A suite of light harvesting and photo-protective pigments were quantified across transects  and shown to increase in concert with algal biomass. Ice algal communities drove net autotrophy of surface ice, with maximal rates of net production averaging 0.52 ± 0.04 mg C l−1 d−1, and a total accumulation of 1.306 Gg C (15.82 ± 8.14 kg C km−2) predicted for the 2016 ablation season across an 8.24 × 104 km2 region of the GrIS. By advancing our understanding of ice algal bloom development, this study marks an important step toward projecting bloom occurrence and impacts into the future.It is fundamental to understand the development of Zygnematophycean (Streptophyte) micro-algal blooms within Greenland Ice Sheet (GrIS) supraglacial environments, given their potential to significantly impact both physical (melt) and chemical (carbon and nutrient cycling) surface characteristics. Here, we report on a space-for-time assessment of a GrIS ice algal bloom, achieved by sampling an ∼85 km transect spanning the south-western GrIS bare ice zone during the 2016 ablation season. Cell abundances ranged from 0 to 1.6 × 10 Space for time assessment of an ice algal bloom occurring in surface ice of the Greenland ice sheet. Micro-algal residence within supraglacial environments has been reported from numerous polar and high-altitude locations, including Antarctica . A modelling approach demonstrated ice algal communities to be the primary contributors to supraglacial carbon fixation, contributing significantly more than cryoconite-associated communities  for time (duration since snow line retreat), permitting a quasi-temporal assessment of bloom development. We focus here predominantly on the bare ice zone (see Hodson R) data following the approach of Tedstone et al. (R < 0.6) images and the first day of the melt season at which each sampling site became snow free was identified by applying a 7-day rolling window in which a minimum of 3 days had to be classified as snow-free, 0 as snow-covered and a maximum of 4 days could be cloudy  of the surface to be calculated. Subsequently, the surface was imaged with a scale, and the top 2 cm collected using a metal ice saw and trowel into a sterile Whirl-Pak bag. Sampling tools were washed between samples with MiliQ water and Whirl-Pak bags were immediately placed into the dark and transported to a primary ice camp  were sampled in order to constrain ice algal abundance, species diversity and pigmentation. Sample locations were chosen randomly across an area ∼50 × 50 m upwind of the helicopter landing position. Whilst no formal sampling strategy was performed, sampling was conducted to be representative of the surface types present at each site. For each surface sample, a spectral reflectance measurement was taken prior to disturbance of the surface. This was achieved using a Field Spec Pro 3 spectrometer  with a collimating lens limiting the instantaneous field of view of the fibre optic to 8°, held 30 cm above the sample surface at nadir viewing angle to provide a ground ‘footprint’ of ∼35 cmet al. (3 cell−1) were converted to biomass (pg C cell−1) using the relationship of Montagnes et al. (−1) was subsequently calculated as the sum of cell counts (cells ml−1) multiplied by average cell biomass (pg C cell−1) for each species present.All samples were melted in the dark over a ∼24 h period at the primary ice camp. Following melting, samples were thoroughly homogenised before sub-sampling. To assess algal cell numbers, species diversity and biovolume, 15 ml of homogenised sample was fixed with 25% glutaraldehyde at 2% final concentration. Samples were stored in the dark under ambient ice sheet temperatures until transport to the University of Bristol, UK, for counting. Counts were performed using a Fuchs-Rosenthal haemocytometer  on a Leica DM 2000 epifluorescence microscope with attached MC120 HD microscope camera . For those samples containing sufficient cell abundance, a minimum of 300 cells were counted to ensure adequate assessment of assemblage diversity. Imaging for quantification of cell volumes was performed in parallel to counts and measurements of cell diameter and height made using ImageJ software, with cell volume calculated considering ice algal cells as regular cylinders after Hillebrand et al. . Cell vos et al.  between For characterisation of major chlorophyll and carotenoid pigments, ∼100–200 ml of each homogenised sample was filtered onto a glass fibre filter  by mild vacuum filtration. Filters were immediately wrapped in foil and frozen in a Biotrek 10 cryoshipper  filled with liquid nitrogen. Filters remained under these conditions during transport to the University of Bristol where upon they were stored at −80°C prior to analysis by high-performance liquid chromatography (HPLC). Following freeze-drying for 24 h, pigments were extracted from filters in 100% acetone containing vitamin E as internal standard. Extractions were analysed using a modified version of the method of Van Heukelem and Thomas , using aet al.  using the wave scan function from 200–750 nm. To provide an estimate of the relative concentration of water-soluble pigments derived from samples, absorbance spectra were normalised to extraction and filtration volumes. Peak height of the dominant peak identified across spectra (λ335nm) was subsequently taken as representative of relative concentration and normalised to algal biomass for comparisons between sites.For characterisation of water-soluble pigments, a further ∼100–200 ml of n = 5 samples collected at each site during T2 was filtered onto a separate GF/F filter, frozen and transported back to the University of Bristol, as described above. Filters were subsequently freeze-dried for 24 h, and water-soluble pigments extracted in 5 ml Miliq water following the method of Remias et al. . To remoet al.  or respiration  (mg O2 l−1 d−1) were calculated as the difference between initial and final oxygen concentrations (mg O2 l−1), normalised to incubation time (d), as ΔO2/Δt. Gross production (GP) was subsequently calculated as NP—R. Data were converted into units of mg C l−1 d−1 assuming a 1:1 stoichiometry between moles of CO2 stored for each mole of O2 released, i.e. 0.375 g of C per g O2 . Following incubations, 15 ml subsamples were taken as above for determination of algal cell abundance and biovolume, and calculation of algal biomass as previously detailed. Given the absence of cyanobacteria within incubated samples (determined by microscopy), rates of productivity were assumed representative of the specific contribution of ice algae to organic carbon accumulation/respiration within surface ice. Whilst measured R included contributions from both autotrophic and heterotrophic community members, secondary production on the GrIS surface is ∼30-times lower than that of ice algal production  was assessed at the primary ice camp given the greater duration of time required for incubations and measurements. To assess the relationship between ice algal biomass and productivity, oxygen evolution incubations were performed following the method of Telling et al.  with melNP was modelled over the 2016 ablation period in a ∼8.24 × 104 km2 ablation zone area, at 7.5 × 7.5 km resolution. The model area spanned ∼1000 km of latitude along south-western Greenland, intersected by our study transect ∼600 km from the lower limit. Given uncertainties regarding microbial activity within the GrIS marginal zone  fell to zero metres, with the latter determined as the final day of the year in which cumulative meltwater production (ith day + the preceding 4 days) exceeded 3 mm water equivalent. For the duration between these start and end points, daily increases in ice algal abundance were calculated per pixel using the relationship between duration since snow line retreat (d) and ice algal biomass (μg C ml−1) determined from field data, and converted to NP (mg C l−1 d−1) using the relationship between ice algal biomass and NP determined during the productivity incubations detailed above.To estimate the contribution of ice algal assemblages to net carbon fixation in south-western Greenland, NP estimates per pixel. To allow conversion from units of volume (l−1) to area (km2), triplicate 0.5 × 0.5 m surface ice samples containing a low, medium or high biomass of ice algae were sampled at the primary ice camp in an identical fashion to surface sampling described previously, melted over 24 h and the melt volume quantified. Melt volumes did not differ significantly between surface ice of different ice algal loadings, averaging 265.33 ± 16.31 ml across all samples, with 1.061 ± 0.065 l of melt water apparent per m2 of sampled ice. The total contribution of ice algal assemblages to net carbon fixation across the south-western GrIS ablation zone was thus calculated as the sum of NP per km2, multiplied by the extent of our model region.Net carbon assimilation over the 2016 ablation period was thus calculated as the sum of daily All analysis and plotting of data was performed in R v.3.4.1   Figs  and 2, wChlamydomonas and Chloromonas spp.  during the present study, followed by Mesotaenium (∼35%), though deviations from this trend were apparent at sites S1b and S2 during T2 , though highly comparable to those from site S6 in close proximity to our transect sites . Strong spatial patterning is thus evident in ice algal biomass during blooms in GrIS supraglacial environments, with implications for algal population dynamics.Cell abundances ranged from 0 to 1.6 × 10les Fig. , with ma. et al.  from samet al. et al. et al.  and respiration (R) of surface ice containing a high, medium, or low biomass of ice algae demonstrated consistent net autotrophy for surface ice samples ; though no assessment of algal abundance was undertaken during the latter two studies. GP was approximately double R across all samples (GP/R = 2.3 ± 0.2), supporting assumptions built into previous modelling approaches , highly comparable to the 5.5 ± 1.7 days determined by Stibal et al. . As comparison, average NP of 0.0022 ± 0.0026 Gg C was reported for carbon accumulation driven by cryoconite alone (∼1.37 kg C km2, Model 3, Cook et al. et al. (et al. et al. (in-situ carbon fixation potential of ice algal assemblages across the bare ice zone.Total ice algal organic carbon accumulation over the 2016 ablation period was estimated as 1.306 Gg C across our ∼8.24 × 10km2 Fig. . Strong k et al.  modelled. et al. , though . et al. . Given t. et al.  did not et al. et al. Despite their potential importance for surface albedo and carbon flux estimates (Yallop


Satellite imagery reveals flowstripes on Foundation Ice Stream parallel to ice flow, and meandering features on the ice-shelf that cross-cut ice flow and are thought to be formed by water exiting a well-organised subglacial system. Here, ice-penetrating radar data show flow-parallel hard-bed landforms beneath the grounded ice, and channels incised upwards into the ice shelf beneath meandering surface channels. As the ice transitions to flotation, the ice shelf incorporates a corrugation resulting from the landforms. Radar reveals the presence of subglacial water alongside the landforms, indicating a well-organised drainage system in which water exits the ice sheet as a point source, mixes with cavity water and incises upwards into a corrugation peak, accentuating the corrugation downstream. Hard-bedded landforms influence both subglacial hydrology and ice-shelf structure and, as they are known to be widespread on formerly glaciated terrain, their influence on the ice-sheet-shelf transition could be more widespread than thought previously. Subglacial landforms, formed by glacial processes operating over long timescales, influence ice dynamics. Here, the authors show how mega-scale landforms at an Antarctic ice stream grounding zone modulate basal water flow, causing extensive channels in the ice shelf downstream that may impact its structure. In this paper, we refer to these surface channels as M-channels  ice-surface imagery, and linked to upward-incised channels at the ice-shelf base; their cause being the surface elevation differences of buoyant thick versus thin iceels Fig. . MODIS i1, or sedimentary landforms (eskers) that route water3. Because of this, M-channels are thought to be evidence of a well-organised subglacial-hydrological system, channelized by upward melting into the grounded ice by the basal water. As it exits the grounded ice the subglacial water forms a buoyant plume due to being fresher than water within the ice shelf cavity. It then entrains the warmer cavity water, and melts the underside of the floating ice to form an upward-incised channel5, which we refer to as U-channels. Hence, M-channels and U-channels are co-existent. Similar findings have been reported across a variety of ice-stream grounding lines across Antarctica2. The flow of ocean water within the cavity has also been shown to lead to upward-incised ice-shelf channels in, for example, the floating ice at the margin of Pine Island Glacier6.It has previously been demonstrated that M-channels correspond with the likely exit point of subglacial water at the mouths of ice streams that are inferred to have flat beds7. Given this, and the importance of maintaining ice shelf integrity in support of ice-sheet stability in the context of atmospheric warming9, it is necessary to identify and understand the hydrological supply of water responsible for the sizable selective upward linear erosion observed, particularly in deep marine settings in West Antarctica.While surface ice-shelf melting does not cause M-channels, if such melting occurred the water produced would be preferentially routed into and along M-channels due to the linear depression in the surface topography, potentially melting them downwardsHere we inspect ice-penetrating radar data across the Foundation ice stream (FIS) and Filchner-Ronne Ice Shelf (FRIS) to link ice-surface lineations observed in MODIS imagery to basal features in the ice sheet and ice shelf. Our aim is to better understand processes at the transition between grounded and floating ice, and the role that subglacial conditions can have in determining ice-shelf morphology and, potentially, structural integrity. We reveal mega-scale hard-bedded subglacial landforms at the grounding zone of FIS, which cause the ice base to become corrugated as it starts to float. The basal landforms also modulate the flow of basal water, feeding it into a corrugation peak which then develops the corrugation for more than 130 km from the grounding zone. Similar hard-rock basal landforms are known to exist across formerly glaciated terrain in many regions of Antarctica, and landscapes beneath former northern hemisphere ice sheets, suggesting their influence on ice dynamics may be more significant than appreciated both in terms of contemporary ice dynamics and past ice-sheet behaviour.11. The FIS has its trunk in West Antarctica and a wide complex drainage basin with about half its ice supplied by the Academy Glacier and Support Force ice stream in East Antarctica and the other half from West Antarctica. Location of the grounding line of FIS, which is around 2 km below sea level, is ambiguous depending on whether surface slope or tidal flexure is used14. The separation between two of the proposed grounding lines is up to 18.5 km, indicating a possible complex transition between grounded and floating ice .The main trunk of FIS, and its grounding line with the FRIS, lies ~2 km below sea level. The bed beneath the ice stream trunk occupies a deep U-shaped fjord24 , two near the grounding zone (L–L’ and O–O’), and two others parallel to the axis of FIS (M–M’ and N–N’)  as a part of the NASA Operation IceBridge (OIB) mission in 2012, 2014, and 2016; and a survey of the Institute and Möller ice streams undertaken in 2010/2011 (IMAFI)s29 Fig. .Fig. 2Ra12 and tidal flexure14. However, in N–N’, the proposed grounding lines are separated by ~20 km . Furthermore, the amplitude of the bedforms remains largely unchanged upstream of the grounding line in contrast to ‘tadpole’ shaped eskers proposed elsewhere that have higher amplitudes only near the grounding line3. It is interesting to also note that, to our knowledge, no eskers have been identified across the formerly glaciated Antarctic continental shelf. This strongly suggests that the FIS bedforms are erosional landforms with a lithified core40. Any actively deforming till layer may be superficial at the scale of the bedforms, though may likely be present between them (as is evident in the very flat bed separating bedforms). The cross-sectional dimensions of the FIS bedforms match well to large streamlined linear ridges that have been mapped across a number of formerly glaciated regions in Antarctica, where they are carved in hard crystalline bedrock41, and which are plastered by a till sheet at least several metres thick . Between the bedforms, which are separated by less than 1 km in almost all cases, we observe the bed to be very flat, indicative of weak water-saturated sediments. While subglacial morphology on the WAIS-sourced side of the FIS trunk appears more subdued than that of the EAIS-sourced side, a similar glacial geological morphology is apparent with small bedrock highs (up to ~100 m high) interspersed by flat, presumably sediment-draped, surfaces.Orthogonal to ice flow in the trunk of FIS, three radar transects show significant bed roughness due to massive flow-parallel ridges  will be less than the surrounding ice shelf surface. Hence, we are able to confirm U-channels from this additional diagnostic  U-channel . Basal power was extracted from the radar data43 along profiles i-i’, j-j’ and k-k’. Relative basal reflectivity values  were then obtained performing a separate attenuation correction for radar traces in East and West Antarctica  are likely to be present. The bed of the trunk of the FIS is likely to be above pressure melting point and comprised (in between the hard-bedded landforms) of subglacial till. In this scenario, the basal reflectivity hierarchy is likely to correspond to basal water greater than ~5 m deep , saturated till, hard-bedded regions, and dry till 45. The oscillations in basal reflectivity along transects i-i’, j-j’ and k-k’ are therefore consistent with material transitions from drier surrounding regions to water channels (the reflectivity peaks). Supporting evidence for the water channel locations comes from the correlation between the reflectivity peaks and the abruptness peaks. Specifically, high values of both are consistent with water being present43.The radar reflectivity characteristics of the FIS bed are consistent with significant well-organised flow of water within FIS Fig. . The oveWhile the subglacial flow routing model shows the path of basal water is consistent with flow alongside the bedforms in the trunk of the ice stream, we regard the most compelling evidence of water at the bed to come from the radar data. Within the region of grounding zone uncertainty, where the highest (~800 m) U-channel is measured, reflectivity is above +10 dB in three regions; one over a flat region of the bed, and two associated with the fringes of the U-channel (Profile i-i’). The U-channel in profile i-i’ Fig.  is an ofUpstream, across the permanently grounded ice-stream trunk, points where reflectivities reach ~+10 dB are observed (Profile j-j’). Further upstream, an additional three points with reflectivities ~+10 dB are seen, and in one location on the fringe of the trunk reflectivities are >+10 dB (Profile K–K’). Basal reflectivities above 5 dB are mapped in Fig. 46, as the ice-sheet becomes afloat the base of the ice sheet will inherit a “corrugated” morphology from the bedforms. The bedforms thus modulate the upstream flow of subglacial water, allowing a focused stream of water to flow upwards into the peak of a U-channel corrugation across the grounding zone  in 2010–2011. The survey used the BAS Polarimetric radar Airborne Science Instrument (PASIN), operating at a centre frequency of 150 MHz, with a 10 MHz bandwidth and a pulse-coded waveform acquisition rate of 312.5 Hz. Second, a series of geophysical flights conducted by the CReSIS during the NASA Operation Ice Bridge programme in 2012, 2014, and 2016, which used the Multichannel Coherent Radar Depth Sounder system developed at the University of Kansas. The system operated with a carrier frequency of 195 MHz and a bandwidth of 10 MHz in 2012 and 50 MHZ in 2014 onward.Chirp compression was applied to the along track data. Unfocused synthetic aperture (SAR) processing was used by applying a moving average of 33 data points, whereas two-dimensional SAR  processing based on the Omega-K algorithm was used to enhance both along track resolution and echo signal noise. Doppler filtering was used to remove the backscattering hyperbola in the along track direction. The bed echo was depicted in a semi-automatic manner using ProMAX seismic processing software.The data were processed in three steps to improve the signal-to-noise ratio and increase the along-track resolution. The raw data were first converted from a digital quantization level to a receiver voltage level. The surface was captured using the low-gain data, microwave radar or laser altimeter. A normalized matched filter with frequency-domain windowing was then used for pulse compression. Two-dimensional SAR processing was used after conditioning the data, which is based on the frequency wave number (F–K) algorithm. The F–K SAR processing requires straight and uniformly sampled data, however, which in the strictest sense are not usually met in the raw data since the aircraft’s speed is not consistent and its trajectory is not straight. The raw data were thus spatially resampled along track using a sinc kernel to approximate a uniformly sampled dataset. The vertical deviation in aircraft trajectory from the horizontal flight path was compensated for in the frequency domain with a time-delay phase shift. The phase shift was later removed for array processing as it is able to account for the nonuniform sampling; the purpose is to maintain the original geometry for the array processing. Array processing was performed in the cross-track flight path to reduce surface clutter as well as to improve the signal-to-noise ratio. Both the delay-and-sum and minimum variance distortionless response (MVDR) beamformers were used to combine the multichannel data, and for regions with significant surface clutter the MVDR beamformer could effectively minimize the clutter power and pass the desired signal with optimum weights.In both datasets, the waveform was retrieved and sequenced according to its respective transmit pulse type. The modified data were then collated using MATLAB data binary files. A nominal value of 10 m is used to correct for the firn layer during the processing of ice thickness, which introduces an error of the order of ~3 m across the survey field. This is small relative to the total error budget of the order of ~1%. Finally, the GPS and RES data were combined to determine the ice thickness, ice-surface and bed elevation datasets. Elevations are measured with reference to WGS 84. The ice-surface elevation was calculated by subtracting terrain clearance from the height of the aircraft, whereas the bed elevation was computed by subtracting the ice thickness from the ice-surface elevation.Supplementary InformationPeer Review File


Oxidative stress has been shown to play a crucial role in the pathophysiology of the neurodegenerative disease Ataxia Telangiectasia. We have recently demonstrated that Dexamethasone treatment is able to counteract the oxidative state by promoting nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation. However, substantial gaps remain in our knowledge of the underlying molecular mechanism(s) according to which Dexamethasone acts as an NRF2 inducer. Herein we investigate the possible effects of the drug on the main NRF2 activation pathways by initially focusing on key kinases known to differently affect NRF2 activation. Neither AKT nor ERK1/2, known to be NRF2-activating kinases, were found to be activated upon Dexamethasone treatment, thus excluding their involvement in the transcription factor nuclear shift. Likewise, GSK3 inactivating kinase was not inhibited, thus ruling out its role in NRF2 activation. On the other hand, p38 MAPK, another NRF2-inhibitory kinase, was indeed switched-off in Ataxia Telangiectasia cells by Dexamethasone-mediated induction of DUSP1 phosphatase, and therefore it appeared that it might account for NRF2 triggering. However, this mechanism was excluded by the use of a selective p38 inhibitor, which failed to cause a significant NRF2 nuclear shift and target gene induction. Finally, dexamethasone effects on the classical oxidative pathway orchestrated by KEAP1 were addressed. Dexamethasone was found to decrease the expression of the inhibitor KEAP1 at both mRNA and protein levels and to induce the shift from the reduced to the oxidized form of KEAP1, thus favouring NRF2 translocation into the nucleus. Furthermore, preliminary data revealed very low levels of the negative regulator Fyn in Ataxia Telangiectasia cells, which might account for the prolonged NRF2-activated gene expression. Ataxia Telangiectasia (AT) is a rare, incurable, neurodegenerative disease caused by biallelic mutations in the ATM gene , 2, whicIn our laboratory, we have discovered a new ATM transcript induced by Dexamethasone (DEX). This transcript can prompt the translation of a short ATM variant with residual kinase activity, suggesting that drug treatment can provide some gain of functions by directly replacing the kinase that is lacking , 28. In Hence, the aim of the present investigation was to determine how DEX exerts its effect on the NRF2-ARE system, focusing on the main pathways that are known to be involved in NRF2 activation. First, we investigated the effect of DEX on the PI3K/AKT and ERK1/2 pathways, excluding the involvement of these activating kinases. We subsequently investigated the effects of DEX on the inhibitory kinases GSK3 and p38 MAPK, finding evidence that p38 inhibition by DEX could be involved. However, experiments with p38 inhibitors suggested that p38 inhibition may not account for full NRF2 activation. Hence, we refocused our investigation on the oxidative branch of NRF2 regulation, via the KEAP1/NRF2 axis. Surprisingly, it was found that the greatest effects of DEX could be accounted for by direct inhibition of KEAP1 at both transcriptional and post-translational levels. In short, we showed that DEX decreased both KEAP1 gene transcription and protein expression and that, interestingly, it was able to induce the shift from the reduced to the oxidized form of KEAP1, thus favouring NRF2 translocation into the nucleus.2 and treated with 100 nM DEX for 24h prior to RNA and protein extractions. Dimethylsulfoxide (DMSO) was used as the drug vehicle and thus administered in untreated cells as a control. All the cells used in this study were established for diagnostic purposes under informed consent and stored at the Department of Clinical and Molecular Medicine, Sapienza University of Rome. The cell lines were anonymized and made available only by a coding number, so that we had no access to the name of the patients. All the cell lines were used with the approval of the ethical committee of the Sant’Andrea Hospital.EBV-transformed lymphoblastoid B cell lines (LCLs) established from AT patients (ATM-/-) and healthy donors (ATM+/+) were kept in RPMI1640 medium supplemented with 2 mmol/l L-glutamine, 50 mg/ml gentamycin, and 10% fetal calf serum. The LCL AT cell lines AT28RM, AT50RM and AT129RM were already described in . All celTotal RNA was extracted from LCLs treated or not treated with DEX using the RNeasyPlus mini kit (Qiagen), and cDNA was obtained by PowerScript reverse transcriptase (Clontech). Quantitative PCRs for gene expression were performed by TaqMan Gene Expression Assays using the TaqMan Gene Expression Master Mix (Life Technologies) and run on a 7500 Real-Time PCR System (Applied Biosystems). HPRT1 was used as a housekeeping gene, as specified.Western blots were performed on WT and AT cell protein extracts, essentially as described in . After 22, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4) with freshly added protease inhibitors and 40 mM NEM. The suspensions were incubated on ice for 5 min, and Nonidet P40 was added to a final concentration of 0.6%. Following centrifugation at 16000 g, the pellet (nuclei) and the supernatant (cytosol) were separated. Nuclear extracts were then obtained by mild sonication in NEM lysis buffer. Both the cytosolic and nuclear extracts were diluted in 4X Sample buffer and analysed by Western blot or Redox Western blot, as described below. Half of the sample was reduced by the addition of 200 mM DTT and loaded on 4–15% SDS-PAGE to detect reduced forms of KEAP1, while the remaining extracts were loaded on a separate 4–15% SDS-PAGE under nonreducing conditions to evaluate oxidized forms of KEAP1.Protein extracts for redox western were obtained in non-reducing lysis buffer, essentially as described in , but on For Immunoprecipitation (IP), cytosolic and nuclear extracts were incubated with NRF2 rabbit polyclonal antibody (Santa Cruz Biotechnology), and the immune complexes were pulled-down by incubation with protein A Sepharose Fast Flow Beads . Briefly, equal amounts of cytosolic and nuclear extracts belonging to the different samples  were first denatured with 1% SDS to disrupt association of NRF2 with specific interactors. They were then subjected to IP protocol before being electrophoresed on SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed with Phospho-Tyrosine Mouse mAb  or anti-Ub mouse monoclonal antibody (Santa Cruz Biotechnology) for phosphorylation and ubiquitination status, respectively.Statistical analyses were performed with GraphPad Prism 5 software for Windows using the paired non-parametric Wilcoxon signed-rank test. P-values < 0.05 were considered significantly different.Fig 1A. Moreover, we recently published, and herein confirmed, that DEX is able to strongly promote the nuclear translocation of NRF2, especially in AT cells [). Our initial approach to investigate the molecular pathway(s) underlying the DEX effect was to focus on three different pathways for NRF2 activation. These pathways are thoroughly reviewed in [Fig 1A.NRF2 could be activated by different pathways, which are summarized in AT cells , . Neither AKT nor ERK 1/2 were found to be activated after 24h treatment with DEX, thus excluding their involvement in NRF2 activation . Not only were ERK1/2 not activated, but we also found a significant reduction in the phosphorylation status. The de-phosphorylation observed in all cells (both wild type and AT) after DEX may be imputable to the short-term overexpression of several dual-specificity phosphatase genes (DUSP1-3-6-7-8-11-14-16-22) as reported in [We first investigated the “activating kinase” branch involving the AKT and ERK1/2 pathways . Accordingly, we hypothesized that DEX could have an indirect activating effect on NRF2 by blocking one or both of these inhibitory kinases.As mentioned in the introduction, in pathological conditions, the persistence of inflammatory or oxidative statuses can lead to the chronic activation of inhibitory kinases, such as GSK3 and/or p38 MAPK, which exert a negative regulation on the transcription factor NRF2 (Evidence suggests that GSK3 is able to directly (or indirectly by Fyn) phosphorylate NRF2 in specific residues, leading to NRF2 degradation in a KEAP1-independent manner, in particular via the adaptor protein β-TrCP , 36. ActAs shown in the fourth branch of S1 Fig). To the best of our knowledge, no correlations exist between ATM and Fyn protein expression; however, this point certainly warrants further investigation. Fig 4A shows that, in AT cells, Fyn is only cytoplasmic, while in WT cells, where the Fyn content is much higher, a portion of protein is indeed shifted into the nucleus. This is consistent with the establishment of the above-mentioned delayed response and the shutdown of transcription in WT cells. It is noteworthy that in our previous work we observed that 24 hours after DEX treatment, while NRF2 was still nuclear in both, in WT cells it was not transcribing ARE genes, while in AT cells it was [Fig 4C and 4D). This is consistent with the pre-induction phase in which unknown kinases phosphorylate Fyn to mediate its nuclear export and degradation [In particular, in the post-induction phase, activation of Fyn by phosphorylation, leads to the nuclear shift of Fyn itself. Once inside the nucleus, Fyn phosphorylates the tyrosine 568 of NRF2, resulting in the detachment of the transcription factor from target genes and subsequent nuclear export . As ments it was . In suppradation .However, even if the lack of Fyn recruitment in AT cells can explain the prolonged activation of NRF2 and the non-appearance of the post-induction phase, it does not account for the observed triggering of the “induction phase”. Therefore, we shifted our attention to the second inhibitory kinase, p38.ATM(-/-) cells show enhanced activity of the p38 mitogen-activated protein kinase (MAPK) [The p38 subfamily of MAPKs regulates gene expression in response to various extracellular stimuli and exerts a critical role in inflammation and stress response . P38 is e (MAPK) . In partFig 5A and 5B). In addition, we found that 24-hour DEX treatment was able to reduce this activation significantly. In addition, the time-dependent inhibition of p38 by DEX in AT129RM cells clearly suggests that the greatest effect is after 24 hours .To verify this hypothesis we evaluated the p38 activation status in WT and AT cells with or without DEX. In agreement with the findings of Kim and Wong , we obseDUSP1), which codes for the corresponding MAPK phosphatase 1 (MKP1) protein [, consistent with the previously observed p38 inhibition . Panels D, F and G in Fig 5 show the time-dependent inactivation of p38 accompanied by the concomitant DUSP1 mRNA induction and the corresponding MKP1 protein expression in the AT129RM cell line.Inactivation of MAPK in mammalian cells is achieved by a family of dual-specificity MAPK phosphatases (MKP), which target the two critical phosphorylation sites in the activation loop of MAPK. Among the characterized MKPs, MKP1 is known to be responsible for the de-phosphorylation and consequent inactivation of p38 , 48. As  protein , 45. We To verify whether NRF2 activation was to some extent attributable to the observed p38 inhibition by DEX, we investigated whether a p38 inhibitor could mimic the same effect of the glucocorticoid analogue on NRF2 activation, in the WT cell line and in one AT cell line as models.Fig 6A and 6B we can observe how DEX was able to significantly inactivate p38 only in the AT cell line, and this occurred in an MKP1-dependent manner, as supported by the concomitant DUSP1 mRNA induction , while the SB202190 inhibitor was not effective in significantly lowering p-p38 levels in either cell model. Nevertheless, the SB202190 inhibitor acts in a different way than DEX . We therefore used a different approach to demonstrate effective p38 inhibition. Considering that active p38 is able to favour the stability of some pro-inflammatory transcripts (such as IL-23p19) [Fig 6D shows that the inhibitor actually decreased the levels of the IL-23p19 transcript in a dose-dependent manner. After having verified the efficacy of both drugs in inhibiting p38, we compared the expression of a selected NRF2 target gene used as a control for transcription factor activation. We found that DEX was able to significantly increase the glutamate-cysteine ligase catalytic subunit (GCLC) mRNA, whereas SB202190 was not .To this end, we treated WT and AT129RM cells with increasing doses (50 and 100 nM) of the p38 inhibitor, SB202190. SB202190, which does not affect other MAKPs , is a highly selective potent inhibitor of all p38 isoforms, with an IC50 of 50–100 nM . In paraFig 6F and 6G); this is consistent with the low (not significant) induction of model target genes . Taken together, these results led us to rule out the contribution of p38 in NRF2 activation after DEX treatment. We therefore refocused our attention on the first branch of our outline in Fig 1A, which addresses the nuclear-cytoplasmic shuttling of NRF2 under oxidative conditions.In light of these results, we turned our attention to the nuclear shift of NRF2 demonstrated in  and repeFig 1A and 1B). KEAP1 is a relatively cysteine-rich protein that is mainly located in the cytosol as a dimer targeting NRF2 to proteasome degradation [One of the most studied mechanisms of activation of NRF2 are the redox stressors, which put the Kelch-like ECH-associated protein 1 (KEAP1) out of play. KEAP1 is an NRF2 repressor that is inactivated by stress-stimulated modification of thiols elicited by oxidants and/or electrophiles . Modificradation .As we can observe in Indeed, we found a net decrease in the KEAP1 protein after DEX treatment, but only in AT cells . To confFig 7F show that oxidized forms of KEAP1 (namely Ox1 and Ox2) are indeed detectable in non-reducing SDS-PAGE. These two higher isoforms are supposed to be formed by an intermolecular disulphide bridge between the cysteine residues of two KEAP1 molecules (homodimer) or KEAP1 together with another protein (heterodimer) [, we found an increase in WT and especially in AT cells after DEX treatment, which is consistent with a further inactivation of KEAP1 and consequent activation of NRF2. To directly prove that KEAP1 is indeed responsible for modulating NRF2 after DEX treatment, we investigated whether the increase in the oxidized form of KEAP1 corresponds to alterations in NRF2 ubiquitination levels. To this end, NRF2 protein was immunoprecipitated from both cytosolic and nuclear extracts and then immunoblotted with anti-ubiquitin antibody. The results obtained show that NRF2 ubiquitination status was indeed decreased after DEX, which is consistent with the increased oxidation of KEAP1 . To the best of our knowledge, this is the first report addressing the effect of DEX as an NRF2 inducer that acts by disrupting the NRF2-KEAP1 complex. Other authors [Highly reactive cysteine residues, within the domains of KEAP1, set up at least three different redox sensor systems. Cys151 is crucial for oxidation by tBHQ and sulphoraphane, and can form a transient disulphide bridge with Cys151 in another KEAP1 subunit ; Cys273 rodimer) . However authors –58 have In conclusion, in the present investigation we have shown that in AT cells, the activation of NRF2 by the glucocorticoid analogue DEX does not occur via activating or inactivating kinases, as commonly described in the literature. In fact, neither AKT nor ERK1/2 activating kinases were activated and GSK3 inactivating kinase was not inhibited, thus excluding their involvement in NRF2 activation. Regarding the effect of DEX on the p38 MAPK inactivating kinase, despite a significant p38 inhibition, this effect does not seem to have a role in NRF2 activation or stabilization. Our results suggest that the effect of DEX is more likely exerted directly on the KEAP1 inhibitory protein and that this effect involves the oxidative pathway. As we have shown, both KEAP1 mRNA and protein levels are reduced after drug treatment. In addition, an increase in oxidized and inactive forms of KEAP1, which corresponds to a decrease in NRF2 ubiquitination status, was observed. This result is consistent with a recent study describing the transcriptomic and proteomic profile of AT cells treated with DEX. The study described the upregulation of several proteins involved in protein metabolism and processing in the endoplasmic reticulum, protein folding and post-translational modifications . Taken tS1 FigA),B) Western blot analysis for Fyn in the total extracts of WT (238RM) and AT  cells treated with DEX or not treated, as indicated. β-ACTIN served as a loading control. C), D) Quantification of the relative amount of Fyn protein in the total cell extracts of WT and AT cell lines tested in A and B. Blots shown are representative and the histograms are the means and SEM of three independent experiments.(DOCX)Click here for additional data file.S2 FigA) KEAP1 mRNA level in AT28RM and AT50RM cells treated with DEX or not treated, as indicated. HPRT1 was used as the housekeeping gene for data normalization. B) Western blot analysis for KEAP1 in the total extracts derived from the same cell lines analysed in A. β-ACTIN served as a loading control. C) Quantification of the relative amount of KEAP1 protein in the total cell extracts of AT cell lines tested in B. Blots shown are representative and the histograms are the means and SEM of four independent experiments .(DOCX)Click here for additional data file.


In another treatment using non-differentiated HSG cells, it wasevident that both expression and secretion of cystatin C increased upon additionof the β-adrenergic agonist, and these effects were essentially eliminated bythe antagonist propranolol. The HSG cell line appears to have potential as amodel for exploring the mechanism of cystatin secretion, particularly the S-typecystatins that originate primarily in the submandibular glands.Physical exercise is known to activate the sympathetic nervous system, whichinfluences the production of saliva from salivary glands. Our examination ofsaliva collected from highly trained athletes before and after a number ofphysical competititions showed an increase in the secretion of S-type cystatinsand cystatin C as a subacute response to aerobic and anaerobic exercise. Theelevation in salivary cystatins was transient and the recovery time coursediffered from that of amylase and other salivary proteins. An  Saliva is a complex fluid containing proteins, carbohydrates, inorganic molecules,lipids, nucleic acids and water . It can The autonomic nervous system responds differentially to physical exercise . While tin-vitro model system was set upusing a human submandibular gland cell line to explore the association betweencystatin secretion and a β-adrenergic stimulus. Evidence is presented forco-migration of type-S cystatins and cystatin C, suggesting that they may travel inthe same packet.The multiple observations that exercise can alter the protein profile in salivaprompted us to explore the changes detectable in saliva from trained athletes. Wereadily identified proteins of the cystatin family, a group of cysteine proteases of~16 kD, because they increased markedly with exercise, and are much smaller proteinsthan amylase. Previous studies have reported cystatins as a product primarily ofsubmandibular glands, while amylase is derived mainly from parotid glands . Intriguad libitum. Experimentalprotocols were approved by the Ethics Committee of Clementino Fraga FilhoHospital of the Federal University of Rio de Janeiro (Protocol Number:030/10).Twenty male Brazilian marines  was used as an anaerobic test thatrequires maximum effort. It was applied individually in Phase 4 , outdoorAn aerobic incremental intensity test consisted of a single session dividedinto four bouts, at 65%, 75%, 85% and 95% of exercise  –phase 5g for 10 min at 4°C) and the supernatant transferred to anew tube for storage at -80°C. In phase 5 (see g for 20 min at 4°C) and stored at -80°C for analysis oflactate.Biological samples were collected by trained professionals. Because of theintensity of some of the exercises, athletes required a period of recoverybefore they could breathe without gasping, a problem also noted by others . Therefose 5 see , blood  (n = 6 for each set). Protein from HSG cell lysates and culturemedium was quantified by BCA assay kit .Blood for plasma lactate determinations was collected from an antecubital veinbefore and after each increment of intensity in phase 5 . Saliva For Western blots, proteins separated on gradient SDS-PAGE gels were transferredat constant voltage (100 V) to a polyvinylidene difluoride (PVDF) membrane for90 min at 4°C in transfer buffer  methanol). Next, membranes were dried and then rehydrated for an overnightincubation with primary antibodies for cystatin.We were unable to obtainantibodies that were specific to each of the S-type cystatins, which are 90%identical in their primary sequence, so we used a monoclonal antibody for S-typecystatins as a group  diluted 1:1000, and/or a polyclonal antibody against cystatin C (P14)  diluted 1:1000. The nextmorning, membranes were washed and then incubated 1 h with the appropriatesecondary antibodies, diluted 1:10,000: for S-type cystatins, this was goatanti-mouse antibody conjugated with IRDye 800CW  and for cystatin C, this was goat anti-rabbit antibodyconjugated with IRDye 680RD , diluted 1:10.000. Afterwashing, fluorescence was captured and quantified on an Odyssey infrared scanner(LI-COR).The quantity of cystatin C in cell lysates and culture medium was determined byan ELISA assay, which was available for cystatin C but not cystatins type S  as instructed by the manufacturer.Optical density was measured at 450 nm in a VersaMax ELISA Microplate Reader. The results are presented in ng ofcystatin C per μg of total protein.4)2CO3 (100 mM). Next, proteins weredigested with trypsin at a final concentration of 10 ng/μL in(NH4)2CO3 (25 mM). Resulting peptides wereextracted with 50% acetonitrile/0.1% trifluoroacetic acid (ACN/TFA) and appliedto a C18 ZipTip desalting column of 0.6 μL (EMD-Millipore). The final sampleeluted from ZipTip with ACN/TFA was subjected to fractionation and desalting byliquid chromatography in-line to an electrospray ionization quadrupoletime-of-flight mass spectrometer (ESI-Q-TOF) as described [www.matrixscience.com) by comparison to the tandem mass spectraof the National Center for Biotechnology Information proteins and MassSpectrometry protein sequence database for Homo sapiens. Onemissed cleavage was permitted per peptide and a peptide mass tolerance of ± 0.1kDa was allowed. Cysteines were assumed to be carbamidomethylated with avariable modification in methionine by oxidation.For a more detailed confirmation of low-molecular-weight peptides that increasein saliva with exercise, we turned to mass spectrometry. Electrophoresis (1DSDS-PAGE) was performed in a Bio-Rad system . Threemicrograms of protein obtained from pooled saliva drawn before and after threepentathlon events (Phase 2 in escribed . Proteinin-vitro assay. A humansubmandibular gland (HSG) cell line [2 and 95% air. For differentiation into acinus-like structures,cells were seeded at a density of 6 x 103 cells/cm2 intissue-culture dishes coated with 10.8 mg/mL GFR Matrigel  and maintained in complete medium for 3 days.For insight into cystatin secretion in response to adrenergic stimuli at thecellular level, we used an ell line  was a ge°C. Treatmentof cells differentiated into acini was carried out with 100 μM IPR or vehiclefor 60 min without a prior medium exchange.For experimental treatment without differentiation into acini, HSG cells wereseeded in 6-well culture plates that received fresh culture medium when thecells reached 80% to 85% confluence. After 24 h, culture medium was supplementedwith isoproterenol (IPR) and/or propranolol (PRO) at 100 μM. After 60 min,culture medium was harvested separately and stored at -80 ºC for later analysis.Cells were washed with PBS, collected and stored at -80The HSG cell line used in this study originated in Japan in 1981 from irradiatedcells of a human submandibular gland. It exhibits various features expected forcells of salivary gland origin, such as formation of acinus-like structures,response to isoproterenol, and synthesis and release of S-type cystatin, typicalof submandibular glands . PreviouSpecific primary antibodies to cystatin C and (as a group) to the 3 salivarytype-S cystatins  make it possible to identify cystatins inmixtures of cell proteins and quantify them using fluorescent secondaryantibodies that are visible in a confocal fluorescence microscope. Stimulatedand unstimulated HSG cells in culture were fixed with 2% paraformaldehyde and0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 5 min, rinsed threetimes with PBS and then permeabilized with 0.5% Triton-X100 in PBS for 3 min.Next, specimens were blocked with 5% BSA in PBS for 1 hr prior to an overnightincubation with primary antibody, either the monoclonal human anti-mousecystatin S/SA/SN or rabbit anti-goat cystatin C at a dilution of 1:400. The nextday, specimens were washed three times in PBS with 0.1% Tween 20 for 5 min eachfollowed by incubation for 2 h with a 1:400 dilution of a rabbit anti-goatsecondary antibody labeled with Alexa 594  for cystatin C and a donkey anti-mouse secondary antibodylabeled with Alexa 488  for cystatins type S. DNA wasdetected directly with DAPI .Prior to mounting, coverslips were washed an additional three times in PBS-Tween20 for 5 min each.For quantification of the cystatin distributions following an adrenergicstimulus, raw fluorescence images were obtained from multiple fields of HSG cellcultures that were fixed, permeabilized and labelled as described in theprevious paragraph and examined with an LSM510 confocal microscope . Excitation of Alexa 594 was at 590 nm and emission at 620 nm,while Alexa 488 was excited at 500 nm and emission collected at 520 nm. Todetermine the total surface area of an acinus-like structure, each of whichcontained a different number of individual cells, the software ImageJ v.1.51h was usedData are shown as the median ± S.D, except as noted. Differences between samplegroups were compared by a Mann-Whitney Rank Sum Test or one-way ANOVA, asindicated in the figures. Statistical significance was defined as p<0.05.Initially, the protein pattern in saliva was compared between six pooled samplesobtained from 20 athletes before and after the first event each day of athree-day pentathlon competition  in all four columns, four individualswere exceptions to the trend and did not show an increase in cystatin secretionafter specific physical exercises: one in cystatins type S-Ergo; two incystatins type S-RAST; and one in cystatin C-RAST . For cysSince an increase in cystatin concentration could occur if there were ageneralized decrease in salivary volume (for example by evaporation or a lowerflow rate), we also measured volume and total protein concentration. cf. ratios Post/Pre for protein inOn the other hand, tests executed by the athletes at maximum or near-maximumeffort raised the overall protein concentration at 5 min , and theTo evaluate the influence of exercise intensity and the time course ofpost-exercise recovery we examined cystatins S/SA/SN in athletes performing anaerobic cycloergometer protocol at four different load increments that lasted 4min and reached 65, 75, 85 and 95% of In the experiment of  pmol/mL . Thus we pmol/mL ,32.A second indication of a strong sympathetic stimulus was provided by measurementsof salivary α-amylase, a recognized sympathetic response component. inset), then fell slowly over 15 minutes. The main panel showsthat total protein and type-S cystatins were also maximal at 5 min, butcystatins fell immediately to 54.9% of the resting value at 10 min beforerecovering, while total protein stayed up  for preliminaryexperiments.In salivary glands, agonists of the β-adrenergic system bind to β-adrenergicreceptors that activate adenylyl cyclase to generate intracellular cAMP, whichin turn activates cAMP-dependent protein kinase (PKA) to increase exocytosisfrom acinar cells ,34,35. TAn ELISA assay confirmed the response to a β-adrenergic stimulus in HSG cellsplated on plastic. The secretion of cystatin C to the culture medium increased11 fold with the addition of isoproterenol (IPR) (left). Staining of the nuclei by DAPI (blue) shows the presenceof multiple cells in each acinus-like cluster. The immunofluorescentlocalization of cystatin C (red) and S-type cystatins (green) in thenon-stimulated state (upper panels) reveals a generalized intracellulardistribution, and the yellow of the merged images within the cell clusters atleft in panel A is consistent with co-localization of the two cystatins in thesame intracellular compartments.When immortalized HSG cells are cultured on an appropriate organic substrateinstead of unadorned plastic, they spontaneously differentiate into acinus-likestructures that assume morphological and functional features of submandibularglands, including the expression of cystatin SN . In the left), thissuggestion of co-localization was reduced but there was a substantial release ofcystatin C and S-type cystatins to the medium, now visible in yellow in themerged image outside as well as inside the acinus-like structures followingfixation and exposure to fluorescent antibodies. At a higher magnification(center and right panels), a comparison ofthe distribution of the cystatins between the center and periphery of eachmulticellular acinus was carried out by measuring the fluorescence in aninterior circle (I) and a peripheral ring (P), which were used to calculate afluorescence ratio (P/I). In the absence of IPR , both cystatins showed a preferential distributiontowards the interior of acinus-like structures. For cystatin C, this ratio (P/I)increased from 6.4 ± 2.4 without IPR to 19.3 ± 4.2 with the addition of IPR; andfor S-type cystatins, P/I increased from 3.3 ± 2.9 fold without IPR to 15.8 ±3.0 with IPR (median ± S.D.).With the addition of 100 μM isoproterenol , left, tOf the more than 2,000 proteins identified in human saliva, only a subset of 200 to300 are secreted by the salivary glands . The genTo our knowledge, our study is the first description of cystatins as proteins whoseconcentration in saliva responds to exercise. Our measurements of salivary amylaseand plasma lactate in trained athletes are consistent with the premise of exercisewith a high β-adrenergic component. While cystatin C is produced in many differentorgans, the S-type cystatins are mainly expressed in salivary glands. The S-typecystatins are of particular interest since they originate primarily from thesubmandibular glands and not, like amylase, from the parotids. In our experimentsthe cystatins appeared as a low-molecular-weight protein band in whole saliva thatshowed a clear increase in intensity after athletes performed a variety of strenuousphysical activities. Mass spectrometry of the excised band identified two componentsof the cystatin-2 family, cystatin C and cystatins type S, as proteins sub-acutelymodulated by exercise. Commercial antibodies were available for cystatin C and also(as a group) for cystatins type S. Using these antibodies in quantitative Westernblots, we found that both aerobic and anaerobic exercises performed at or nearmaximal intensity led to an increased presence of both cystatins in saliva. Bothshowed statistically significant changes when analyzing the 12 athletes as agroup.in vitro.In recent decades, saliva has gained acceptance as a readily accessible body fluidthat provides indirect access to physiological changes accompanying exercise, aswell as oral health at rest and the monitoring of therapeutic drugs circulating inthe blood . This stNevertheless, it is interesting to examine the question of what purpose might beserved by increased cystatin secretion during exercise; in the last decade anincreasing number of other salivary peptides and proteins have been investigated inthis context. Both cystatins increase in some pathological states and decrease inothers ,18,41,42A potential link between cystatins and exercise appears in recent studies ofautophagy, which occurs in salivary glands under stress but has not beeninvestigated extensively and has not yet been associated with cystatins in theseorgans . CystatiTaken together, these data do not provide an answer, but they do suggest one or moretypes of protective role for cystatins. If salivary cystatins protect the buccalmucosae against microorganisms, why would they increase during exercise? Oftenreferred to as AMPs , many of the defense elements found insaliva  increase in concentration or secretion rate immediately followingshort, intense exercise ,48,49, aSalivary secretion is highly variable and each gland produces a different volume, aswell as a different pattern of proteins . The incWhen the saliva from physical tests was analyzed for each athlete at the same timepoint, a large variance in the response of S-type cystatins and cystatin C wasobserved, ranging from as much as a 9.8-fold increase for one individual to adecrease below resting value for three others . This heathletes . Howeverathletes , and proathletes ,16,40.A limitation of our study design is the absence of data on the increase incatecholamines that accompanied our chosen exercises. However, salivary amylase andplasma lactate have both been demonstrated repeatedly to respond during exercise inparallel with the release of catecholamines ,31,39. Win vitro with HSG, a cell line originally derived fromhuman submandibular glands. Initial experiments with non-differentiated cells showedthat the cellular release of cystatin C was stimulated 11 fold by a beta-agonisttreatment with IPR. Conversely, the antagonist propranolol decreased production by9-fold. When cells were plated on Matrigel and allowed to differentiate intoacinus-like structures, changes in the subcellular distribution of cystatins type Sand cystatin C were tracked by calculating the ratio of peripheral to internalcystatins. Even in unstimulated cell groups, the periphery had a higher fluorescencelabeling of cystatin C and cystatins S, suggesting the presence ofcystatin-containing vesicles. Activating the β-adrenergic signalling pathway led toa much higher ratio of cystatins at the periphery. It could not be determinedwhether the IPR treatment accelerated the normal secretion pathway, acted on aseparate pool of vesicles or reflected de novo production throughincreased transcription and translation. In rats, IPR increases the exocytosis ofα-amylase through a significant increase in interaction between the accessoryproteins v- and t-SNARE, which mediate vesicle fusion [Supporting evidence for cystatin secretion as a response to β-adrenergic stimulationwas obtained e fusion . It is ae fusion . More inin-vitro model showed that the release phase for bothcystatins was associated with redistribution of internal stores toward the peripheryof acinus-like structures derived from human submandibular glands.Increased concentrations of both cystatin C and cystatins type S were measured insaliva as a response to both aerobic and anaerobic exercise, in association withathletes´ exerting near-maximal effort. The increases in cystatin were rapid andtransient, returning to basal levels within 15 min. While increased salivary amylaseas well as plasma lactate were consistent with activation of the sympathetic nervoussystem by the physical activity, the time course of recovery was much faster forcystatin than for amylase or total protein, suggesting different post-secretoryprocesses. An


Bothrops lanceolatus snakebite, although relatively uncommon (~30 cases/year), may result in serious complications such as systemic thrombosis and local infections. Infections have been hypothesized to be related to bacteria present in the snake’s oral cavity. In this investigation, we isolated, identified, and studied the susceptibility to beta-lactams of bacteria sampled from the oral cavity of twenty-six B. lanceolatus specimens collected from various areas in Martinique. Microbiota from B. lanceolatus oral cavity was polymicrobial. Isolated bacteria belonged to fifteen different taxa; the most frequent being Aeromonas hydrophyla (present in 50% of the samples), Morganella morganii, Klebsiella pneumoniae, Bacillus spp., and Enterococcus spp. Analysis of antibiotic susceptibility revealed that 66.7% of the isolated bacteria were resistant to amoxicillin/clavulanate. In contrast, the majority of isolated bacteria were susceptible to the third-generation cephalosporins . Microbiota from B. lanceolatus oral cavity is polymicrobial with bacteria mostly susceptible to third-generation cephalosporins but rarely to amoxicillin/clavulanate. In conclusion, our findings clearly support that first-line antibiotic therapy in the B. lanceolatus-bitten patients, when there is evidence of infection, should include a third-generation cephalosporin rather than amoxicillin/clavulanate.In Martinique,  Bothrops lanceolatus , Streptococcus spp., Aeromonas spp., Staphylococcus aureus, and Clostridium spp., as reported in several observational studies from snakebites worldwide [The oral microbiota of snakes comprises a wide range of aerobic and anaerobic microorganisms, including orldwide ,6,7,8,9.B. lanceolatus oral cavity. Then, based on the nature and susceptibility of the isolated bacteria to beta-lactams, our objective was to propose the most adequate preemptive antibiotic therapy that should be administered in the suspicion of infection in B. lanceolatus-bitten patients in Martinique.Therefore, we designed an experimental study aiming to identify the main bacterial microbiota from B. lanceolatus snakes captured by personnel of the National Office of Forests in Martinique were studied. Snakes were grouped according to their geographical origin, i.e., wet (forest and near water areas) versus dry regions (peri-urban areas). The snake’s oral cavity was opened and sampled from the vicinity of the fangs using sterile cotton swabs. Samples were subjected to Gram staining and examined for bacterial growth. They were plated on non-selective blood agar and chocolate agar and cultured at 37 °C for 2–7 days and the color and shape of the colonies were observed. Species identification was performed with API-20E and API-20NE systems . Antimicrobial susceptibilities of all isolates to beta-lactams were determined by the disk diffusion methods based on the definitions of the Antibiogram Committee of the French Microbiology Society [This experimental study was conducted at the Microbiology Laboratory of the University Hospital of Martinique. During eighteen months, all  Society . The inhp-Values less than 0.05 were considered to be statistically significant.Data are expressed as absolute values and percentages. Comparisons between the subgroups were performed using Chi-2 tests. B. lanceolatus were captured and their oral cavity sampled for bacteriological culture. Twenty snakes were captured from the “wet” zones versus six from the “dry” zones. All samples obtained from the snake mouths tested positive for bacterial growth. During the study period, twenty-six specimens of Aeromonas hydrophila, Morganella morganii, and Klebsiella pneumonia (A. hydrophila was isolated in 50% of the samples (13 cases). In four cases (15.4%), pure cultures were obtained or the bacterium was quantitatively predominant. A. hydrophila was isolated in 60.0% of cases from snakes captured from a “wet” zone compared to 16.7% from snakes captured from a “dry” zone (p = 0.06). In 20 cases (76.9%), the sample was polymicrobial. The most frequently isolated bacteria were neumonia . A. hydrIn 66.7% of the cases, the isolated microorganisms were resistant to amoxicillin/clavulanate, while bacteria isolated were more frequently susceptible to the third-generation cephalosporins including cefotaxime (73.3%) and ceftazidime (80.0%).B. lanceolatus oral cavity in Martinique were A. hydrophila, followed by other members of the Enterobacteriaceae family. The cultures were polymicrobial and bacteria were susceptible to third-generation cephalosporins in most cases, coinciding with the majority of studies that focused on other snakes from different regions of the Americas [In this study, the most frequently isolated microorganisms from Americas ,10,11,12B. lanceolatus is endemic to Martinique in the French West Indies. Its venom can cause severe local damage, ranging from blistering and tissue necrosis to secondary cellulitis and abscess in severe cases [B. lanceolatus oral flora is polymicrobial, combining Gram-positive, Gram-negative bacteria were isolated, with varying proportions across the Martinique sub-regions. re cases . As prevre cases ,4,5,6,7,A. hydrophila was the most commonly isolated bacterial species, especially when the snake was captured from a “wet” zone. This bacterium is known to cause tissue damage and necrotizing fasciitis [B. lanceolatus bite may result from the tissue-damaging properties of the venom combined with the necrosis potency of A. hydrophila. It is obvious that tissue damage induced by the venom is responsible for perfusion abnormalities in the zone of the bite. This is due to microvascular damage and thrombosis induced by the venom itself, in addition to microcirculatory compression due to local edema and compartment syndrome in some cases. In these conditions, tissue damage and ischemia, with an associated decrease in the local innate immunity, is an excellent medium for the growth of bacteria ejected from the snake’s oral cavity at the moment of the bite, as previously demonstrated for Staphylococcus aureus in an experimental model [asciitis ,14,15, ral model . B. lanceolatus species have a high incidence of bite abscesses and, thus, represent an ideal candidate to use antibiotics, if they are given when there is the evidence of infection soon enough after the incident and when appropriate antibiotics are employed.In the case of a moderate to severe snakebite, most authors and guidelines recommend the use of antibiotics to reduce complications by preventing secondary infection. However, the 2010 World Health Organization statements advised against the use of preemptive antibiotics in snake bites except in certain circumstances . Our stuBothrops snakebites in the Western Brazilian Amazon region [The most recommended antibiotic for snakebite treatment is amoxicillin/clavulanate, albeit without strong evidence supporting this recommendation. The Infectious Diseases Society of America (IDSA) guidelines for the diagnosis and management of skin and soft-tissue infections recommended amoxicillin/clavulanate as the preemptive antibiotic of choice to reduce complications by preventing secondary infection from animal bites other than snakes . Howevern region . Similarn region . In thisB. lanceolatus-bitten patients with signs of local infection. However, since the systematic prophylactic antibiotic use in snakebite patients remains a matter of debate [B. lanceolatus cases should be restricted to cases where there is evidence of infection. Our findings were consistent with these two studies ,20, sincf debate , the useB. lanceolatus oral cavity is polymicrobial with bacteria mostly susceptible to third-generation cephalosporins and more rarely to amoxicillin/clavulanate. In the absence of definitive international guidelines for the management of snakebite-associated infections, our findings support that the first-line antibiotic therapy should include a third-generation cephalosporin rather than amoxicillin/clavulanate in patients suffering infections as a consequence of B. lanceolatus snakebites in Martinique.Microbiota from


We found that Pertussis Toxin (PTX) treatment or sequestering Gβγ reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gβγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/βγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-Δ386 mutant, which has impaired agonist-dependent β-arrestin-2 recruitment; however, this situation may have resulted from the low β-arrestin expression in the COS-7 cells. When β-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/βγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF The pre pathway . Subsequormation . Besidesreceptor , 6. Seveal cells , 8 whileHO cells . In huma kinases , 11. Mos kinases , 12, 13.s-coupled GPCRs has been proposed to mediate upstream activation of the ERK cascade in cells with high B-Raf expression -stimulated phosphorylation of ERK1/2 in CRF1R-expressing human myometrial, CHO, and HEK293 cells -PKA pathway by Gpression . Consistf levels . Kageyamf levels  found, hHO cells , 12, 17.93 cells , and in 93 cells . PKC inheceptors . These f202 and Tyr204 during CRF1R and CRF2R signaling in various cell lines has been confirmed by inhibiting ERK1/2 activation with PD98059 (1R-expressing CHO and HEK293 cells (2R activation by urocortin 2 (Ucn2) and urocortin 3 (Ucn3) has also been shown to signal via the Ras→C-Raf→MEK1/2 cascade in rat cardiomyocytes, based on the ability of manumycin A (a Ras inhibitor) and R1-K1 to abolish ERK1/2 phosphorylation R can stimulate phosphorylation of Akt (2(b)R Akt signaling in HEK293 cells is mediated by pertussis-sensitive G proteins and PI3K but not by cAMP-stimulated activation of PKA or EPAC, or by PKC , total ERK1/2, total EGFR and phospho-c-Src (Tyr416) from Cell Signaling Technology ; (ii) total ERK2, total c-Src and total Akt from Santa Cruz Biotechnology ; (iii) phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173) and phospho-EGFR (Tyr845) from Biosource-Invitrogen ; (iv) phospho-Akt (Ser473) from Biosource International ; (v) phospho-PYK2 (Tyr402) from Calbiochem ; (vi) polyclonal anti-human HB-EGF antibody from R&D Systems ; (vii) secondary antibodies conjugated to horseradish peroxidase from Zymed Laboratories .General reagents utilized were as follows: (i) DMEM, fetal bovine serum (FBS), antibiotic solutions and other cell culture reagents from Invitrogen/GIBCO ; (ii) Pertussis Toxin, reagents for electrophoresis and other highly pure chemicals from Sigma-Aldrich . Human/rat CRF was purchased from Bachem . Phorbol 12-myristate 13-acetate (PMA), and the following specific inhibitors were purchased from Calbiochem : Src inhibitor PP2; EGFR tyrosine kinase inhibitor AG1478; PKA inhibitor H89; PKC inhibitor bisindolylmaleimide I, BIM; PI3K inhibitors wortmannin and LY294002; MMP inhibitor GM6001, and Protease Inhibitor Cocktail Set III. BB-94 (batimastat) was obtained from British Biotechnology Ltd . Antibodies for Western blots were obtained from the following sources: (i) phospho-p44/42 MAP kinase (Thr1R (HA-CRF1R), the HA-CRF1R-Δ386 mutant, and the β-arrestin-2 constructs were previously described . Transient transfections were performed using LipofectAMINE  as described previously ; HA-CRF1R and ct-βARK; HA-CRF1R and dn-Src; HA-CRF1R and dn-Akt, or HA-CRF1R and full-length β-arrestin-2. After replacing the transfection medium with fresh growth medium, transfected COS-7 cells were cultured for 1 day. Subsequently, cells were re-seeded in 6-well plates and cultured for an additional day prior to the experiment.COS-7 cells (from the American Type Culture Collection) were cultured at 37°C in a humidified atmosphere of 95% air, 5% COeviously . Cells wThe protocols for measuring total and phosphorylated ERK1/2, c-Src, PYK2, Akt, and EGFR have been previously published , 29. Aft1R were grown in 10-cm dishes and serum-deprived for 24 h before treatment with 100 nM CRF for 10 min at 37°C. Cells were washed twice with ice-cold PBS and lysed in Nonidet P-40 solubilization buffer . After immunoprecipitation of HA-CRF1Rs with anti-HA monoclonal antibody  and protein A/G PLUS-Agarose , the proteins were resolved by SDS-PAGE, Western blotted, and probed with anti-EGFR polyclonal or anti-HA monoclonal antibodies, followed by a horseradish peroxidase conjugate to identify co-immunoprecipitated proteins. Blots were also stripped with stripping buffer  and reprobed with anti-c-Src polyclonal antibody. Western blot detection of co-immunoprecipitated Src was carried out as described above. Blots were visualized and quantified, as indicated above.COS-7 cells transfected with HA-CRFpost-hoc analyses were performed using Dunnet or Bonferroni's multiple comparison tests to determine individual group differences.Data are presented as mean ± S.E.M. Analyses of variances (ANOVAs) across experimental groups were performed using PRISM™, Version 8.0 for macOS . If the one-way ANOVA was statistically significant, planned 1R caused transient phosphorylation of ERK1/2 that reached a peak at 5–10 min and declined thereafter toward the basal level over the next 30 min (1R-expressing HEK293 and CHO-K1 cells (data not shown), but the rate and magnitude of CRF-induced ERK1/2 activation was considerably less in these cell lines compared to COS-7 cells. In contrast, CRF1Rs expressed in SK-N-MC neuroblastoma cells  and maximal effect over the 0.1–1 μM range  of COS-7 cells transiently transfected with HA-CRFt 30 min . CRF  for 30 min prior to stimulation with CRF (100 nM). As shown in 1R-mediated activation of ERK1/2 was similar in COS-7 cells pretreated for 30 min with the highly selective PKA inhibitor Rp-cAMP (0–100 μM) or vehicle , increased (~1.6-fold increase over CRF stimulation) rather than decreased CRF-stimulated ERK1/2 activation  and by co-transfecting COS-7 cells with plasmids encoding the carboxyl terminus of βARK containing its βγ-binding domain (ct-βARK)  , which srylation , suggest by ~40% . Thus, o1R-mediated ERK signaling. In COS-7 cells transiently transfected with HA-CRF1R, EGF stimulation of the endogenous EGFRs caused ERK1/2 phosphorylation in a time-dependent manner, reaching a maximum effect after 5 min of stimulation, which persisted for at least 30 min , especially the EGFR, is often an important mechanism used by GPCRs to activate ERK1/2 , 34, we 1R were pretreated with the EGFR tyrosine kinase inhibitor AG1478 , a significant inhibition (~80%) of CRF-induced maximal ERK1/2 phosphorylation was observed   . Tyr1173with CRF . Togethetivation , 37. ThuMMPs catalyze the release of extracellular heparin-binding EGF (HB-EGF) ligand, which, in turn, binds to and activates the EGFR, thereby stimulating ERK1/2 phosphorylation –40. Alth416, which is a requirement for Src activation . To further evaluate the role of Src in CRF1R ERK1/2 signaling, COS-7 cells were co-transfected with the CRF1R and a dn-Src. Overexpression of inactive Src prevented ERK1/2 activation by CRF (50 = 2 μM)  . These f1R-induced transactivation of EGFRs. In this context, previous research has established that Src can activate EGFR signaling by phosphorylating Tyr845 of the EGFR protein . Interestingly, it was also observed that under the same immunoprecipitation conditions, the EGFR is also present in the CRF1R/co-precipitated complex, even in the absence of CRF stimulation (1R/EGFR interaction (~2.5-fold increase over control). These observations suggest that CRF promotes the formation of a multiprotein complex that would allow rapid EGFR phosphorylation at Tyr845 by Src, present in this complex.We next determined if CRF-stimulated Src activation is required for CRF protein . As showwith PP2 . In a rewith PP2 , it was mulation . After s1R leads to PYK2 phosphorylation in COS-7 cells. As shown in It has been shown that, in parallel to Src activation by many GPCRs, the proline-rich tyrosine kinase 2, PYK2, is also phosphorylated and activated, and in association with Src is required for the subsequent transactivation of EGFR , 48. Thei protein and Gβγ subunits in this process. It is important to note that none of the observed effects of PTX and ct-βARK on CRF actions were present on EGF-stimulated ERK1/2 and Akt phosphorylation  , or LY29F on Akt , therebyHO cells , we invee ERK1/2 –53. Howee ERK1/2 –56. In te ERK1/2  in a conignaling . To examignaling , suggest845  EGFR transactivation (1R may involve two mechanisms: (i) a strong dependence on upstream Src activating PI3K and then Akt  a then Akt .1R, which also shows a diminished agonist-dependent β-arrestin-2 recruitment  phosphorylation and transactivation of the EGFR, (ii) activation of PI3K. Interestingly, CRF1R-induced Akt phosphorylation also requires Src-mediated activation of PI3K as the main mechanism, but it is mostly independent of EGFR transactivation.In the present study, we investigated the molecular mechanisms associated with the activation of ERK1/2 and Akt signaling cascades by the human CRF1R and CRF2R. ERK1/2 is widely distributed in the brain and is considered an essential regulator of the molecular processes involved in response to stress R in HEK293 R-stimulated ERK1/2 activation in CHO, A7r5, and mouse neonatal cardiomyocyte cells  EGFR activation of the classical Ras/Raf/MEK/ERK pathway, and (ii) PI3K regulation and subsequent activation of ERK1/2  and anti-sauvagine (a CRF2R antagonist). Although CRF1R was reported to be expressed in the human heart (2(b)R is the major CRF receptor expressed in the HL-1 mouse atrial cardiomyocyte cell line with no measurable level of CRF1R mRNA. Their data detecting only CRF2R expression in HL-1 cells is consistent with previous and more recent studies demonstrating only CRF2R expression in rat and mouse cardiomyocytes , located in the carboxyl terminus of the CRF1R (Pro398Thr399Ser400Pro401) may provide a site for the direct interaction between Src and CRF1R after agonist stimulation. However, the CRF1R-Δ386 mutant, which lacks the ProXXPro motif, induces a similar degree of ERK1/2 activation that is induced by the wild-type CRF1R, which suggests this putative region may not participate in the binding to Src  with isoproterenol, results in phosphorylation of the receptor on Tyr305  located at the end of the third intracellular loop (Tyr309Arg310Lys311Ala312), which may be a site where Src can directly interact with CRF1R. Further work is needed to establish the importance of this putative site in the agonist-induced CRF1R/Src interaction and Src activation.Moreover, there is evidence that Tyr phosphorylation of GPCRs plays a role in mediating GPCR-Src interactions . For insn Tyr305 , 85. The1R homologous desensitization and endocytosis is well-recognized, particularly for β-arrestin-2 (1R signaling is still not fully understood.β-arrestins are a small family of cytosolic proteins initially identified for their central role in GPCRs desensitization. Furthermore, β-arrestins act as adaptors in clathrin-mediated receptor endocytosis . In thisrestin-2 , 63, 87.restin-2 , 86. β-a1R-mediated ERK1/2 activation, β-arrestin-2-mediation of CRF1R internalization participates in the late phase of sustained ERK1/2 activation after G protein activation and B-Raf mediate the early phase of ERK1/2 activation (1R-interacting protein, inhibited CRF-induced-CRF1R internalization in a PDZ-binding motif-dependent manner by suppressing β-arrestin-2 recruitment. Intriguingly, neither the overexpression of PSD-95 nor the knockdown of endogenous PSD-95 affected CRF-mediated activation of ERK1/2 (Regarding β-arrestin regulation of CRF/CRFtivation . Howeverf ERK1/2 .1R-mediated ERK1/2 activation observed in COS-7 cells. Using a phosphorylation-deficient mutant CRF1R, which has a decreased interaction with β-arrestin-2 (1R-mediated ERK1/2 activation observed in COS-7. This finding, however, can be explained in part by the low expression level of β-arrestins in COS-7 cells (1R-mediated ERK1/2 activation (1R internalization (1R MAP kinase activation in cells with sufficient β-arrestin expression.Under this experimental evidence and due to the importance of β-arrestins in the scaffolding and activation of Src and regulation of MAP kinase cascades, it was decided to evaluate their role in the CRF/CRFrestin-2 , no signrestin-2 , suggest-7 cells , 62. Thitivation . Likewislization . Thus, o1R help to elucidate the molecular mechanisms involved in response to stress mediated by this receptor. For instance, kinases in the ERK1/2-MAP kinase cascade, including Src and PYK2, are highly expressed in extended amygdala and forebrain neurons regulating anxiety defensive behavior and stress responsiveness (1Rs can also signal through other cellular pathways that may be involved in post-traumatic stress disorder pathophysiology. As we showed here, CRF1R activated by CRF stimulated rapid phosphorylation of Akt at Ser473 that is mediated by upstream Src and PI3K. Preclinical research has shown that activated Akt in the ventral tegmentum promotes resilience to anxiety- and depressive-like responses to stress (1R Akt signaling during trauma and severe stress may differ depending on the brain region. Hence, ERK1/2-MAP kinase and Akt cascade signaling by CRF1R regulated by Src, PYK2, and EGFR may have critical roles in stress-induced anxiety and depression.Our findings on signaling pathways activated by CRFsiveness –92. Acutsiveness –95. Furto stress , while ho stress . Therefo1R in COS-7 cells. Although CRF1R coupling to G proteins strongly activates PKA and PKC pathways, neither second messenger kinases were involved in CRF1R-mediated ERK1/2 signaling. However, Gβγ released during activation of CRF1R by CRF, particularly from Gi, stimulates phosphorylation of Src and PYK2, which in turn promotes transactivation of the EGFR through the formation of a heterotrimeric complex formed by the association of CRF1R, Src, and EGFR. EGFR transactivation, which occurred independent of MMP generation of the HB-EGF ligand, was essential for CRF-stimulated ERK1/2 phosphorylation while having only a small role in CRF1R-mediated Akt activation. Although PI3K activation contributes to CRF-stimulated ERK1/2 phosphorylation, CRF1R-mediated EGFR transactivation is independent of the PI3K/Akt pathway. In contrast, CRF1R Akt signaling while also being mediated by generation of Gβγ and phosphorylation of Src is weakly dependent on EGFR transactivation.In summary, the data presented herein establish that the tyrosine kinase Src serves as a central upstream regulator of ERK1/2-MAP kinase and Akt cascade signaling by the human CRFAll datasets generated for this study are included in the article/JO-R, RH, and KC conceived the project. JO-R, FD, and RH designed the experiments. GP-M, AF-G, JH-A, and MD-C carried out the experiments. JO-R, GP-M, and AF-G analyzed and discussed the data. JO-R and RH wrote the manuscript. All authors read and approved the final version of the manuscript and took a due care to ensure the integrity of the work.This work is dedicated to Dr. Kevin J. Catt, who was an extraordinary scientist, mentor, and friend who passed away on October 1, 2017.FD was employed by Novaliq GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


Toxoplasma gondii, the coccidian protozoan parasite with worldwide distribution, is the agent of toxoplasmosis. The disease is life threatening in congenital form and in immunocompromised patients. The present study was carried out in 2016 to evaluate the in vitro effects of nanosilver colloid on tachyzoites and bradyzoites of T. gondii, RH and Tehran strains.T. gondii, RH strain (type I) and bradyzoites and tissue cysts of T. gondii, Tehran strain (type II) and incubated for 30, 60, 90 and 120 minutes. The mortality rates of tachyzoites and bradyzoites were evaluated by trypan blue dye and MTT assay. Then SEM carried out to show the changes between control and exposed parasites.Different concentrations  of nanosilver colloid were added to tachyzoites of The greatest mortality rate was seen in 20 ppm concentration and after 120 minutes of exposure. By electron microscopy, the structural changes were seen in tachyzoites of RH and tissue cyst of Tehran strain in comparison with control groups.T. gondii, RH and Tehran strains.Nanosilver colloid was effective on both tachyzoites and bradyzoites of  Toxoplasma gondii. If a woman is infected during the pregnancy, it could be life threatening for developing fetus  were inoculated in peritoneal cavity of BALB/c mice as previously described  were inoculated in peritoneal cavity of BALB/c mice. After 2 month of infection, the mice were sacrificed and their brains were examined for the presence of tissue cysts by light microscopy. By using percoll 45%  tissue cysts were isolated from the brain tissues. Some of the tissue cysts remained intact and the others were ruptured to release the bradyzoites.This study was carried out in 2016. Briefly, tachyzoites of escribed . After 3Ethics Committee of Tehran University of Medical Sciences approved the study.Commercial nanosilver colloid  with the size of 70 nm was used. The concentrations of 5, 10, 20 ppm from commercial nanosilver were prepared .T. gondii RH and Tehran strains (1×104) were exposed with these concentrations for 30, 60, 90 and 120 minutes at room temperature, respectively. At the same time tachyzoites and bradyzoites of these strains were exposed with PBS (pH 7.2) as control group. Trypan blue dye  and observing with light microscopy was performed to estimate the number of dead parasites and then, the mortality rates were calculated. The intact tissue cysts of Tehran strain were incubated with the mentioned nanosilver concentrations, dyed with trypan blue and examined with light microscopy. Experiments were carried out duplicated.Tachyzoites and bradyzoites of T. gondii strains based on cytotoxicity assay. Briefly, after incubation of parasites with different concentrations  of nanosilver colloid, 10 μl of MTT was added and incubated for 4 hours in 37 °C, then 50 μl of DMSO solution  was added. The optical density was determined at 630 nm by an automated ELISA-reader . The viability rate was estimated for each sample according to the formula: OD test- OD blank / OD control- OD blank ×100. Then the mortality rate was calculated. Experiments were carried out duplicated.The MTT was performed to estimate the viability rate of tachyzoites and bradyzoites of For the scanning electron microscopy (SEM) the suspension of tachyzoites with the concentration of 20 ppm of nanosilver colloid was incubated for 2 hours. This procedure was done for tissue cysts as well. Samples were fixed in glutaraldehyde for 1 hour, stained with osmium tetroxide (OSO4), dehydrated, stained with gold and examined using scanning electron microscopy .T. gondii, RH and Tehran strains. The highest mortality rate was seen in 20 ppm and after 120 minutes of exposure. The results nanosilver exposed tachyzoites that stained by trypan blue and counted by light microscopy and the results of MTT assay are shown in Nanosilver colloid was effective in concentrations of 5, 10 and 20 ppm after 30, 60, 90 and 120 min of exposure both for tachyzoites and bradyzoites of T. gondii, Tehran strain that exposed with the concentrations of nanosilver colloid, stained with trypan blue and counted by light microcroscopy and the results of MTT assay were shown in The results of bradyzoites of The SEM of the intact tissue cyst of control group is shown in T. gondii infection is usually effective on active tachyzoites form whereas treatment is ineffective for the chronic form of infection with the presence of bradyzoites  were incubated with nanosilver colloid too, then dyed with trypan blue and examined by light microscopy. The bradyzoites in tissue cyst were dyed gradually and after 120 min the whole organisms were dyed blue that indicates the death of bradyzoites.In the present study, tachyzoites of The results of SEM showed the obvious structural changes in the tachyzoites of nanosilver exposed parasites in comparison with the control group. The crescent shape of tachyzoites changed to an atypical shape with large surface dimples and ridges. The SEM of tissue cyst showed the round and well organized shape in control group in comparison with a dimpled and damaged shape in nanosilver exposed one.T. gondii. Moreover this nano-particle has the ability to enter and kill the organisms inside the tissue cyst. Further studies for silver nanoparticle effects on T. gondii in cell culture and experimental models are suggested.Nanosilver colloid could kill tachyzoites and bradyzoites of