitivity D1000 ScreenTape system. RNA-seq was carried out on an Illumina HiSeq platform, with the coverage of the six samples ranging from 18 million to 24 million reads each. Analysis of the data was performed using CLC Bio Genomics Workbench software, with the reads being mapped to the genome sequence of E. coli BW25113. Duplicate sample data were averaged, and the complete comparative data for RNA-seq are provided in Tables S1 and S2 in the supplemental material. Low-level expression data were eliminated using the formula for expression cutoff in terms of the number of reads per million (For transcriptome sequencing (RNA-seq), 2 system from Eppendorf. Changes in transcript levels relative to the levels in the untreated control cultures were calculated. The growth and exposure for RNA extractions for the qPCR experiments with 30-min compound exposures were performed twice  on different days.For determination of RNA levels by quantitative PCR (qPCR), various concentrations of antibiotic were first tested in cultures grown in 20-ml volumes. The concentrations of antibiotics that were twice the lowest level required to inhibit growth, determined by measurement of the optical density , were selected, and in a subsequent experiment cells were incubated with those concentrations for 30 min. RNA was then prepared from these cultures with the hot acid phenol procedure as described above. Primers were designed by use of the GenScript real-time PCR primer design tool. cDNA was synthesized with a SuperScript III first-strand synthesis system for reverse transcription-PCR (Invitrogen) and random hexamer primers. The qPCR was carried out using PerfeCTa SYBR Green FastMix (Quanta Biosciences) in a Mastercycler RealplexE. coli ΔacrB at an OD600 of 0.5 (5 × 108 CFU/ml) for 30 min prior to RNA isolation by addition of a portion of the culture to hot acid phenol. These concentrations of the compound were just below and just above the levels where effects on growth were detected (Previous work has established that the inhibitor (compound 2)  19) blo blo19) bdetected . The culdetected .lol genes  were also checked by qPCR (see below).The data for genes that were upregulated 5-fold or more by treatment with compound 2 are listed in In terms of decreased levels of RNA in response to pharmacological inhibition of lipoprotein transport, the number of genes for which major reductions in RNA levels were seen was much smaller than the number for which increases in RNA levels were seen. As can be seen in E. coli ΔacrB growth under conditions analogous to those employed in the RNA-seq experiments with the higher cell numbers and higher concentrations of the LolCDE inhibitor. The E. coli strain was exposed to these compounds at concentrations 2 times the inhibitory levels  for 30 min, conditions identical to those used in the RNA-seq experiments. RNA was extracted, purified, and converted to cDNA for qPCR determinations. Results for individual selected genes are presented in Next we selected several genes that demonstrated significant upregulation or downregulation and determined the concentrations of mRNA for those genes by quantitative PCR (qPCR). Their transcript levels were also compared to those obtained following treatment of cells with seven other antibiotics with several distinct mechanisms of action, in order to determine if a subset of the genes under study was uniquely regulated by the LolCDE inhibitor or whether these were also affected by antibiotic disruption of other cell physiological properties. Preliminary experiments with high-volume shaking cultures established the minimal concentrations of the various antibiotics that were inhibitory to rpsL, encoding the ribosomal protein S12, as a housekeeping gene whose level of transcription should not vary significantly from that for the control cells to determine the efficiency and variability of the RNA extractions in the different experiment using cells treated with the LolCDE inhibitor or various antibiotics. All of the rpsL transcript levels were within ∼2-fold of those for the control cells , as well as with polymyxin B. A similar pattern emerged with the hyperosmosis-associated gene ycfJ, whose transcript levels were notably higher following treatment with the β-lactams and polymyxin B and were similar to the levels observed with the LolCDE inhibitor. The levels of the mRNAs for the putative toxin-antitoxin pair ymgD and ymgG were also elevated in the presence of the LolCDE inhibitor, the two β-lactam compounds, and polymyxin B. A similar pattern in response to antibiotic treatment was observed for ivy, which protects cells permeabilized by chemical or physical stresses  was examined by qPCR. In addition to being affected by the LolCDE inhibitor, its concentration was also reduced by treatment with the other antibiotics tested, with the exception of fosfomycin, the inhibitor of MurA, an enzyme involved in the early stages of the peptidoglycan biosynthetic pathway. It appears that a number of different cellular perturbations have the ability to affect the highly orchestrated and hierarchical regulation of flagellar genes.Among the genes whose mRNAs showed a decrease when lipoprotein transport to the outer membrane was blocked, one . These results were compared to those obtained with control cells, which received no compound. While RNA-seq can measure only transcript levels, the compound and antibiotics tested likely directly or indirectly affected the transcriptional regulation of particular genes; however, we cannot exclude the possibility that the effect was posttranscriptional at the level of mRNA stability.Earlier work  establisnhibitor . Our goaE. coli was treated with the compound. There was, however, a modest 3.5-fold increase in lolA transcript levels at the higher concentration of the inhibitor, a result also seen in past experiments employing LolCDE depletion (––rpoS (σS)-mediated stress responses -controlled genes are primarily induced by protein misfolding in association with heat shock. The genes known to be controlled by σ32 include dnaK, dnaJ, and grpE, the mRNAs for which also do not show altered levels during LolCDE inhibition , ponhibitor . Other σc stress , 53. The and wzc –57, wereabilized .S and Rcs (Several of the RNAs responding to lipoprotein transport inhibition encode lipoproteins: the previously mentioned OsmB, an osmotic stress protein whose expression is controlled by both σ and Rcs . Other l and Rcs , 35, 37.ompF, previously shown to be controlled by CpxA/R (ompF responds differently from other CpxA/R-regulated genes that are adversely affected by LolCDE inhibition.Many fewer large-magnitude changes in the levels of expression genes showing decreases in their transcript levels following interference with lipoprotein trafficking were found. The levels of expression of virtually the entire set of mRNAs for genes associated with flagellum formation were markedly lower, and these genes comprised the group with the largest negative changes in expression , 60. Thiy CpxA/R . It is uecnB, coding for the bacteriolytic toxin part of a toxin-antitoxin pair which has been implicated in σS-regulated osmolarity stress and bacteriolysis (ecnA, was not upregulated in this case; this should lead to cell death. Another gene was katE, which encodes a hydroperoxidase also regulated by σS and which is induced in response to oxidative stress (clsB gene, which encodes one of three cardiolipin synthases in E. coli (ycfT, which encodes an inner membrane protein implicated in biofilm control and which is located immediately adjacent to and divergent from the genes encoding LolCDE, were also highly responsive to LolCDE inhibition. Among the group of antibiotics tested by qPCR at the inhibitory concentrations determined in this study, only the LolCDE inhibitor had an impact on the transcript levels of these four genes, although the level of transcription of ycfT mRNA also displayed a modest elevation with polymyxin B. The apparent specificity of these responses to pharmacological inhibition of LolCDE merits further future characterization of the genes for these proteins as possible candidates for use in cell-based reporter screens to find additional Lol pathway inhibitors. Other tested genes, such as ycfJ, implicated in biofilm formation, were also affected by polymyxin B and, to a lesser extent, by the two β-lactam antibiotics imipenem and meropenem, all of which have impacts on the bacterial cell envelope. The levels of both ymgD and ymgG mRNAs were elevated by the same compounds as well. The same was true of the expression of ivy, encoding another periplasmic protein that acts to protect peptidoglycan from hydrolysis by lysozyme and is induced by outer membrane permeabilization (To determine if any of the responses observed were also induced by exposure to other antibiotics with different mechanisms of action, bacteria were exposed to several different antibiotics, and their mRNAs were analyzed by qPCR. Compared to a group of selected genes from the RNA-seq experiment, it was found that the transcript levels for four of the genes tested were elevated only by exposure to the LolCDE inhibitor. One of these genes was riolysis , 63. Inte stress , 65 The  E. coli , and ycflization .E. coli BW25113 ΔacrB. The results obtained with cells of these strains were compared with those obtained with cells of the untreated parent. Quantitative PCR of representative genes involved in the different pathways clearly demonstrated that the transcript expression effects observed were through the inhibition of LolCDE by compound 2.The question as to whether the responses are a direct result of LolCDE inhibition by the compound was addressed by isolating a resistant LolC mutant with the N256K mutation and repeating the compound exposure at the higher concentration employed for RNA-seq and qPCR with this mutant and identically treated parental strain E. coli to treatment with a compound that inhibits the function of LolCDE. Direct inhibition of LolCDE function led to a loss of cell viability and, ultimately, cell lysis. The blockade of lipoprotein transport was found to lead to compound dose-dependent levels of induction of selected cell stress pathways. Many of these are envelope stress responses that have previously been observed under various cell stimuli, such as disturbances in metabolism, periplasmic protein misfolding, or overproduction of lipoprotein NlpE (The purpose of this study was to examine the responses of ein NlpE , 68. Of